Cross-Reference to Related Applications

This application claims priority to U.S. Provisional Patent Application no. 63/101,640, filed on May 8, 2020, entitled OroVAX: Al PREDICTION OF SARS-CoV-2 IMMUNE-EPITOPE PEPTIDES FOR THE DEVELOPMENT OF AN ORAL VACCINE, naming Maurizio Chiriva-lnternati et al. as inventors, and designated by attorney docket number KIR-1004-PV, and to U.S. Provisional Patent Application no. 63/094,528, filed on October 21, 2020, entitled PEPTIDE COMPOSITIONS FOR THE TREATMENT OF PATHOGENIC INFECTIONS, naming Maurizio Chiriva-lnternati et al. as inventors. The entire content of each of the foregoing patent applications is incorporated herein by reference for all purposes.

Field

The technology relates in part to peptide compositions for the treatment of a disease or condition caused by a pathogen, such as a virus, to methods of selecting peptides for preparing the compositions, and to methods of treatment, including prophylactic treatment, using the compositions.

Background

Pathogenic diseases affect millions of people worldwide. These diseases often manifest as epidemics and can lead to serious medical conditions or even death in millions of people over a span of 1-2 years. The process of drug discovery from the bench to the market is long and expensive: taking 10 or more years and costing one to two billion dollars or more, making it sometimes onerous and impractical to implement the search for cures. Vaccines, as preventive means, offer a more attractive alternative as development costs and time frames are relatively lower. Vaccines arguably have been the most successful biomedical advance in the prophylactic treatment of pathogenic diseases or conditions. Each year, over 100 million children globally receive vaccinations to prevent diseases that were once widespread and linked to serious medical conditions or even death. Globally distributed childhood vaccines include those for measles, mumps, rubella, seasonal influenza virus, tetanus, polio, Hepatitis B, cervical cancer, diphtheria, pertussis, and others. Additionally, vaccines for diseases that are endemic to certain regions, such as Yellow fever virus whose mosquito vectors circulate in tropical and subtropical regions year- round, are administered to the general population. Altogether, it the World Health Organization (WHO) estimates that vaccination prevents between 2 and 3 million deaths annually (WHO).

Traditional vaccines, such as attenuated vaccines, also known as “live-attenuated” vaccines, are created by altering the genome, such that they have low pathogenicity or are harmless. Vaccines against measles, mumps, rubella, and others have been created this way and have had a good success rate, but they have sometimes reverted to pathogenic (virulent) status through mutation. Another conventional vaccine is the inactivated vaccine, which is produced by killing the original pathogen (e.g., virus) through heat or chemicals and then introducing the remaining shell (e.g., virus shell) into the host body. The shell, when properly manufactured, retains enough of the original pathogen to elicit an immune response. Some varieties of polio vaccines and influenza vaccines have been produced in this manner; however, improper manufacturing can lead to retention of some of the original pathogen and cause infections and other problems, e.g., the need for booster doses, etc.. A third type of vaccine manufactured from an original pathogen, such as a virus, is the viral-like particle (VLP) vaccine. VLPs are constructed out of surface proteins that can self-assemble to a virus-like structure, which mimics the original virus structure and can elicit a strong immune response with adjuvants. VLP vaccines have been manufactured for the hepatitis B virus, the human papillomavirus (the main cause of cervical cancer), and the hepatitis E virus.

Vaccine compositions containing peptides as active agents can be produced, and peptides can be generated by chemical synthesis, approaches suitable for large-scale and cost-effective production (e.g., solid-phase peptide synthesis approaches). Peptides can be modified (e.g., with lipids, carbohydrates, phosphate, acetyl, and amide groups) to increase stability, immunogenicity, and solubility.

Summary

Provided herein in certain aspects is a pharmaceutical composition that includes:

(a) one or more polypeptides each independently containing an amino acid sequence consisting of, or 95% or more identical to, an amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5; and

(b) a pharmaceutically acceptable excipient.

The term “95% or more identical to,” as used in any of the aspects provided herein (e.g., methods, compositions, pharmaceutical compositions, kits), means any percentage, as a whole number or fraction thereof, between 95% and 100%, such as, for example, about or equal to 95%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%.

In some aspects, the pharmaceutical composition contains 2, 3, 4 or 5 polypeptides whose sequences differ from each other and each of which independently contains an amino acid sequence that is, or is 95% or more identical to, the amino acid sequence set forth in SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID N0:4 or SEQ ID NO:5. In aspects, the pharmaceutical composition contains 5 polypeptides each independently containing an amino acid sequence that is, or is 95% or more identical to, the amino acid sequence set forth SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

In certain aspects, the pharmaceutical composition contains 5 polypeptides that include: a polypeptide containing the amino acid sequence set forth in SEQ ID NO:1, a polypeptide containing the amino acid sequence set forth in SEQ ID NO:2, a polypeptide containing the amino acid sequence set forth in SEQ ID NO:3, a polypeptide containing the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide containing the amino acid sequence set forth in SEQ ID NO:5. In aspects, the pharmaceutical composition contains 5 polypeptides that include: a polypeptide having the amino acid sequence set forth in SEQ ID NO:1, a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, a polypeptide having the amino acid sequence set forth in SEQ ID NO:3, a polypeptide having the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide having the amino acid sequence set forth in SEQ ID NO:5.

Any of the pharmaceutical compositions provided herein can be formulated for systemic, parenteral, topical, oral, mucosal, intranasal, subcutaneous, aerosolized, intravenous, bronchial, pulmonary, vaginal, vulvovaginal, esophageal, or oroesophageal administration. In certain aspects, the pharmaceutical compositions provided herein are formulated for oral administration. In aspects the pharmaceutical compositions can be formulated as a gel, ointment, liquid, suspension, aerosol, tablet, pill, powder or lyophile.

In certain aspects, the pharmaceutical compositions provided herein are formulated as microparticles. In aspects, the microparticle contains a sustained-release polymeric matrix. In some aspects, the microparticle contains b-cyclodextrin, ethyl cellulose (EC) or b-cyclodextrin and ethyl cellulose (EC).

In certain aspects, the pharmaceutical compositions containing the polypeptides provided herein are formulated as microparticles that include between about or equal to 10% to about or equal to 20% w/w ethylcellulose, such as about or equal to 10%, 11%, 12%, 13%, 14%, 15%, 16%, 16%, 17%, 18%, 19% or 20% w/w cellulose, based on the weight of the microparticles. In certain aspects, the microparticle contains about or equal to 15% w/w ethylcellulose, based on the weight of the microparticles. In certain aspects, the microparticle contains about or equal to 20% w/w ethylcellulose, based on the weight of the microparticles.

In certain aspects, the pharmaceutical compositions containing the polypeptides provided herein are formulated as microparticles that include between about or equal to 50% to about or equal to 70% w/w b- cyclodextrin, such as about or equal to 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70% w/w b-cyclodextrin, based on the weight of the microparticles. In certain aspects, the microparticle contains about or equal to 60% w/w b-cyclodextrin, based on the weight of the microparticles.

In certain aspects, the pharmaceutical compositions provided herein contain an agent that protects the components of the composition against degradation in the acidic environment of the stomach.

In aspects, the agent is hydroxypropyl-methyl cellulose acetate succinate (HPMCAS) and in certain aspects, the composition is formulated as a microparticle. In some aspects, the composition is a microparticle and the microparticle includes between about or equal to 20% to about or equal to 40% w/w HPMCAS, based on the weight of the microparticles, such as about or equal to 20%,

21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39% or 40% w/w HPMCAS, based on the weight of the microparticles. In certain aspects, the microparticle contains about or equal to 30% w/w HPMCAS, based on the weight of the microparticles. In certain aspects, the microparticle contains about or equal to 20% w/w HPMCAS, based on the weight of the microparticles.

In certain aspects, the pharmaceutical compositions provided herein include microparticles of a size between about or equal to 1 pm and about or equal to 5 pm in diameter, as determined by dynamic light scattering (DLS). In aspects, any of the pharmaceutical compositions provided herein are formulated as microparticles and the microparticles include a lectin. In certain aspects, the lectin is selected from among Aleuria aurantia lectin (AAL), wheatgerm agglutinin and Ulex europaeus- 1. In some aspects, the lectin is AAL.

In certain aspects, the pharmaceutical compositions containing the polypeptides provided herein are formulated as microparticles that include between about or equal to 0.1% to about or equal to 0.5% w/w lectin, based on the weight of the microparticles, such as about or equal to 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45% or 0.5% w/w lectin, based on the weight of the microparticles. In aspects, the microparticles include about or equal to 0.25% w/w lectin, based on the weight of the microparticles. In certain aspects, the lectin is AAL.

In aspects, the pharmaceutical compositions provided herein include about or equal to 1% to about or equal to 10% w/w of the total amount of polypeptides, such as about or equal to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% w/w polypeptides, based on the total weight of the composition.

In aspects, the pharmaceutical compositions provided herein include about or equal to 1% to about or equal to 10% w/w of each polypeptide in the composition, such as about or equal to 1%, 2%,

3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% w/w of each polypeptide in the composition, based on the total weight of the composition. In aspects, the pharmaceutical compositions are formulated as microparticles and the percent weight of the polypeptides is based on the weight of the microparticles. In aspects, the pharmaceutical compositions provided herein include equimolar amounts of each polypeptide in the composition. In aspects, the pharmaceutical compositions provided herein include about or equal to 1% w/w of each polypeptide in the composition.

In certain aspects, the pharmaceutical compositions provided herein of are formulated for prophylactic treatment. In aspects, the pharmaceutical compositions provided herein are formulated as vaccines. In aspects, the pharmaceutical compositions include one or more suitable pharmaceutically acceptable adjuvants and/or one or more suitable pharmaceutically acceptable carriers. In certain aspects, the pharmaceutical compositions are for treatment of a disease or condition caused by a coronavirus. In aspects, the coronavirus is SARS-CoV-2. In some aspects, the disease or condition is COVID-19.

Also provided herein is a method of selecting a polypeptide for treatment of a disease or condition caused by a pathogen in a subject, which includes:

(a) selecting a protein that is expressed by the pathogen and is associated with entry of the pathogen into the subject and/or infectivity of the pathogen in the subject;

(b) processing the sequence of the selected protein into amino acid subsequences;

(c) encoding the amino acid subsequences into numerical strings;

(d) computing a binding affinity value for each of the amino acid subsequences for one or more HLA alleles from the numerical strings according to bias values and weight values associated with each of the one or more HLA alleles, thereby generating computed binding affinity values for the amino acid subsequences for each of the one or more HLA alleles, where the computing is performed by a convolutional neural network (CNN) that contains a plurality of virtual neurons arranged in capsules;

(e) based on the computed binding affinity values, selecting at least one amino acid subsequence as having a binding affinity value that is above a threshold binding affinity value; and

(f) selecting a polypeptide that includes an amino acid sequence that has at least one amino acid subsequence of (e), or an amino acid sequence that is 95% or more identical to the at least one amino acid subsequence of (e), for treatment of a disease or condition caused by the pathogen in the subject. In aspects, the one or more HLA alleles include an HLA Class 1 allele or an HLA Class 2 allele. In some aspects, the binding affinity values for each of the amino acid subsequences are computed for two or more HLA alleles. In certain aspects, at least one HLA allele is an HLA Class 1 allele and at least one HLA allele is an HLA Class 2 allele.

In any of the polypeptide selection methods provided herein, in certain aspects, at least one HLA allele is selected from among HLA-A*11:01, HLA-DRB1*01:01, HLA-B*18:01 and HLA-B*58:01. In aspects, at least one selected amino acid subsequence binds to an HLA Class 2 allele. In certain aspects, the polypeptide selection methods provided herein additionally include, in (e): analyzing the accessibility of the at least one amino acid subsequence on the protein surface as an epitope; and based additionally on the accessibility of the at least one amino acid subsequence on the protein surface as an epitope, selecting the at least one amino acid subsequence for subsequent selection of the polypeptide in (f).

In certain aspects, two or more amino acid subsequences are selected in (e) and two or more polypeptides are selected in (f). In aspects, at least one polypeptide binds to an HLA Class 1 allele and at least one polypeptide binds to an HLA Class 2 allele. In some aspects, at least one polypeptide binds to an HLA Class 1 allele and also binds to an HLA Class 2 allele.

In certain aspects of the polypeptide selection methods provided herein, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more amino acid subsequences are selected in (e) and 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more polypeptides are selected in (f). In aspects, 5 amino acid subsequences are selected in (e) and 5 polypeptides are selected in (f).

In certain aspects of the polypeptide selection methods provided herein, (e) includes, additionally: identifying whether the at least one amino acid subsequence is conserved among species of the pathogen; and based additionally on identifying the at least one amino acid subsequence as being conserved among species of the pathogen, selecting the at least one amino acid subsequence for subsequent selection of the polypeptide in (f). In aspects, (e) includes, additionally: identifying whether the at least one amino acid subsequence is resistant to mutations; and based additionally on identifying the at least one amino acid subsequence as being resistant to mutations, selecting the at least one amino acid subsequence for subsequent selection of the polypeptide in (f).

In certain aspects of the polypeptide selection methods provided herein, the pathogen is a virus or a bacterium. In aspects, the protein that is expressed by the pathogen is associated with entry of the pathogen into the subject. In some aspects, the pathogen is a virus. In aspects, the virus is a coronavirus. In certain aspects, the coronavirus is SARS-CoV-2. In aspects, the protein is selected from among a spike protein, a membrane protein and an envelope protein. In some aspects, the protein is a spike protein. In certain aspects, the at least one amino acid subsequence is selected from among SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.

In certain aspects of the polypeptide selection methods provided herein, the treatment of a disease or condition caused by the pathogen in the subject is a prophylactic treatment. In aspects, at least one of the polypeptides selected in (f) is immunogenic. In certain aspects, the polypeptide selection methods provided herein further include preparing a polypeptide composition containing the polypeptide(s) selected in (f) for treatment of a disease or condition caused by the pathogen in the subject. In aspects, the composition contains at least one polypeptide that includes an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. In certain aspects, the composition contains at least one polypeptide having the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, or an amino acid sequence 95% or more identical to the amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. In some aspects, the composition contains at least one polypeptide whose sequence is the sequence set forth in SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. In certain aspects, the composition includes 5 polypeptides each independently containing an amino acid sequence that is, or is 95% or more identical to, the amino acid sequence set forth SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. In aspects, the composition contains the following 5 polypeptides: a polypeptide that includes the amino acid sequence set forth in SEQ ID NO:1, a polypeptide that includes the amino acid sequence set forth in SEQ ID NO:2, a polypeptide that includes the amino acid sequence set forth in SEQ ID NO:3, a polypeptide that includes the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide that includes the amino acid sequence set forth in SEQ ID NO:5. In some aspects, the composition includes the following 5 polypeptides: a polypeptide whose sequence is the amino acid sequence set forth in SEQ I D NO: 1 , a polypeptide whose sequence is the amino acid sequence set forth in SEQ ID NO:2, a polypeptide whose sequence is the amino acid sequence set forth in SEQ ID NO:3, a polypeptide whose sequence is the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide whose sequence is the amino acid sequence set forth in SEQ ID NO:5.

In certain aspects, the compositions prepared by the methods provided herein include a microparticle. In certain aspects, the microparticle includes a sustained-release polymeric matrix. In aspects, the microparticle includes b- cyclodextrin, ethyl cellulose (EC) or b-cyclodextrin and ethyl cellulose (EC). In certain aspects, the composition is formulated for systemic, parenteral, topical, oral, mucosal, intranasal, subcutaneous, aerosolized, intravenous, bronchial, pulmonary, vaginal, vulvovaginal, esophageal, or oroesophageal administration. In aspects, the composition is formulated for oral administration. In certain aspects, the composition includes microparticles that are formulated for oral administration. In some aspects, the composition or microparticle includes an agent that protects the components of the composition or microparticle against degradation in the acidic environment of the stomach. In aspects, the agent is hydroxypropyl-methyl cellulose acetate succinate (HPMCAS). In certain aspects, the composition is a microparticle of a size between about or equal to 1 pm and about or equal to 5 pm in diameter, as determined by dynamic light scattering (DLS).

In certain aspects, the compositions prepared by the methods provided herein include a lectin. In aspects, the composition is formulated as a microparticle. In certain aspects, the lectin is selected from among Aleuria aurantia lectin (AAL), wheatgerm agglutinin and Ulex europaeus- 1. In aspects, the lectin is AAL. In aspects, the composition is formulated for prophylactic treatment. In some aspects, the composition is formulated as a vaccine. In certain aspects, the composition includes one or more suitable pharmaceutically acceptable adjuvants and/or one or more suitable pharmaceutically acceptable carriers.

Also provided herein are compositions prepared by any of the methods provided herein. In aspects, the composition is formulated for prophylactic treatment. In certain aspects, the composition is formulated as a vaccine. In aspects, the composition includes one or more suitable pharmaceutically acceptable adjuvants and/or one or more suitable pharmaceutically acceptable carriers. In certain aspects, the composition is formulated for oral administration. In aspects, at least one polypeptide is encoded by a coronavirus. In certain aspects, at least one polypeptide includes an amino acid sequence that is, or is 95% or more identical to, an amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. In certain aspects, the composition includes 2, 3, 4 or 5 polypeptides whose sequences differ from each other and each of which independently includes an amino acid sequence that is, or is 95% or more identical to, an amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. In certain aspects, the composition includes 5 polypeptides each independently including an amino acid sequence that is, or is 95% or more identical to, an amino acid sequence set forth SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. In certain aspects, the composition includes the following 5 polypeptides: a polypeptide whose sequence includes the amino acid sequence set forth in SEQ ID NO: 1 , a polypeptide whose sequence includes the amino acid sequence set forth in SEQ ID NO:2, a polypeptide whose sequence includes the amino acid sequence set forth in SEQ ID NO:3, a polypeptide whose sequence includes the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide whose sequence includes the amino acid sequence set forth in SEQ ID NO:5. In aspects, the composition includes the following 5 polypeptides: a polypeptide whose sequence is the amino acid sequence set forth in SEQ ID NO:1, a polypeptide whose sequence is the amino acid sequence set forth in SEQ ID NO:2, a polypeptide whose sequence is the amino acid sequence set forth in SEQ ID NO:3, a polypeptide whose sequence is the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide whose sequence is the amino acid sequence set forth in SEQ ID NO:5. In certain aspects, the composition includes a microparticle. In aspects, the microparticle includes a sustained-release polymeric matrix. In some aspects, the microparticle includes b-cyclodextrin, ethyl cellulose (EC) or b-cyclodextrin and ethyl cellulose (EC).

In certain aspects, the compositions prepared by the methods provided herein are formulated as microparticles that include between about or equal to 10% to about or equal to 20% w/w ethylcellulose, such as about or equal to 10%, 11%, 12%, 13%, 14%, 15%, 16%, 16%, 17%, 18%, 19% or 20% w/w cellulose, based on the weight of the microparticles. In certain aspects, the microparticle contains about or equal to 15% w/w ethylcellulose, based on the weight of the microparticles.

In certain aspects, the compositions are formulated as microparticles that include between about or equal to 50% to about or equal to 70% w/w b-cyclodextrin, such as about or equal to 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70% w/w b- cyclodextrin, based on the weight of the microparticles. In certain aspects, the microparticle contains about or equal to 60% w/w b-cyclodextrin, based on the weight of the microparticles.

In certain aspects, the compositions prepared by the methods provided herein contain an agent that protects the components of the composition against degradation in the acidic environment of the stomach. In aspects, the agent is hydroxypropyl-methyl cellulose acetate succinate (HPMCAS) and in certain aspects, the composition is formulated as a microparticle. In some aspects, the composition is a microparticle and the microparticle includes between about or equal to 20% to about or equal to 40% w/w HPMCAS, based on the weight of the microparticles, such as about or equal to 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39% or 40% w/w b-cyclodextrin, based on the weight of the microparticles.

In certain aspects, the microparticle contains about or equal to 30% w/w HPMCAS, based on the weight of the microparticles.

In certain aspects, the compositions include microparticles of a size between about or equal to 1 pm and about or equal to 5 pm in diameter, as determined by dynamic light scattering (DLS). In aspects, any of the compositions prepared by the methods provided herein are formulated as microparticles and the microparticles include a lectin. In certain aspects, the lectin is selected from among Aleuria aurantia lectin (AAL), wheatgerm agglutinin and Ulex europaeus- 1. In some aspects, the lectin is AAL. In certain aspects, the compositions are formulated as microparticles that include between about or equal to 0.1% to about or equal to 0.5% w/w lectin, based on the weight of the microparticles, such as about or equal to 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45% or 0.5% w/w lectin, based on the weight of the microparticles. In aspects, the compositions prepared by the methods provided herein include about or equal to 1% to about or equal to 10% w/w of the total amount of polypeptides, such as about or equal to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% w/w polypeptides, based on the total weight of the composition. In aspects, the compositions include about or equal to 1% to about or equal to 10% w/w of each polypeptide in the composition, such as about or equal to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% w/w of each polypeptide in the composition, based on the total weight of the composition. In aspects, the compositions are formulated as microparticles and the percent weight of the polypeptides is based on the weight of the microparticles.

In some aspects, the compositions prepared by the methods provided herein contain 2, 3, 4 or 5 polypeptides whose sequences differ from each other and each of which independently contains an amino acid sequence that is, or is 95% or more identical to, the amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. In aspects, the composition contains 5 polypeptides each independently containing an amino acid sequence that is, or is 95% or more identical to, the amino acid sequence set forth SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. In certain aspects, the composition contains 5 polypeptides that include: a polypeptide containing the amino acid sequence set forth in SEQ ID NO: 1 , a polypeptide containing the amino acid sequence set forth in SEQ ID NO:2, a polypeptide containing the amino acid sequence set forth in SEQ ID NO:3, a polypeptide containing the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide containing the amino acid sequence set forth in SEQ ID NO:5. In aspects, the composition contains 5 polypeptides that include: a polypeptide having the amino acid sequence set forth in SEQ ID NO:1, a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, a polypeptide having the amino acid sequence set forth in SEQ ID NO:3, a polypeptide having the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide having the amino acid sequence set forth in SEQ ID NO:5.

Also provided herein are isolated polypeptides selected by the methods provided herein. In certain aspects, the isolated polypeptides are derived from the protein, e.g., by digestion with proteases. In aspects, the isolated polypeptides obtained by digestion are subjected to further purification prior to isolation. In aspects, the isolated polypeptides are synthesized. In aspects, the isolated polypeptides obtained by digestion are subjected to further purification prior to isolation.

Also provided herein are polynucleotides encoding the isolated polypeptides provided herein. In aspects, provided herein are compositions containing two or more isolated polypeptides or polynucleotides encoding the two or more isolated polypeptides, where the polypeptides are selected by any of the methods provided herein. In certain aspects, the isolated polypeptide is encoded by a virus or a bacterium. In certain aspects, at least one isolated polypeptide of a composition containing two or more polypeptides is encoded by a virus or a bacterium. In aspects, the isolated polypeptide or at least one isolated polypeptide is encoded by a virus. In certain aspects, the virus is a coronavirus. In aspects, the coronavirus is SARS-CoV-2. In certain aspects, the isolated polypeptide is derived from a protein is selected from among a spike protein, a membrane protein and an envelope protein. In aspects, the protein is a spike protein. In aspects, the isolated polypeptide includes an amino acid sequence that is, or is 95% or more identical to, the amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. In certain aspects, the isolated polypeptide has the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

Also provided herein are polynucleotides encoding any of the isolated polypeptides provided herein. In aspects, the polynucleotide is an expression vector or expression plasmid. In certain aspects, the polynucleotide is a DNA plasmid or vector, or RNA plasmid or vector. In some aspects, the polynucleotide is a DNA plasmid or vector and a portion of the DNA plasmid or vector includes a DNA virus or portion thereof. In aspects, the DNA virus is a herpesvirus, an adenovirus or a poxvirus.

In certain aspects, the polynucleotide is a RNA plasmid or vector and a portion of the RNA plasmid or vector includes a RNA virus. In aspects, the RNA virus is a retrovirus or a ssRNA virus.

In certain aspects, any of the isolated polypeptides provided herein, including isolated polypeptides in compositions containing two or more isolated polypeptides, is encoded by a virus. In aspects, the virus is a coronavirus. In certain aspects, the coronavirus is SARS-CoV-2. In certain aspects, the protein from which an isolated polypeptide is derived is selected from among a spike protein, a membrane protein and an envelope protein. In aspects, the protein is a spike protein. In some aspects, the isolated polypeptide is a portion of the S1 subunit of the spike protein.

In certain aspects, in any of the compositions containing two or more isolated polypeptides provided herein, at least one polypeptide contains an amino acid sequence that is, or is 95% or more identical to, the amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. In some aspects, a composition that includes 2 or more isolated polypeptides as provided herein contains 2, 3, 4 or 5 isolated polypeptides whose sequences differ from each other and each of which independently contains an amino acid sequence that is, or is 95% or more identical to, the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. In aspects, the composition contains 5 polypeptides each independently containing an amino acid sequence that is, or is 95% or more identical to, the amino acid sequence set forth SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. In certain aspects, the composition contains 5 polypeptides that include: a polypeptide containing the amino acid sequence set forth in SEQ ID NO: 1 , a polypeptide containing the amino acid sequence set forth in SEQ ID NO:2, a polypeptide containing the amino acid sequence set forth in SEQ ID NO:3, a polypeptide containing the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide containing the amino acid sequence set forth in SEQ ID NO:5. In aspects, the composition contains 5 polypeptides that include: a polypeptide having the amino acid sequence set forth in SEQ ID NO:1, a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, a polypeptide having the amino acid sequence set forth in SEQ ID NO:3, a polypeptide having the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide having the amino acid sequence set forth in SEQ ID NO:5. Also provided herein, in certain aspects, is a vaccine composition that includes an isolated polypeptide or polynucleotide provided herein, and further including one or more suitable pharmaceutically acceptable adjuvants and/or one or more suitable pharmaceutically acceptable carriers.

Also provided herein, in certain aspects, is a composition that includes an antigen presenting cell (APC) and an isolated polypeptide or polynucleotide encoding the polypeptide as provided herein.

In aspects, the composition includes a polynucleotide and the polynucleotide resides within the APC. In certain aspects, the polypeptide or a portion thereof is presented on the surface of the APC. In certain aspects, the APC is a monocyte. In aspects, the APC is a dendritic cell. In certain aspects, the composition includes two or more isolated polypeptides or one or more polynucleotides encoding the two or more isolated polypeptides. In certain aspects, at least one polypeptide contains an amino acid sequence that is, or is 95% or more identical to, the amino acid sequence set forth in SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

In some aspects, a composition that includes 2 or more isolated polypeptides and an APC as provided herein contains 2, 3, 4 or 5 isolated polypeptides whose sequences differ from each other and each of which independently contains an amino acid sequence that is, or is 95% or more identical to, the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. In aspects, the composition contains 5 polypeptides each independently containing an amino acid sequence that is, or is 95% or more identical to, the amino acid sequence set forth SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. In certain aspects, the composition contains 5 polypeptides that include: a polypeptide containing the amino acid sequence set forth in SEQ ID NO:1, a polypeptide containing the amino acid sequence set forth in SEQ ID NO:2, a polypeptide containing the amino acid sequence set forth in SEQ ID NO:3, a polypeptide containing the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide containing the amino acid sequence set forth in SEQ ID NO:5. In aspects, the composition contains 5 polypeptides that include: a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 , a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, a polypeptide having the amino acid sequence set forth in SEQ ID NO:3, a polypeptide having the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide having the amino acid sequence set forth in SEQ ID NO:5. The terms “having” and “whose sequence is” are used interchangeably in reference to any of the aspects provided herein.

Also provided herein is a device that contains any of the compositions, pharmaceutical compositions, isolated polypeptides or polynucleotides provided herein. In certain aspects, the contents of the device is/are in unit dosage form, liquid dosage form, solid dosage form, oral dosage form, a tablet, a capsule, a topical patch, a syringe, an inhaler, a dosage cup, a dropper, a pump, a spray bottle, an aerosol container, a wound dressing, or an applicator for administering the contents.

Also provided herein is a kit that contains any of the compositions, pharmaceutical compositions, isolated polypeptides or polynucleotides provided herein and a device for administration of the contents of the kit other than the device. In aspects, the contents of the kit other than the device are contained in the device. In some aspects, the contents of the kit other than the device are present as a separate component that is distinct from the device. In certain aspects, the device is a dressing, a topical patch, a pump, a spray bottle, an aerosol container, a syringe, an inhaler, a dosage cup, a dropper, or an applicator.

Also provided herein are methods for treating a disease or condition associated with a pathogen in a subject by administering any of the compositions, pharmaceutical compositions, isolated polypeptides or polynucleotides provided herein in an amount sufficient to induce an immune response in the subject. Also provided herein are methods for treating a disease or condition associated with a pathogen in a subject by administering a therapeutically effective amount of any of the compositions, pharmaceutical compositions, isolated polypeptides or polynucleotides provided herein. In certain aspects of the methods of treatment provided herein, the pathogen is a virus. In aspects, the virus is a coronavirus. In certain aspects, the coronavirus is SARS-CoV-2. In some aspects, the treatment is a prophylactic treatment. In aspects, the disease or condition is COVID-19.

Also provided herein is a method of inducing an immune response in a subject by administering any of the compositions, pharmaceutical compositions, isolated polypeptides or polynucleotides provided herein in an amount sufficient to induce an immune response. In aspects, the method further includes obtaining polyclonal antibodies from the subject and/or antiserum that immunospecifically binds to a polypeptide, or a polypeptide encoded by, the compositions, pharmaceutical compositions, isolated polypeptides or polynucleotides provided herein. In certain aspects, the method further includes: isolating spleen cells from the subject, and combining the spleen cells with myeloma cells under conditions that produce monoclonal antibody generating hybridomas. In aspects, the method further includes screening the hybridomas for those that produce monoclonal antibodies that immunospecifically bind to a polypeptide, or a polypeptide encoded by, the compositions, pharmaceutical compositions, isolated polypeptides or polynucleotides provided herein. In aspects, the method further includes isolating or otherwise obtaining the monoclonal antibodies identified by screening the hybridomas.

Also provided herein are monoclonal antibodies isolated or obtained by the methods provided herein. Also provided herein are chimeric antigen receptor (CAR) molecules (CAR binding molecules, i.e., antigen-binding molecules that are CARs) that include any of the monoclonal antibodies provided herein.

Also provided herein are methods of treating a disease or condition associated with a pathogen by administering, to a subject in need thereof, the monoclonal antibodies or CAR molecules provided herein. In certain aspects, the pathogen is a virus. In aspects, the virus is a coronavirus. In certain aspects, the coronavirus is SARS-CoV-2. In aspects, the disease or condition is COVID-19.

Certain implementations are described further in the following description, examples and claims, and in the drawings.

Brief Description of the Drawings

The drawings illustrate certain implementations of the technology and are not limiting. For clarity and ease of illustration, the drawings are not made to scale, and, in some instances, various aspects may be shown exaggerated or enlarged to facilitate an understanding of particular implementations.

Figure 1 depicts the cytotoxic effect of microparticles at various given concentrations.

Figure 2 depicts uptake of the microparticles in mice. Figure 2A depicts uptake in the small intestine. Figure 2B depicts uptake in Peyer’s Patches (PP).

Figure 3 depicts flow-cytometry results of the immunophenotype of human dendritic cells exposed to the microparticles or to control stimuli.

Figure 4 depicts a study of the immune stimulant function of the microparticles. Figures 5A & 5B illustrate 3D mapping of the CPFGEVFNATRFASV epitope (SEQ ID NO: 1 ), with wire frame (Figure 5A) and sphere (Figure 5B) models. Darker shading depicts the location of the epitope.

Figures 6A & 6B illustrate 3D mapping of the GEVFNATRF epitope (SEQ ID NO:2), with wire frame (Figure 6A) and sphere (Figure 6B) models. Darker shading depicts the location of the epitope.

Figures 7 A & 7B illustrate 3D mapping of the ASVYAWNRK epitope (SEQ ID NO:3), with wire frame (Figure 7A) and sphere (Figure 7B) models. Darker shading depicts the location of the epitope.

Figures 8A & 8B illustrate 3D mapping of the VGGNYNYLYRLFRKS epitope (SEQ ID NO:4), with wire frame (Figure 8A) and sphere (Figure 8B) models. Darker shading depicts the location of the epitope.

Figures 9A & 9B illustrate 3D mapping of the VGGNYNYLY epitope (SEQ ID NO:5), with wire frame (Figure 9A) and sphere (Figure 9B) models. Darker shading depicts the location of the epitope.

Figure 10 depicts the potency of BSK-02 microparticles in a dendritic cell (iDC) model.

Figure 11 depicts immunogenicity testing of the polypeptides having the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.

Figure 12 depicts T-cell mediated selective killing of antigen presenting cells (APCs) that present the polypeptides having the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.

Detailed Description

There are many diseases for which the development of a safe and effective vaccine remains elusive. For example, microbial pathogens that have exceptionally broad sequence diversity among their constituent family members (e.g., HIV-1), or pathogens such as influenza virus that undergo significant annual antigenic drift, have been especially difficult to approach from a vaccine perspective. Malaria has also been a challenging vaccine target due to the many stages of the parasite life cycle. Dengue virus is the most globally distributed arbovirus with ~390 million infections worldwide each year, but the development of a Dengue vaccine has been challenging due to a complex immunopathology in which induction of sub-neutralizing antibody levels contributes to an enhanced form of the disease.

Infectious disease vaccines aim to induce a protective immune response in a naive host by exposing the immune system to epitopes contained on the pathogen prior to exposure to the infectious agent itself. The major challenges that confront infectious disease vaccines stem from the nature of the epitopes against which the immune response is directed; in some cases, immunodominant epitopes arising from natural infection may not be those that are most desirable (e.g., susceptible to neutralization and/or highly conserved). In contrast, vaccines targeting diseases that involve “self” antigens (e.g., cancer or neurodegenerative disease) provide an additional complication in that the immune system suppresses responses to “self” antigens.

Viral diseases affect millions of people worldwide. Annually, dengue virus disease affects about 50 to 100 million people globally with 9000+ fatalities, rotavirus infects about two million children under five years of age, of whom about 527,000 die, seasonal influenza epidemics cause severe illness in three to five million people and a quarter to a half million deaths, and the most recent SARS-CoV-2 virus has caused over a million deaths within the space of just a few months, not to mention countless cases of serious illness with lingering long-term effects such as organ damage and cardiac disease. Thus, there is a continuous hunt for new drugs and vaccines, and this is compounded by the fact that new viruses are coming up to attack human hosts with higher frequency, while mutability of viral sequences rapidly render existing drugs and vaccines obsolete. Provided herein are artificial intelligence-guided methods of identifying immunogenic epitopes that are predicted to be most effective for prophylactic treatment of diseases or conditions caused by pathogens and, in certain aspects, predicted to be resistant to mutations. Also provided herein are compositions containing the immunogenic polypeptides so identified, or polynucleotides that encode the immunogenic polypeptides so identified. In aspects, the compositions are for oral administration, such as an oral vaccine. In certain aspects, the composition is formulated as a microparticle. In aspects, the microparticle is for oral administration.

Artificial Intelligence Guided Selection of Immunogenic Polypeptides

DIAMOND™ is a computational platform and a neural network that can identify novel immunologic targets for T- and B-lymphocytes. It uses proprietary SpliceDiff™ software, which is part of an integrated bioinformatic and artificial intelligence (A. I.) system such as that described in PCT application PCT/US20/35183, filed on May 29, 2020, the contents of which are expressly incorporated by reference herein and the contents of which are outlined herein. This proprietary platform can use public and proprietary databases to identify target polypeptides for the treatment of diseases or conditions caused by pathogens, such as infectious diseases and can expand into the tumor or the infectious disease target space. The platform addresses several challenges in today’s clinical pipeline: target identification and the selection of immunologically significant peptides for activating T and B lymphocytes, e.g., for prophylactic treatment or for generating antibodies for treatment. The A. I. system used to identify and select polypeptides for the treatment of diseases or conditions associated with or caused by pathogens is an Immunotherapy Builder System (IBS) that includes multiple modules. An IBS can narrow a multitude of amino acid sequence variants to a subset of predicted disease-associated variants, or immunogenic variants. In certain aspects, an IBS can narrow a multitude of amino acid subsequences in an input amino acid sequence to a subset identified as having immunogenic potential (e.g., for an immunotherapy). An IBS can facilitate in silico (i) discovery of novel disease-associated targets, and/or (ii) narrowing of a large number of targets to a significantly smaller subset of targets having high immunogenic potential, thereby facilitating resource-efficient development of novel immunotherapies. Portions of disease- associated targets (e.g., amino acid subsequences) identified by systems and processes described herein as having immunogenic potential, and/or longer amino acid sequences each containing one or more of such portions, can be considered predicted disease-associated antigens. Portions of predicted disease-associated targets can be identified by systems and processes described herein as having immunogenic potential according to assessment of major histocompatibility complex (MHC) interaction and/or T-cell receptor (TCR) interaction and/or B-cell receptor (BCR) interaction, for example.

An IBS can include two or more of the following modules: a Differential Expression Module (DEM), a MHC Allele Affinity Determination Module (MAAM), a MHC Composite Feature Module (MCFM), a MHC Fragment Locator Module (MFLM), a T-Cell Receptor Immunogenicity Determination Module (TIM); and a B-Cell Receptor Epitope Determination Module (BEM). An IBS can include a Sequence Acquisition Interface (SAI), which can be implemented to acquire an amino acid sequence of interest. A MAAM, MCFM, MFLM, BEM and/or a DEM can be configured to receive an amino acid sequence from an SAI.

An IBS can be implemented to (i) identify a disease-associated amino acid sequence variant or an immunogenic amino acid subsequence (e.g., by implementation of a DEM or a TIM) among variants or subsequences of a particular gene/protein; and/or (ii) compute MHC binding affinity values for amino acid subsequences within an amino acid sequence of interest (e.g., by implementation of a MAAM, MCFM and/or a MFLM); and/or (iii) compute a T-cell receptor (TCR) immunogenicity score for each of a plurality of amino acid subsequences having an estimated MHC binding affinity value above or below a threshold (e.g., by implementation of a TIM); and/or (iv) identify B-cell receptor (BCR) epitopes in an amino acid sequence of interest (e.g., by implementation of a BEM), for example.

A MAAM can compute a MHC binding affinity value for amino acid subsequences of an input amino acid sequence, for one or more MHC alleles or MHC supertypes, by implementation of a convolutional neural network (CNN) that contains a plurality of virtual neurons arranged in capsules. A MAAM can compute a MHC binding affinity value with an advantageously low error rate, and can narrow amino acid subsequences to a subset predicted to exhibit strong and/or intermediate MHC binding affinity. These features are useful for identifying a subset of amino acid subsequences of high immunogenic potential for an immunotherapy, for example.

For amino acid subsequences outputted by a MAAM, a MFLM can output a graphic representation of the amino acid subsequences, or subset thereof, mapped to an input amino acid sequence. These features are useful for narrowing amino acid subsequences to a subset located in one or more regions in the amino acid sequence that can be presented by multiple MHC alleles. Such a narrowing process is useful for building an immunotherapy that is potentially effective across a broad population, for example.

A MCFM can compute a composite MHC binding affinity value for amino acid subsequences of an input amino acid sequence, for one or more MHC alleles or MHC supertypes, where the composite binding affinity value is based on (i) a proteasome cleavage score for the amino acid subsequence, and/or (ii) a transporter affinity score for the amino acid subsequence, and/or (iii) a MHC allele or MHC supertype binding affinity value for the amino acid subsequence (e.g., a normalized MHC allele or MHC supertype binding affinity value). A composite binding affinity value is useful for narrowing amino acid subsequences to a subgroup having high immunogenic potential for an immunotherapy, for example.

A TIM can compute a T-cell receptor (TCR) immunogenicity score based on estimation of interaction of amino acid subsequences of an input amino acid with a TCR. A TIM often is implemented after a subset of amino acid subsequences characterized by high immunogenicity potential and/or high multiple MHC allele-binding potential is identified by one or more modules that assess MHC interaction (/.e., a MAAM, a MFLM and/or a MCFM). Immunogencity scores computed by a TIM are useful for narrowing amino acid subsequences to a smaller subset of high T-cell- mediated immunogenic potential, for example.

A BEM can compute a B-cell receptor (BCR) epitope score for each amino acid in an input amino acid sequence, where the score is indicative of the probability that the amino acid exists within a BCR epitope scores computed by a BEM are useful for narrowing amino acid subsequences to a smaller subset of high B-cell-mediated immunogenic potential, for example.

A DEM can identify a disease-associated amino acid sequence variant among variants encoded by a particular gene based on an analysis of expression level of the variant in disease samples and non-disease samples from multiple tissues. A DEM is useful for identifying disease-associated alternatively-spliced variants. In certain instances, a disease-associated alternatively-spliced variant includes an insert of an amino acid or two or more consecutive amino acids relative to other variants encoded by a gene, for example. A DEM is useful for identifying disease-associated variants that can be targeted by an immunotherapeutic, for example. An amino acid sequence of a disease-associated variant identified by a DEM, or portion thereof, can be utilized as an input amino acid sequence for one or more of a MAAM, a MFLM, a MCFM, a TIM and a BEM, for example.

The IBS can generate a multi-sequence alignment (MSA) to identify the possible presence of emerging non-synonymous mutational hotspots in the immunogenic epitopes (subsequences) that are identified, or to identify immunogenic epitopes that are conserved between multiple species of the same pathogen, e.g., two or more coronaviruses, to further identify immunogenic epitopes that are resistant to mutations and/or offer cross protection of multiple species in immunogenic therapy (e.g., vaccines). A MSA often aligns a variant amino acid sequence with an amino acid sequence of at least one other variant encoded by the same gene. A MSA can be generated using any suitable sequence alignment algorithm, non-limiting examples of which include Clustal (e.g., ClustalW, ClustalW2, Clustal Omega), Multiple Alignment using Fast Fourier Transform (MAFFT), T- COFFEE, M-COFFEE, LALIGN, PSAIign, PRRN, PRRP, DIALIGN, MUSCLE, MergeAlign, Partial- Order Alignment (POA), Sequence Alignment and Modeling System (SAM), HMMER, PRANK, PAGAN, ProGraphMSA, MEME, MAST and EDNA. A DEM can generate a MSA based on a gene identifier, which can involve synching amino acid sequence databases having disparate gene identifier information.

The prediction of peptide affinity for HLA molecules is currently performed using standard artificial neural networks (ANN), while DIAMOND™ is equipped with a state-of-the-art capsule neural network (CNN), which applies an iterative routing-by-agreement mechanism, and therefore when multiple capsules agree, the probability of correct prediction is expected to be higher than that obtained with other network architectures.

Pathogens

The methods provided herein can be used to identify and select polypeptides for the treatment of any disease or condition caused by or associated with a pathogen, include, but are not limited to, diseases caused by pathogens such as viruses, bacteria, fungi, protozoa, and parasites. Infectious diseases can be caused by viruses including coronaviruses, adenovirus, cytomegalovirus, dengue, Epstein-Barr, hanta, hepatitis A, hepatitis B, hepatitis C, herpes simplex type I, herpes simplex type II, human immunodeficiency virus, (HIV), human papilloma virus (HPV), influenza, measles, mumps, papova virus, polio, respiratory syncytial virus, rinderpest, rhinovirus, rotavirus, rubella, SARS virus, smallpox and viral meningitis. Infectious diseases can also be caused by bacteria including Bacillus anthracis, Borrelia burgdorferi, Campylobacter jejuni, Chlamydia trachomatis, Clostridium botulinum, Clostridium tetani, Diphtheria, Escherichia coli, Legionella, Helicobacter pylori, Mycobacterium rickettsia, Mycobacterium tuberculosis, Mycoplasma Neisseria, Pertussis, Pseudomonas aeruginosa, Streptococcus pneumoniae, Streptococcus, Staphylococcus, Vibrio cholerae and Yersinia pestis. Infectious diseases can also be caused by fungi such as Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum and Penicillium marneffei. Infectious diseases can also be caused by protozoa and parasites such as chlamydia, kokzidiose, leishmania, malaria, rickettsia, and trypanosoma.

Using the selection methods provided herein, the immunology prediction modules from the DIAMOND™ artificial neural network can be applied to select immunogenic epitopes of a protein expressed by any pathogen associated with a disease or condition. In aspects, the protein that is analyzed using the DIAMOND™ artificial neural network is identified based on its resistance to mutations, or based on whether it is conserved between species of the pathogen, e.g., conserved between strains of the influenza virus or between strains of coronaviruses. In aspects, the protein that is identified for analysis is associated with entry of the pathogen into the target host. In certain aspects, the selected immunogenic epitopes include amino acid sequences that are subsequences of a protein associated with entry of a pathogen into a target host. In aspects, the immunogenic epitopes containing amino acid sequences that are subsequences of a protein associated with entry of a pathogen into a target host can neutralize entry of the pathogen into the host.

In certain aspects, the pathogen is a virus. In aspects, the protein identified for analysis using selection methods provided herein is selected from among a spike protein, a membrane protein and an envelope protein. In certain aspects, the protein is a spike protein.

The selection methods provided herein can, in certain aspects, select immune-dominant polypeptides that are predicted to bind to the most common HLA class I and class II alleles and would be most likely to elicit neutralizing antibodies. From the predicted immune-dominant polypeptides, in some aspects, the polypeptides that are selected include one or more polypeptides that are accessible on the protein surface. In aspects, immune hotspot regions can be identified as follows:

1) Sequences predicted to bind with high affinity to the most common HLA alleles in the world’s population.

2) Regions of the protein where sequences in point 1) cluster and/or overlap.

3) Regions predicted to be antibody epitopes, containing at least 1 HLA class II binder peptide. 4) Regions where the predicted antibody peptides are accessible in 3D space.

Also provided herein are any of the compositions provided herein that include immunogenic polypeptides (epitopes) selected according to the methods provided herein, and/or that include polynucleotides that encode the immunogenic polypeptides (epitopes) selected according to the methods provided herein. Provided herein, in certain aspects, are immunogenic polypeptides selected by the methods provided herein, and related compositions, that include or encode capsid surface antigens of a pathogen. In aspects, the immunogenic polypeptides and related compositions are for treatment, including prophylactic treatment, of a disease or condition caused by or associated with a pathogen. In certain aspects, the immunogenic polypeptides and related compositions can cross protect against more than one strain of a pathogen.

In certain aspects, the immunogenic polypeptides selected by the methods provided herein are predicted by the method as being the most immunogenic. In aspects, the immunogenic polypeptides are predicted to bind to the most common HLA class I and class II alleles that would be most likely to elicit neutralizing antibodies. In aspects, at least one of the selected the immunogenic polypeptides is accessible on the surface of the pathogen.

Coronaviruses

In certain aspects, provided herein are immunogenic polypeptides, polynucleotides encoding the polypeptides or compositions containing the immunogenic polypeptides and/or polynucleotides for treating a disease or condition caused by a coronavirus.

Novel coronaviruses (nCoV) are part of a family of beta coronaviruses that can cause illnesses such as the common cold, or create more life threatening conditions such as SARS-CoV (Severe Acute Respiratory Syndrome) or MERS-CoV (Middle East Respiratory Syndrome). As a family of viruses, the coronaviruses are transmitted from animals to humans (zoonotic transmission) followed by human-to-human transmission. SARS-CoV was initially spread from civet cats to humans, and MERS-CoV was transmitted from dromedary camels to humans.

SARS-CoV-2 is an enveloped positive single strand RNA virus that the WHO (World Health Organization) has declared to have caused a global pandemic, causing world-wide distress and economic hardship. COVID-19 disease (SARS-CoV-2) can present with flu like symptoms such as fever, cough, shortness of breath, breathing difficulties, nausea, diarrhea, pneumonia, severe acute respiratory syndrome, coagulation dysfunction, septic shock, kidney failure, and even death.

The corona protein spikes situated at the exterior surface of the virus are the binding point for the Angiotensin-converting enzyme 2 (ACE2) receptor in lung tissue. Since patients treated with ACEIs will have increased numbers of ACE2 receptors in their lungs, an increase in ACE2 receptor mediated antigenic stimuli may therefore increase the risk of the corresponding inflammation-related adverse effects associated with COVID-19 infections. Consequently, pneumonia, acute myocardial injury, and/or chronic damage to the cardiovascular system are potential severe complications of this viral disease, and the symptoms of the infection may be more pronounced in patients with hypertension and diabetes. Therefore, affected patients may be at an increased risk of more severe disease outcomes mediated by infection-induced endothelial dysfunction.

Therefore, given the highly infectious nature of this coronavirus and the significant morbidity and mortality associated with the development of potential complications such as Acute Respiratory Distress Syndrome (ARDS) that is considered to be secondary to a cytokine storm, urgent approaches are needed to address this global crisis with treatments that not only are effective at protecting against infection, but are also safe, well tolerated, and have global commercial feasibility. Given that the virus displays high infectivity and high morbidity and mortality rates and given the absence of an effective treatment or vaccine, the only currently available preventive measures are social distancing and the use of masks. Based upon historical precedent, even after an initial reduction in the epidemic peak, the virus could result in a seasonal disease, such as that associated with the influenza virus. This scenario makes it vital to develop methods of treatment of diseases or conditions associated with SARS-CoV-2, including prophylactic treatment.

Currently there is no known cure for this devastating disease, so novel preventative and therapeutic options are needed, including vaccine development. The SARS-CoV-2 virus is highly polymorphic, both on HLA and on Single Nucleotides (SNP). The 47 key point mutations are likely host dependent (host-dependent RNA editing in the transcriptome), with 910 identified Single Nucleotide Variations (SNVs) in addition to other novel missense mutations. This creates complexity in developing an effective vaccine. A low or absent immune response is therefore possible, and the risk of severe adverse effects is high.

The presentation of target antigens to effector T lymphocytes by antigen-presenting cells (APCs), such as dendritic cells (DCs), can help effect an active immunotherapy. APCs are capable of presenting viral peptide antigens (VPAs), eliciting adaptive immune responses against VPA infected cells. The Diamond™ artificial intelligence antigen discovery platform used in the selection methods provided herein has revealed key conserved peptide domains within the SARS-CoV-2 protein spikes, membrane, and viral envelope proteins that are most likely to induce a potentially immunogenic response.

Provided herein, in certain aspects, are immunogenic polypeptides selected by the methods provided herein, and related compositions, that include or encode capsid surface antigens of a coronavirus. In certain aspects, the coronavirus is SARS-CoV-2. In aspects, the immunogenic polypeptides and related compositions are for treatment, including prophylactic treatment of COVID-19. In certain aspects, the immunogenic polypeptides and related compositions can cross protect against more than one strain of coronavirus.

In certain aspects, the immunogenic polypeptides selected by the methods provided herein are predicted by the method as being the most immunogenic. In aspects, the immunogenic polypeptides are predicted to bind to the most common HLA class I and class II alleles that would be most likely to elicit neutralizing antibodies. In aspects, at least one of the selected the immunogenic polypeptides is accessible on the surface of the viral protein.

In aspects, the immunogenic polypeptides selected by the methods provided herein are derived from a coronavirus glycoprotein. In aspects, the protein is a SARS-CoV-2 surface glycoprotein. In certain aspect, the glycoprotein is a spike glycoprotein. Like other coronaviruses, SARS-CoV-2 uses the spike glycoprotein (S), the main target for neutralization antibody, to bind to its receptor, and mediate membrane fusion and virus entry. Without being bound by theory, in certain aspects, when the immunogenic polypeptides are derived from the coronavirus S protein, the risk for off-target immunotoxicity is extremely low as this protein is not found on human cells. Each monomer of trimeric S protein is about 180 kDa, and contains two subunits, S1 and S2, mediating attachment and membrane fusion, respectively. In aspects, the immunogenic polypeptides selected by the methods provided herein are in (and are derived from) the S1 subunit of the spike protein (first 600 amino acids). In aspects, the immunogenic polypeptides are accessible to neutralizing antibodies.

In aspects, the immunogenic polypeptides contain or have a sequence selected from among those set forth in SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, or a sequence that is 95% or more identical to a sequence selected from among those set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

Binding Molecules

In certain aspects, provided herein are binding molecules that specifically bind to a polypeptide having the sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, or to a sequence that is 95% or more identical to SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

In certain aspects, the binding molecules provided herein are antibodies, or antigen-binding fragments thereof. In aspects, the VH and VL domains of the antibodies provided herein are humanized and/or deimmunized so as to exhibit a reduced immunogenicity upon administration to recipient subjects. In aspects, the binding molecules provided herein can include, but are not limited to, bispecific diabodies, BiTEs, bispecific antibodies, trivalent binding molecules and the like that include: (i) Variable Domains (VH and VL) and (ii) a domain capable of binding to an epitope of a molecule present on the surface of an effector cell. Also provided herein are pharmaceutical compositions that contain any of the binding molecules provided herein, and methods involving the use of any of such binding molecules in the treatment of diseases or conditions associated with a coronavirus, such as COVID-19. In certain aspects, the binding molecules are monoclonal antibodies. In aspects, the binding molecules are chimeric antigen receptors (CARs).

As used herein, the terms “antibody” and “antibodies” refer to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, polyclonal antibodies, camelized antibodies, single-chain Fvs (scFv), single-chain antibodies, Fab fragments, F(ab’) fragments, disulfide-linked bispecific Fvs (sdFv), intrabodies, and epitope-binding fragments of any of the above. The term “antibody” includes immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an epitope-binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGi, lgG2, lgG3, lgG4, IgAi and lgA ₂ ) or subclass. Antibodies are capable of “immunospecifically binding” to a polypeptide or protein or a non-protein molecule (or of binding to such molecule in an “immunospecific manner”) due to the presence on such molecule of a particular domain or moiety or conformation (an “epitope”). In the context of antibodies or antigen binding fragments thereof, or CAR molecules, the terms “immunospecific” or “immunospecifically binding” are used interchangeably herein with “specific” or “specifically binding,” respectively.

An epitope-containing molecule can have immunogenic activity, such that it elicits an antibody production response in an animal; such molecules are termed “antigens”. Examples of epitopes in the SARS-CoV-2 spike protein include those having the sequences set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5. As used herein, an antibody, diabody or other epitope-binding molecule is said to “immunospecifically” bind a region of another molecule (i.e., an epitope) if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with that epitope relative to alternate epitopes. It also is understood by reading this definition that, for example, an antibody (or moiety or epitope) that immunospecifically binds to a first target may or may not bind to a second target. As such, “immunospecific binding” does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to antibody (or CAR molecule) binding means “immunospecific” binding.

The term “monoclonal antibody,” as used herein, refers to a homogeneous antibody population wherein the monoclonal antibody contains amino acids (naturally occurring or non-naturally occurring) that are involved in the selective binding of an antigen. Monoclonal antibodies are specific, being directed against a single epitope (or antigenic site or determinant). The terms “antibody” or “monoclonal antibody,” as used herein, encompass not only intact antibodies / monoclonal antibodies and full-length antibodies / monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv, etc.), single-chain (scFv) binding molecules, mutants thereof, fusion proteins comprising an antibody portion, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immunoglobulin molecule that contains an antigen recognition site of the required specificity and the ability to bind to an antigen. It is not intended to be limited as regards to the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term also includes whole immunoglobulins as well as the fragments etc. described above under the definition of “antibody.”

Antibodies, such as polyclonal antibodies and monoclonal antibodies, can be prepared using standard methods (see, e.g., Kohler et al., Nature 256:495-497 (1975); Kohler et al., Eur. J. Immunol. 6:511-519 (1976); and WO 02/46455). For example, to generate polyclonal antibodies, an immune response is elicited in a host animal, such as mice, rats or rabbits, to an antigen of interest. Blood from the host animal is then collected and the serum fraction containing the secreted antibodies is separated from the cellular fraction, using methods known to those of skill in the art. To generate monoclonal antibodies, an animal is immunized by standard methods to produce antibody-secreting somatic cells. These cells then are removed from the immunized animal for fusion to myeloma cells. Somatic cells that can produce antibodies, particularly B cells, can be used for fusion with a myeloma cell line. These somatic cells can be derived from the lymph nodes, spleens and peripheral blood of primed animals. Specialized myeloma cell lines have been developed from lymphocytic tumors for use in hybridoma-producing fusion procedures (Kohler and Milstein, Eur. J. Immunol. 6:511-519 (1976); Shulman et al., Nature, 276:269-282 (1978); Volk et al., J. Virol., 42:220-227 (1982)). These cell lines have three useful properties. The first is they facilitate the selection of fused hybridomas from unfused and similarly indefinitely self-propagating myeloma cells by having enzyme deficiencies that render them incapable of growing in selective medium that support the growth of hybridomas. The second is they have the ability to produce antibodies and are incapable of producing endogenous light or heavy immunoglobulin chains. A third property is they efficiently fuse with other cells. Other methods for producing hybridomas and monoclonal antibodies are well known to those of skill in the art. It is routine to produce antibodies against any polypeptide, e.g., antigenic marker on an immune cell population.

The binding molecules provided herein can be assayed for the ability to bind to their cognate immunogenic polypeptide epitopes by any method known to those of skill in the art. Binding assays can be performed in solution, suspension or on a solid support. Negative controls also can be included in such assays as a measure of background binding. Binding affinities can be determined using quantitative ELISA, Scatchard analysis (Munson et aL, (1980) Anal. Biochem., 107:220), surface plasmon resonance, isothermal calorimetry, or other methods known to one of skill in the art (e.g., Liliom et aL (1991) J. Immunol Methods. 143(1 ): 119-25).

Such assays also can be performed, for example, in solution (e.g., Houghten (1992) Bio/Techniques 13:412-421), on beads (Lam (1991) Nature 354:82-84), on chips (Fodor (1993) Nature 364:555-556), on bacteria (U.S. Pat. No. 5,223,409), on spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), on plasmids (Cull et aL (1992) Proc. Natl. Acad. Sci. USA 89:1865- 1869) or on phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404- 406; Cwirla et aL (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382; and Felici (1991) J. Mol. Biol. 222:301-310).

The binding can be detected using a method that is capable of being quantified such that the level of activity can be assessed. For example, methods of quantitation include, but are not limited to, spectrophotometric, fluorescent and radioactive methods. Such methods measure, for example, colorimetric signals, chemiluminescent signals, chemifluorescent signals or radioactive signals. In aspects, the binding molecules provided herein can be labeled with a detectable moiety or tag to facilitate detection and determination of binding activity. The skilled artisan can select an appropriate detectable moiety or tag for use in the assays described or known in the art. Linkage can be at the N- or C-terminus of the therapeutic antibody. Examples of tags and moieties are provided in Table 13 hereafter.

Solution binding assays, including any solution binding assay known to the skilled artisan, can be used to assess binding activity including equilibrium dialysis, competitive binding assays (e.g., Myers eta!., (1975) Proc. Natl. Acad. Sci. USA), radiolabeled binding assays (e.g., Feau et a!., (2009) J. Biomol. Screen. 14(1):43-48), calorimetry, including isothermal titration calorimetry (ITC) and differential scanning calorimetry (e.g., Alvarenga et al. (2012) Anal. Biochem 421 ( 1 ) : 138- 151, Perozzo et al., (2004) J. Recept Signal. Transduct Res. 24(1-2): 1-52; Holdgate (2001)

Biotechniques 31(1): 164-166, 168, 170, Celej et al. (2006) Anal. Biochem. 350(2):277-284), and spectroscopic fluorescence assays, including fluorescence resonance energy transfer (FRET) assays (Wu et al. (2007), J. Pharm. Biomed. Anal. 44(3):796-801). The conditions for binding assays in can be adapted from conditions discussed above for binding assays performed on a solid support.

Immunoassays include competitive and non-competitive assay systems using techniques such as, but not limited to, western blots or immunoblots, such as quantitative western blots; radioimmunoassays; ELISA (enzyme linked immunosorbent assay); Meso Scale Discovery (MSD, Gaithersburg, Maryland); "sandwich" immunoassays; immunoprecipitation assays; ELISPOT; precipitin reactions; gel diffusion precipitin reactions; immunodiffusion assays; agglutination assays; complement-fixation assays; immunoradiometric assays; fluorescent immunoassays; protein A immunoassays; immunohistochemistry; immuno-electron microscopy or liposome immunoassays (LIA). Such assays are routine and well-known in the art (see, e.g., Ausubel etal., Eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York).

In some examples, immunohistochemistry and/or immunofluorescence can be used to assess binding in animal models. For example, antibody binding to xenograft tumors in a rodent or other animal model can be analyzed. In other examples, immunohistochemistry can be used to assess antibody binding to skin, such as primate skin. In other examples, immunohistochemistry can be used to assess binding to xenograft tumors and primate skin grafts, ex vivo, for example to visually or quantitatively compare binding preferences of the antibody and to determine if the tested antibody exhibits selective or specific binding.

In other examples, an animal model containing a xenograft tumor or skin graft, such as an animal model described herein, can be administered a binding molecule, such as an antibody, provided herein, such as by systemic administration., to assess in vivo binding of the antibody. In such examples, the tissue can be harvested at particular time(s) to assess binding ex vivo by immunohistochemistry or immunofluorescence as described above. In other examples, the administered binding molecule is conjugated to a fluorophore, such as an infrared fluorophore (e.g., DyLight ⁷⁵⁵ ), which is capable of transmitting fluorescence through the skin. In such examples, antibody binding can be visualized in vivo using a fluorescent imaging system such as the I VIS Caliper imaging system, and antibody binding to xenograft tumors and/or primate skin grafts can be assessed. Tissue can subsequently be harvested for ex vivo confirmational immunohistochemical analysis.

Depending on the quantitative assay selected to measure antibody binding, absolute binding can be represented, for example, in terms of optical density (OD), such as from densitometry or spectrophotometry measurements; arbitrary fluorescent units (AFU), such as from fluorescence measurements; or lumens, such as from chemiluminescence measurements. In some examples, the specific activity is calculated by dividing the absolute binding signal by the antibody protein concentration. In some examples, the specific activity is normalized to give a normalized specific activity (NSA) for each antibody by dividing the specific activity of the antibody by the specific activity of a reference antibody, such as an antibody that is not specific for any of the immunogenic polypeptides provided herein (e.g., SEQ ID NOS:1-5), or is a parental antibody from which the antibody of interest is derived.

Binding activity also can be measured in terms of binding affinity, which can be determined in terms of binding kinetics, such as measuring rates of association (k _a or k _on ) and/or dissociation (k _d or /c _off ), half maximal effective concentration (EC50) values, and/or thermodynamic data (e.g., Gibbs free energy, enthalpy, entropy, and/or calculating association (KA) or dissociation (KD) constants. Typically, determination of binding kinetics requires known antibody and antigen protein concentrations. Rates of association ( k _a ) and association constants (KA) are positively correlated with binding affinity. In contrast, rates of dissociation (k _d ), dissociation constants (KD) and EC50 values are negatively correlated with binding affinity. Thus, higher binding affinity is represented by lower k _d , K _D and EC50 values.

Polypeptides

The polypeptides, including immunogenic polypeptides, provided herein can be prepared using the methods provided herein. The terms peptides and polypeptides are used interchangeably herein and can refer to a single peptide unit or to two or more peptide units (e.g., peptide units independently obtained e.g., by recombinant method or by chemical synthesis and then linked together by a peptide bond or other linker). The polypeptides referred to herein, such as immunogenic peptides or immunogenic polypeptides, or peptide vaccines or polypeptide vaccines, also can, in certain aspects, refer to peptides or polypeptides that are conjugated to additional entities, such as polymers, e.g., to increase half-life or stability.

A polypeptide generally refers to a polymer, linked by peptide bonds, that has a sequence of amino acids encoded by a polynucleotide. Proteins or portions thereof (e.g., a subunit of a protein) are generally made up of polypeptides. A peptide generally refers to a portion or fragment of a larger polypeptide. In some instances, a peptide refers to a polymer containing between about 2 amino acids to about 10 amino acids, 2 amino acids to about 20 amino acids, or about 2 amino acids to about 30 amino acids. Peptides can include, for example, dipeptides, tripeptides, tetrapeptides, and oligopeptides. Amino acids that have been incorporated into peptides and/or polypeptides may be referred to as residues. Peptides and polypeptides typically have an N-terminal (amine group) residue at one end and C-terminal (carboxyl group) residue at the opposite end, and amino acid sequences are typically read in the N-terminal to C-terminal direction.

The immunogenic polypeptides selected by the methods provided herein can be produced synthetically, such as using solid phase or solution phase peptide synthesis. For example the immunogenic polypeptides can be produced by direct peptide synthesis using solid-phase techniques (see e.g., Stewart etal. (1969) Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco; Merrifield J (1963) J Am Chem Soc., 85:2149-2154). In vitro protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City CA) in accordance with the instructions provided by the manufacturer. If needed, various fragments of a polypeptide can be chemically synthesized separately and combined using chemical methods. If produced recombinantly, the immunogenic polypeptides provided herein can be obtained using any available methods known in the art for cloning and isolating nucleic acid molecules. Such methods include PCR amplification of nucleic acids and screening of libraries for the identification of polynucleotides that encode the immunogenic polypeptides can be used, including nucleic acid hybridization screening, antibody-based screening and activity-based screening.

In aspects, a nucleic acid containing material can be used as a starting material from which a desired polypeptide-encoding polynucleotide can be isolated. For example, DNA and mRNA preparations, cell extracts, tissue extracts, fluid samples (e.g., blood, serum, saliva), samples from healthy and/or diseased subjects can be used in amplification methods. The source can be from any eukaryotic species including, but not limited to, vertebrate, mammalian, human, porcine, bovine, feline, avian, equine, canine, and other primate sources. Nucleic acid libraries also can be used as a source of starting material. Primers can be designed to amplify a desired polypeptide. For example, primers can be designed based on expressed sequences from which a desired polypeptide is generated.

Additional polynucleotide sequences can be joined to a polypeptide-encoding nucleic acid molecule, including linker sequences containing restriction endonuclease sites for the purpose of cloning the synthetic gene into a vector, for example, a protein expression vector or a vector designed for the amplification of the core protein coding DNA sequences. Furthermore, additional nucleotide sequences specifying functional DNA elements can be operatively linked to a polypeptide-encoding nucleic acid molecule. Examples of such sequences include, but are not limited to, promoter sequences designed to facilitate intracellular protein expression, and secretion sequences, for example heterologous signal sequences, designed to facilitate protein secretion. Such sequences are known to those of skill in the art. In addition, tags or other moieties can be added, for example, to aid in detection or affinity purification of the polypeptide. For example, additional nucleotide residue sequences such as sequences of bases specifying an epitope tag or other detectable marker also can be linked to enzyme-encoding nucleic acid molecules. Examples of such sequences include nucleic acid sequences encoding a His tag or Flag Tag.

The identified and isolated nucleic acids can then be inserted into an appropriate cloning vector. A large number of vector-host systems known in the art can be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as pCMV4, pBR322 or pUC plasmid derivatives or the Bluescript vector (Stratagene, La Jolla, CA). Other expression vectors include the HZ24 expression vector exemplified herein (see e.g., SEQ ID NOS:4 and 5). The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. Insertion can be effected using, for example, TOPO cloning vectors (Invitrogen, Carlsbad, CA). Recombinant molecules can be introduced into host cells via, for example, transformation, transfection, infection, electroporation and sonoporation, so that many copies of polynucleotide are generated, and large quantities of the immunogenic polypeptides expressed.

In certain aspects, the immunogenic polypeptides can be conjugated to polymers. Exemplary polymers that can be conjugated to the polypeptides include natural and synthetic homopolymers, such as polyols (/.e., poly-OH), polyamines (/.e., poly-Nhy and polycarboxylic acids (/.e., poly- COOH), and further heteropolymers, /.e., polymers containing one or more different coupling groups, e.g., hydroxyl groups and amine groups. Examples of suitable polymeric molecules include polymeric molecules selected from among polyalkylene oxides (PAO), such as polyalkylene glycols (PAG), including polyethylene glycols (PEG), methoxypolyethylene glycols (mPEG) and polypropylene glycols, PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG), branched polyethylene glycols (PEGs), polyvinyl alcohol (PVA), polycarboxylates, polyvinylpyrrolidone, poly-D,L-amino acids, polyethylene-co-maleic acid anhydride, polystyrene-co- maleic acid anhydride, dextrans including carboxymethyl-dextrans, heparin, homologous albumin, celluloses, including methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, carboxyethylcellulose and hydroxypropylcellulose, hydrolysates of chitosan, starches such as hydroxyethyl-starches and hydroxypropyl-starches, glycogen, agaroses and derivatives thereof, guar gum, pullulan, inulin, xanthan gum, carrageenan, pectin, alginic acid hydrolysates and bio polymers. In aspects, the immunogenic polypeptides provided herein are isolated. In aspects, the immunogenic polypeptides provided herein are formulated as any of the compositions provided herein.

Polynucleotides

The immunogenic polypeptides selected by the methods provided herein can be produced synthetically, or can be produced by standard recombinant DNA techniques known to one of skill in the art. For example, nucleic acid encoding one or more immunogenic polypeptides can be incorporated into an expression vector and then introduced into a host cell to be expressed heterologously. In certain aspects, polynucleotides encoding the immunogenic polypeptides provided herein can be formulated as any of the compositions provided herein. Hence, also provided herein are polynucleotide molecules encoding any of the immunogenic polypeptides provided herein. In certain aspects, the polynucleotides encoding the immunogenic polypeptides provided herein are synthesized by methods known to those of skill in the art. In aspects, the polynucleotides encoding the immunogenic polypeptides provided herein are isolated.

Pharmaceutical compositions, articles of manufacture, kits

Provided herein are pharmaceutical compositions that include any of the polypeptides, or nucleotides encoding the polypeptides, provided herein, and a pharmaceutically acceptable carrier or excipient. A pharmaceutical composition provided herein can be formulated as a gel, ointment, liquid, suspension, aerosol, tablet, pill, powder or lyophile, and/or can be formulated for systemic, parenteral, topical, oral, mucosal, intranasal, subcutaneous, aerosolized, intravenous, bronchial, pulmonary, vaginal, vulvovaginal, esophageal, or oroesophageal administration. In aspects, a pharmaceutical composition provided herein can be formulated as a microparticle. In aspects, the microparticle is for oral administration. A pharmaceutical composition provided herein can be formulated for single dosage administration or for multiple dosage administration. In certain aspects, a pharmaceutical composition provided herein can be a sustained release formulation. In certain aspects, a pharmaceutical composition provided herein is for prophylactic treatment. In aspects, a pharmaceutical composition provided herein is formulated as a vaccine.

The pharmaceutical compositions provided herein can be packaged as articles of manufacture containing packaging material, a pharmaceutical composition that is effective for treating a disease, such as COVID-19, by administration of an immunogenic polypeptide or a cocktail of 2, 3, 4, 5 or more immunogenic polypeptides, or polynucleotides encoding the polypeptide(s), and a label that indicates the infection, disease or disorder that the polypeptide(s) or polynucleotide(s) is/are to be used for. The pharmaceutical compositions can be packaged in unit dosage forms containing an amount of the pharmaceutical composition for a single dose or multiple doses. In aspects, the packaged compositions can contain a lyophilized powder of the pharmaceutical compositions, which can be reconstituted (e.g., with water or saline) prior to administration.

The pharmaceutical compositions provided herein also can be included in kits. Kits can optionally include one or more components such as instructions for use, devices and additional reagents (e.g., sterilized water or saline solutions for dilution of the compositions and/or reconstitution of lyophilized protein), and components, such as tubes, containers and syringes for practice of the methods. For example, the kits can include one or more immunogenic polypeptides as provided herein, or polynucleotides encoding the one or more polypeptides as provided herein, or a microparticle containing the one or more immunogenic polypeptides as provided herein, and can optionally include instructions for use, a device for administering the antibody to a subject, a device for detecting the immunogenic polypeptides or an antibody response elicited by the immunogenic polypeptides in a subject or in samples obtained from a subject, and a device for administering an additional therapeutic agent to a subject. The kit, optionally, can include instructions. Instructions typically include a tangible expression describing the one or more immunogenic polypeptides, or polynucleotides encoding the one or more polypeptides, or a microparticle containing the one or more immunogenic polypeptides, and, optionally, other components included in the kit, and methods for administration, including methods for determining the proper state of the subject, the proper dosage amount, dosing regimens, and the proper administration method for administering the one or more immunogenic polypeptides, or polynucleotides encoding the one or more polypeptides, or a microparticle containing the one or more immunogenic polypeptides, . Instructions also can include guidance for monitoring the subject over the duration of the treatment time.

For treatment of a disease or condition, including prophylactic treatment such as administering a vaccine, the dosage and the frequency of administration, can vary. The compositions can be administered in a single dose, in multiple separate administrations, or by continuous infusion. For repeated administrations over several days or longer, depending on the condition, the treatment can be repeated until a desired suppression of disease symptoms occurs, the desired improvement in the patient's condition is achieved, or the desired level of immune response is elicited. Repeated administrations can include increased or decreased amounts of the compositions, depending on the progress and/or the desired result. For example, the compositions can be administered to animal or human subjects at a dosage of about or equal to 0.1 mg to about or equal to 10 g or more, such as, for example, about or equal to 0.5 mg to about or equal to 5 g, about or equal to 5 g to about or equal to 50 g, about or equal to 1 mg to about or equal to 20 g, about or equal to 1 g to about or equal to 10 g, about or equal to 1 g to about or equal to 5 g, about or equal to 1 mg to about or equal to 100 mg, about or equal to 5 mg to about or equal to 8 g, about or equal to 10 g to about or equal to 80 g, or about or equal to 50 mg to about or equal to 100 mg, about or equal to 0.1 mg/kg to about or equal to 100 mg/kg, such as, for example, about or equal to 0.5 mg/kg to about or equal to 50 mg/kg, about or equal to 5 mg/kg to about or equal to 50 mg/kg, about or equal to 1 mg/kg to about or equal to 20 mg/kg, about or equal to 1 mg/kg to about or equal to 100 mg/kg, about or equal to 10 mg/kg to about or equal to 80 mg/kg, or about or equal to 50 mg/kg to about or equal to 100 mg/kg or more; or at a dosage of about or equal to 0.01 mg/m ² to about or equal to 800 mg/m ² or more, such as for example, about or equal to 0.01 mg/m ² , about or equal to 0.1 mg/m ² , about or equal to 0.5 mg/m ² , about or equal to 1 mg/m ² , about or equal to 5 mg/m ² , about or equal to 10 mg/m ² , about or equal to 15 mg/m ² , about or equal to 20 mg/m ² , about or equal to 25 mg/m ² , about or equal to 30 mg/m ² , about or equal to 35 mg/m ² , about or equal to 40 mg/m ² , about or equal to 45 mg/m ² , about or equal to 50 mg/m ² , about or equal to 100 mg/m ² , about or equal to 150 mg/m ² , about or equal to 200 mg/m ² , about or equal to 250 mg/m ² , about or equal to 300 mg/m ² , about or equal to 400 mg/m ² , about or equal to 500 mg/m ² , about or equal to 600 mg/m ² , or about or equal to 700 mg/m ² .

Any of the pharmaceutical compositions or kits provided herein can include a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable” means approved by a regulatory agency of a Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant (e.g., Freund’s adjuvant (complete and incomplete), excipient, or vehicle with which the therapeutic is administered. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

A pharmaceutical composition sometimes is provided as a pharmaceutical pack or kit containing one or more containers filled with a therapeutic composition of cells prepared by a method described herein, alone or with such pharmaceutically acceptable carrier. Additionally, one or more other prophylactic or therapeutic agents useful for the treatment of a disease can also be included in the pharmaceutical pack or kit. A pharmaceutical pack or kit may include one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. A pharmaceutical pack or kit sometimes includes one or more other prophylactic and/or therapeutic agents useful for the treatment of a disease, in one or more containers.

Microparticles

In certain aspects, provided herein are compositions that are formulated as microparticles. The microparticles provided herein can be made by spray drying methods by mixing the various matrix and active ingredients, in an aqueous medium spraying in droplets of controlled size and drying the droplets to form microparticles, nanoparticles or both microparticles and nanoparticles. Spray drying is a drying method that was firstly described more than 140 years ago as an improvement in drying and concentrating liquids. But it was not until the beginning of the 20th century that the level of sophistication and knowledge of the process allowed its industrial use.

Spray drying involves the atomization of a liquid feed into very small droplets within a hot drying gas leading to flash drying of the droplets into solid particles. The particles are then separated from the drying gas, using a cyclone and/or a filter bag, as a final spray dried product. The feed can be a solution, a suspension or an emulsion and the resulting product can be classified as a powder, granules or agglomerates.

In a single continuous step, spray drying therefore converts a liquid feedstock into a powder with well-defined properties. Properties such as level of moisture or residual solvent in the powder, particle morphology or size and powder density can be manipulated to a great extent to target levels. The remarkable flexibility in tailoring the properties of the final powder, the gentleness of the process and its economics when compared with competing technologies such as freeze drying led to its proliferation in multiple industrial applications including cosmetics, fine chemicals, detergents, polymers, excipients and pharmaceuticals.

This technique eliminates the use of organic solvents and transforms dissolved components into a dry and stable particulate form. Atomization of dissolved polymeric components and cargo molecules into small droplets increases the surface-to-mass ratio and further evaporation and drying decreases the particle size and increase particle stability. Spray drying has many advantages over other formulation methods including lower residual solvent levels, higher loading, as well as higher encapsulation capacity and particle stability (8, 9). Methods and ingredients for making microparticles as provided herein are described in part in WO 2010/037142 and WO 2016/081,783, the contents of which are incorporated in their entirety by reference herein. The microparticles can be spray-dried, e.g., using a Buchi 191 Spray Dryer. The spray drying procedure aerosolizes the vaccine antigen-polymer matrix, where optimum water removal from the droplet results in nanoparticles containing the vaccine antigen in a polymer matrix. The particle size and zeta potential can be determined, e.g., using a Malvern Zeta Sizer. Physical characteristics of the particles, such as size morphology, distribution and zeta potential and any other characteristic desired to be assessed in the pharmaceutical API or microparticles, can be achieved by any known methods.

In certain aspects, the microparticle compositions provided herein are for oral administration. The vast array of gut-associated lymphoid tissue (GALT) in mammals offers opportunity for exploiting the gastrointestinal tract for active immunotherapy. The murine and human GALT is defined by the presence of highly interactive and specialized lympho-epithelial and lymphoid units known as follicle-associated epithelium (FAE). FAE is arranged in clusters, known as Peyer’s patches (PPs), or isolated lymphoid follicles (I LFs), distributed throughout the small and large intestines. The FAE serves as a barrier between the intestinal lumen and the body, and mediate antigen transport, recognition and clearance by the GALT. FAE contains specialized microfold cells (M cells) that sample and expose luminal antigens to TLR-regulated and GALT-associated multifunctional DCs. Thus, M cells play a critical role in delivering luminal antigens for further processing and immune presentation. Since the frequency and density of M cells varies with age and among species (mice GALT having relatively more M cells than humans), orally-delivered cancer immunotherapeutics intended for human use can incorporate an M cell targeting agent to increase antigen delivery and processing by GALT APCs. In order to enhance targeting of an oral microparticle-based formulation to M cells, in certain aspects, a lectin can be used. In aspects, the lectin is Aleuria aurantia lectin (AAL) (10). In aspects, the AAL in the formulation can function as an immune adjuvant. In certain aspects, the microparticles provided herein can include an immune adjuvant other than AAL. In aspects, the adjuvant other than AAK is selected from the group consisting of CpG ODN, MF59, alum, flagellin, R848, monophosphoryl lipid A, ODN 1826, and combinations thereof. In aspects, the immune adjuvant is CpG ODN. In certain aspects, the microparticles provided herein include AAL and no immune adjuvant other than AAL. In aspects, the microparticles provided herein include AAL and an immune adjuvant other than AAL. In certain aspects, the microparticles include one or more immune adjuvants at a total concentration, or, independently, at a concentration of between about or equal to 0.1% to about or equal to 0.5%, such as about or equal to 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45% or 0.5% w/w.

In aspects, the ingredients for the microparticle matrix include a water-soluble polymer such as beta cyclodextrin and enteric coating materials such as ethyl cellulose (EC) and/or hydroxypropylmethyl cellulose acetate succinate (HPMCAS). In aspects, the microparticles provided herein have immunogenic peptides (e.g., VPAs) associated with the microparticle. In aspects, the VPAs are encapsulated by the microparticle.

Generally, M cells act as sampling ports for any foreign molecules encountered in the small intestine and have been shown to house numerous dendritic cells and immune cells. Following the sampling of the oral vaccine by M cells, the particle is processed by a dendritic/antigen presenting cell (APC) and presented on major histocompatibility complex (MHC) class I or MHC class II molecules. CpG oligodeoxynucleotides, which have been shown to provide signals for the dendritic cell and T- cell activation can be used as an adjuvant in the formulation. In certain aspects, CpG has also been shown to enhance the efficacy of weak tumor antigens and to promote T cell responses.

In certain aspects, the following microparticle formulations are described and/or provided herein:

Unless otherwise specified, the term “OroVAX,” as used herein, refers to a microparticle formulation that includes HPMCAS, b-cyclodextrin, EC, AAL and the five polypeptides (VPAs) having the sequences set forth in SEQ ID NOS: 1-5, in the proportions and percentage ranges described herein. In aspects, the OroVAX formulations provided herein contain equimolar amounts of the five polypeptides (VPAs) having the sequences set forth in SEQ ID NOS: 1-5. In some aspects, the OroVAX formulations provided herein contain, as w/w based on the weight of the microparticles, 20% HPMCAS, 60% b-cyclodextrin, 20% EC, 0.25% AAL and 1% of each of the five polypeptides (VPAs) having the sequences set forth in SEQ ID NOS:1-5. Unless otherwise specified, the terms “BSK” or OroVAX without VPAs,” as used interchangeably herein, refer to a microparticle formulation that includes HPMCAS, b-cyclodextrin, EC and AAL, in the proportions and percentage ranges described herein. In certain aspects, the BSK formulations provided herein contain, as w/w based on the weight of the microparticles, 20% HPMCAS, 60% b- cyclodextrin, 15% EC and 0.25% AAL.

Unless otherwise specified, the terms “BSK02” or “BSK-02,” as used interchangeably herein, refer to a microparticle formulation that includes HPMCAS, b-cyclodextrin, EC, AAL and the SP17 protein or a SP17 polypeptide, in the proportions and percentage ranges described herein. The SP17 protein (GenBank Accession No. CAA88459.1) has the sequence set forth below as SEQ ID NO:6:

MSIPFSNTHY RIPQGFGNLL EGLTREILRE QPDNIPAFAA AYFESLLEKR EKTNFDPAEW GSKVEDRFYN NHAFEEQEPP EKSDPKQEES QISGKEEETS VTILDSSEED KEKEEVAAVK IQAAFRGHIA REEAKKMKTN SLQNEEKEEN K (SEQ ID NO:6)

The SP17 polypeptide (amino acids 111-142 of the SP17 protein) has the sequence set forth below as SEQ ID NO:7:

KEKEEVAAVK IQAAFRGHIA REEAKKMKTN SL (SEQ ID NO:7)

In certain aspects, the BSK02 formulations can contain, as w/w based on the weight of the microparticles, 20% HPMCAS, 60% b-cyclodextrin, 15% EC, 0.25% AAL and 5% SP17 protein or SP17 polypeptide. In certain aspects, the BSK02 formulations can additionally contain CpG as an immune adjuvant. In aspects, the CpG immune adjuvant is present in a weight ratio of 1:500, relative to the weight of the microparticles (e.g., 10 pg CpG / 5 mg dose of microparticles).

Immunotherapy based R&D holds the key to developing the most effective and durable therapies to address this global pandemic. A key step to start the chain of reaction of activating the immune system begins with antigen presenting cells (APCs), such as dendritic cells (DCs). The coronavirus protein spike (S-protein) on the exterior surface of the virus (from which the name corona was derived) is the mechanism by which it binds to the ACE-II receptor on lung tissue (4). Using the Artificial Intelligence (Al) system and methods provided herein the ideal VPA sequences from within the SARS-CoV-2 S-protein were selected (SEQ ID NOS: 1-5), and then incorporated into the OroVAX microparticle formulation. Upon absorption into the Gl tract, the DCs digest the VPAs derived from the S-protein; thus, beginning the chain reaction of immune system activation. Reactogenicity is also a concern with any type of vaccination for Covid-19 based treatment of Covid-19. However, this risk is much greater for vaccinations that are administered by injection versus a more physiologic oral route, since injections are not only more invasive to the patient, but they are also not the normal pathway through which SARS-Cov-2 is transmitted and thus an injectable route might generate a more abrupt/rapid immune response that may be more likely to facilitate more adverse effects. Thus the key to potentially decreasing this type of adverse reaction is to deliver the immunotherapy, or vaccination, in a manner that is more consistent with the mechanism of viral transmission of SARS- CoV-2, which primarily occurs via the mucous membranes of the upper respiratory and enteric tracts. Since OroVAX is delivered orally, it will be absorbed and processed by the patient’s immune system through the mucous membranes of the gastrointestinal system, and will hence more closely mirror the physiologic absorption pathway of SARS-CoV-2 as compared to an injectable formulation, resulting in a more gradual increase in the activity of the immune system and yielding a safer and more durable immunologic response with less expected long-term exhaustion of T-cell immunity. Further, unlike the pathway of immune stimulation via an intact virus, OroVAX incorporates only the key immunogenic proteins as identified by the Diamond Al antigen discovery platform, and thus it is a non-infectious oral vaccine.

Due to the fact that OroVAX is a biodegradable and biocompatible multifunctional polymer microparticle, the adverse effects will likely be minimal at the doses prescribed. Moreover, given the stable matrix of the OroVAX formulation, it is also ideal for low- and middle- income countries (4), as well as medically underserved rural sites in the US. For example, since it can be stored at room temperature, it is easily manufactured and shipped, and requires minimal training of health care providers world-wide, making it an ideal vaccine for LMICs, and for world-wide military distribution and support.

OroVAX is a biodegradable and biocompatible multifunctional polymer microparticle vehicle suitable for the immunotherapy-based treatment of SARS-CoV-2 targeting Viral Peptide Antigens (VPA). In aspects, the microparticles are composed of b-cyclodextrin, hydroxypropyl-methyl cellulose acetate succinate (HPMCAS), and ethyl cellulose (EC). OroVAX manufacturing is more time-and cost-effective compared with other vaccines under development and meets the requirement for broad and rapid deployment. The virus Spike surface protein was selected as the target, due to its role in viral entry and because it is less likely to undergo mutations that would make the vaccine less effective. Within the protein spikes that are present on the surface of the virus, are stable highly immunogenic genomic sequences (e.g., SEQ ID NOS:1-5) that are important for the immune system to be able to effectively fight the Covid-19 pandemic, and it is these peptide sequences which have been incorporated into OroVAX. OroVAX manufacturing is more time-and cost-effective compared with other vaccines under development and meets the requirement for broad and rapid deployment.

Parameters such as cost of production, scalability, and storage at room temperature are fundamental for a vaccine that, if effective, will have to be deployed as widely as possible in a short time, in order to make an impact on the world-wide consequences of a pandemic such as COVID- 19. OroVAX is manufactured in a single-step spray drying process, which allows it to 1) contain production costs, 2) facilitate production scale-up from a few hundred to several million doses, and 3) facilitate transportation and storage, due to its stability at room temperature. We believe that OroVAX, unlike other vaccine strategies currently being evaluated in a clinical setting, has fewer chances of eliciting a non-neutralizing immune response, since it contains only selected epitopes instead of the full-length Spike protein, making OroVAX the first rationally designed anti-COVID-19 vaccine developed for human use.

Methods of use

The immunogenic polypeptides, polynucleotides encoding the immunogenic polypeptides and compositions provided herein can be used to treat any disease or condition associated with a pathogen. In certain aspects, the treatment is a prophylactic treatment. In aspects, provided herein are methods of eliciting an immune response by administering, to a subject in need thereof, any one of the immunogenic polypeptides, polynucleotides encoding the immunogenic polypeptides and compositions provided herein. Also provided herein are methods of treatment by administering, to a subject in need thereof, a therapeutically effective amount of the immunogenic polypeptides, polynucleotides encoding the immunogenic polypeptides and compositions provided herein. In any of the methods provided herein, in certain aspects, the disease or condition is associated with a virus. In aspects, the virus is a coronavirus. In certain aspects, the coronavirus is SARS-CoV-2. In aspects, the disease or condition is COVID-19.

Certain Implementations

Following are non-limiting examples of certain implementations of the technology.

A1. A method of selecting a polypeptide for treatment of a disease or condition caused by a pathogen in a subject, comprising:

(b) selecting a protein that is expressed by the pathogen and is associated with entry of the pathogen into the subject and/or infectivity of the pathogen in the subject;

(b) processing the sequence of the selected protein into amino acid subsequences; (c) encoding the amino acid subsequences into numerical strings;

(d) computing a binding affinity value for each of the amino acid subsequences for one or more HLA alleles from the numerical strings according to bias values and weight values associated with each of the one or more HLA alleles, thereby generating computed binding affinity values for the amino acid subsequences for each of the one or more HLA alleles, wherein the computing is performed by a convolutional neural network (CNN) that contains a plurality of virtual neurons arranged in capsules;

(e) based on the computed binding affinity values, selecting at least one amino acid subsequence as having a binding affinity value that is above a threshold binding affinity value; and

(f) selecting a polypeptide comprising an amino acid sequence consisting of the at least one amino acid subsequence in (e), or an amino acid sequence that is 95% or more identical to the at least one amino acid subsequence in (e), for treatment of a disease or condition caused by the pathogen in the subject.

A2. The method of embodiment A1 , wherein the one or more HLA alleles comprises an HLA Class 1 allele or an HLA Class 2 allele.

A3. The method of embodiment A1 or A2, wherein the binding affinity values for each of the amino acid subsequences are computed for two or more HLA alleles.

A4. The method of embodiment A3, wherein at least one HLA allele is an HLA Class 1 allele and at least one HLA allele is an HLA Class 1 allele.

A5. The method of any one of embodiments A1-A4, wherein at least one HLA allele is selected from among HLA-A*11:01, HLA-DRB1*01:01, HLA-B*18:01 and HLA-B*58:01.

A6. The method of any one of embodiments A1-A5, wherein at least one selected amino acid subsequence binds to an HLA Class 2 allele.

A7. The method of any one of embodiments A1-A6, wherein (e) comprises, additionally: analyzing the accessibility of the at least one amino acid subsequence on the protein surface as an epitope; and based additionally on the accessibility of the at least one amino acid subsequence on the protein surface as an epitope, selecting the at least one amino acid subsequence for subsequent selection of the polypeptide in (f).

A8. The method of any one of embodiments A1-A7, wherein two or more amino acid subsequences are selected in (e) and two or more polypeptides are selected in (f). A9. The method of embodiment A8, wherein at least one polypeptide binds to an HLA Class 1 allele and at least one polypeptide binds to an HLA Class 2 allele.

A10. The method of any one of embodiments A1-A9, wherein at least one polypeptide binds to an HLA Class 1 allele and binds to an HLA Class 2 allele.

A11. The method of any one of embodiments A1-A10, wherein 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more amino acid subsequences are selected in (e) and 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more polypeptides are selected in (f).

A12. The method of embodiment A11, wherein 5 amino acid subsequences are selected in (e) and 5 polypeptides are selected in (f).

A13. The method of any one of embodiments A1-A12, wherein (e) comprises, additionally: identifying whether the at least one amino acid subsequence is conserved among species of the pathogen; and based additionally on identifying the at least one amino acid subsequence as being conserved among species of the pathogen, selecting the at least one amino acid subsequence for subsequent selection of the polypeptide in (f).

A14. The method of any one of embodiments A1-A13, wherein (e) comprises, additionally: identifying whether the at least one amino acid subsequence is resistant to mutations; and based additionally on identifying the at least one amino acid subsequence as being resistant to mutations, selecting the at least one amino acid subsequence for subsequent selection of the polypeptide in (f).

A14. The method of any one of embodiments A1-A13, wherein the pathogen is a virus or a bacterium.

A15. The method of any one of embodiments A1-A14, wherein the protein that is expressed by the pathogen is associated with entry of the pathogen into the subject.

A16. The method of any one of embodiments A1-A15, wherein the pathogen is a virus.

A17. The method of embodiment A16, wherein the virus is a coronavirus.

A18. The method of embodiment A17, wherein the coronavirus is SARS-CoV-2.

A19. The method of any one of embodiments A16-A18, wherein the protein is selected from among a spike protein, a membrane protein and an envelope protein.

A20. The method of any one of embodiments A16-A19, wherein the protein is a spike protein. A21. The method of embodiment A20, wherein the at least one amino acid subsequence is selected from among SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.

A22. The method of any one of embodiments A1-A21 , wherein the treatment of a disease or condition caused by the pathogen in the subject is a prophylactic treatment.

A22.1 The method of embodiment A22, wherein at least one of the polypeptides selected in (f) is immunogenic.

A23. The method of any one of embodiments A1-A22.1 , further comprising, preparing a polypeptide composition comprising the polypeptide(s) selected in (f) for treatment of a disease or condition caused by the pathogen in the subject.

A24. The method of embodiment A23, wherein the composition comprises at least one polypeptide comprising an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

A24.1. The method of embodiment A24, wherein the composition comprises at least one polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, or an amino acid sequence 95% or more identical to the amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

A24.2 The method of embodiment A24.1, wherein the composition comprises at least one polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

A25. The method of embodiment A24, wherein the composition comprises 5 polypeptides each independently comprising an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

A26. The method of embodiment A25, wherein the composition comprises 5 polypeptides comprising: a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:1, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:2, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:3, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:4, and a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:5. A26.1 The method of embodiment A26, wherein the composition comprises 5 polypeptides comprising: a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 , a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:2, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:3, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:5.

A27. The method of any one of embodiments A23-A26, wherein the composition comprises a microparticle.

A28. The method of embodiment A27, wherein the microparticle comprises a sustained-release polymeric matrix.

A29. The method of embodiment A27 or A28, wherein the microparticle comprises b-cyclodextrin, ethyl cellulose (EC) or b-cyclodextrin and ethyl cellulose (EC).

A30. The method of any one of embodiments A23-A29, wherein the composition is formulated for systemic, parenteral, topical, oral, mucosal, intranasal, subcutaneous, aerosolized, intravenous, bronchial, pulmonary, vaginal, vulvovaginal, esophageal, or oroesophageal administration.

A31. The method of embodiment A30, wherein the composition is formulated for oral administration.

A32. The method of any one of embodiments A27-A29, wherein the microparticle is formulated for oral administration.

A33. The method of embodiment A31 or A32, wherein the composition or microparticle comprises an agent that protects the components of the microparticle against degradation in the acidic environment of the stomach.

A34. The method of embodiment A32, wherein the agent is hydroxypropyl-methyl cellulose acetate succinate (HPMCAS).

A35. The method of any one of embodiments A27-A29 and A32-A34, wherein the composition is a microparticle of a size between about or equal to 1 pm and about or equal to 5 pm in diameter, as determined by dynamic light scattering (DLS).

A36. The method of any one of embodiments A27-A29 and A32-A35, wherein the microparticle comprises a lectin.

A37. The method of embodiment A36, wherein the lectin is selected from among Aleuria aurantia lectin (AAL), wheatgerm agglutinin and Ulex europaeus- 1. A38. The method of embodiment A37, wherein the lectin is AAL.

A39. The method of any one of embodiments A31-A38, wherein composition is formulated for prophylactic treatment.

A40. The method of embodiment A39, wherein the composition is formulated as a vaccine.

A41. The method of embodiment A40, wherein the composition comprises one or more suitable pharmaceutically acceptable adjuvants and/or one or more suitable pharmaceutically acceptable carriers.

B1. A composition prepared by the method of any one of embodiments A23-A41.

B2. The composition of embodiment B1 that is formulated for prophylactic treatment.

B3. The composition of embodiment B1 or B2 that is formulated as a vaccine.

B4. The composition of embodiment B3, comprising one or more suitable pharmaceutically acceptable adjuvants and/or one or more suitable pharmaceutically acceptable carriers.

B5. The composition of any one of embodiments B1-B4 that is formulated for oral administration.

B6. The composition of embodiment B5, wherein at least one polypeptide is encoded by a coronavirus.

B7. The composition of embodiment B6, comprising at least one polypeptide comprising an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

B8. The composition of embodiment B7, comprising 2, 3, 4 or 5 polypeptides whose sequences differ from each other and each of which independently comprises an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

B9. The composition of embodiment B8, comprising 5 polypeptides each independently comprising an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

B9.1. The composition of embodiment B9 comprising 5 polypeptides, comprising: a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO: 1 , a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:2, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:3, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:4, and a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:5.

B10. The composition of embodiment B9.1 comprising 5 polypeptides, comprising: a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 , a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:2, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:3, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:5.

B11. The composition of any one of embodiments B1-B10, wherein the composition comprises a microparticle.

B12. The composition of embodiment B11, wherein the microparticle comprises a sustained- release polymeric matrix.

B13. The composition of embodiment B11 or B12, wherein the microparticle comprises b- cyclodextrin, ethyl cellulose (EC) or b-cyclodextrin and ethyl cellulose (EC).

B14. The composition of embodiment B13, wherein the microparticle comprises about or equal to 10% to about or equal to 20% w/w ethylcellulose, based on the weight of the microparticles.

B15. The composition of embodiment B13 or B14, wherein the microparticle comprises about or equal to 15% w/w ethylcellulose, based on the weight of the microparticles.

B16. The composition of any one of embodiments B13-B15, wherein the microparticle comprises about or equal to 50% to about or equal to 70% w/w b-cyclodextrin, based on the weight of the microparticles.

B17. The composition of embodiment B16, wherein the microparticle comprises about or equal to 60% w/w b- cyclodextrin, based on the weight of the microparticles.

B18. The composition of any one of embodiments B1-B17 that is formulated for oral administration.

B19. The composition of embodiment B18, comprising an agent that protects the components of the microparticle against degradation in the acidic environment of the stomach.

B20. The composition of embodiment B19, wherein the agent is hydroxypropyl-methyl cellulose acetate succinate (HPMCAS).

B21. The composition of embodiment B20, wherein the composition is a microparticle and the microparticle comprises about or equal to 20% to about or equal to 40% w/w HPMCAS, based on the weight of the microparticles. B22. The composition of embodiment B21 , wherein the microparticle comprises about or equal to 30% w/w HPMCAS, based on the weight of the microparticles.

B23. The composition of any one of embodiments B11-B22, wherein the size of the microparticle between about or equal to 1 pm and about or equal to 5 pm in diameter, as determined by dynamic light scattering (DLS).

B24. The composition of any one of embodiments B11-B23, wherein the microparticle comprises a lectin.

B25. The composition of embodiment B24, wherein the lectin is selected from among Aleuria aurantia lectin (AAL), wheatgerm agglutinin and Ulex europaeus- 1.

B26. The composition of embodiment B25, wherein the lectin is AAL.

B27. The composition of any one of embodiments B24-B26, wherein the microparticle comprises about or equal to 0.1% to about or equal to 0.5% w/w lectin, based on the weight of the microparticles.

B28. The composition of any one of embodiments B1-B27, comprising about or equal to 1% to about or equal to 10% w/w polypeptides.

B29. The composition of embodiment B28, wherein the composition comprises a microparticle.

B30. The composition of embodiment B29, comprising 2, 3, 4 or 5 polypeptides whose sequences differ from each other and each of which independently comprises an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

B31. The composition of embodiment B30, comprising 5 polypeptides each independently comprising an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

B32. The composition of embodiment B31 comprising 5 polypeptides, comprising: a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:1, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:2, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:3, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:4, and a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:5.

B33. The composition of embodiment B32 comprising 5 polypeptides, comprising: a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:1, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:2, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:3, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:5.

C1. An isolated polypeptide selected by the method of any one of embodiments A1-A22.1.

C2. A polynucleotide encoding the isolated polypeptide of embodiment C1.

C3. A composition comprising two or more isolated polypeptides or polynucleotides encoding the two or more isolated polypeptides, wherein the polypeptides are selected by a method of any one of embodiments A1-A22.1.

C4. The isolated polypeptide of embodiment C1 or polynucleotide of embodiment C2, wherein the isolated polypeptide is encoded by a virus or a bacterium.

C5. The composition of embodiment C3, wherein at least one isolated polypeptide is encoded by a virus or a bacterium.

C6. The isolated polypeptide or polynucleotide of embodiment C4, wherein the isolated polypeptide is encoded by a virus.

C7. The isolated polypeptide or polynucleotide of embodiment C6, wherein the virus is a coronavirus.

C8. The isolated polypeptide or polynucleotide of embodiment C6, wherein the coronavirus is SARS-CoV-2.

C9. The isolated polypeptide or polynucleotide of any one of embodiments C6-C8, wherein the protein is selected from among a spike protein, a membrane protein and an envelope protein.

C10. The isolated polypeptide or polynucleotide of embodiment C9, wherein the protein is a spike protein.

C11. The isolated polypeptide or polynucleotide of any one of embodiments C1, C2, C4 and C6- C10 wherein the polypeptide comprises an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

C11.1 The isolated polypeptide or polynucleotide of embodiment C11, wherein the isolated polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. C12. The polynucleotide of any one of embodiments C2, C4 and C6-C11.1, wherein the polynucleotide is an expression vector or expression plasmid.

C13. The polynucleotide of embodiment C12, wherein the polynucleotide is a DNA plasmid or vector, or RNA plasmid or vector.

C14. The polynucleotide of embodiment C13, wherein the polynucleotide is a DNA plasmid or vector and a portion of the DNA plasmid or vector comprises a DNA virus or portion thereof.

C15. The polynucleotide of embodiment C14, wherein the DNA virus is a herpesvirus, an adenovirus or a poxvirus.

C16. The polynucleotide of embodiment C13, wherein the polynucleotide is in a RNA plasmid or vector and a portion of the RNA plasmid or vector comprises a RNA virus.

C17. The polynucleotide of embodiment C16, wherein the RNA virus is a retrovirus or a ssRNA virus.

C18. The composition of embodiment C5, wherein the isolated polypeptide is encoded by a virus.

C19. The composition of embodiment C18, wherein the virus is a coronavirus.

C20. The composition of embodiment C19, wherein the coronavirus is SARS-CoV-2.

C21. The composition of any one of embodiments C18-C20, wherein the protein is selected from among a spike protein, a membrane protein and an envelope protein.

C22. The composition of embodiment C21, wherein the protein is a spike protein.

C23. The composition of any one of embodiments C18-C22, wherein at least one polypeptide comprises an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth in SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

C24. The composition of embodiment C23, comprising 2, 3, 4 or 5 polypeptides whose sequences differ from each other and each of which independently comprises an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

C25. The composition of embodiment C24, comprising 5 polypeptides each independently comprising an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

C26. The composition of embodiment C25 comprising 5 polypeptides, comprising: a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO: 1 , a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:2, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:3, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:4, and a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:5.

C26.1. The composition of embodiment C26 comprising 5 polypeptides, comprising: a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 , a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:2, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:3, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:5.

C27. A vaccine composition comprising an isolated polypeptide or polynucleotide of any one of embodiments C1, C2, C4 and C6-C17, and comprising one or more suitable pharmaceutically acceptable adjuvants and/or one or more suitable pharmaceutically acceptable carriers.

C28. A composition comprising an antigen presenting cell (APC) and an isolated polypeptide or polynucleotide of any one of embodiments C1 , C2, C4 and C6-C17.

C29. The composition of embodiment C28, wherein the polynucleotide resides within the APC.

C30. The composition of embodiment C28 or C29, wherein the polypeptide or a portion thereof is presented on the surface of the APC.

C31. The composition of any one of embodiments C28-C30, wherein the APC is a dendritic cell.

C32. The composition of any one of embodiments C28-C31, comprising two or more isolated polypeptides or one or more polynucleotides encoding the two or more isolated polypeptides.

C33. The composition of embodiment C32, wherein at least one polypeptide comprises an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

C34. The composition of embodiment C33, comprising 2, 3, 4 or 5 polypeptides whose sequences differ from each other and each of which independently comprises an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

C35. The composition of embodiment C34, comprising 5 polypeptides each independently comprising an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. C36. The composition of embodiment C35 comprising 5 polypeptides, comprising: a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO: 1 , a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:2, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:3, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:4, and a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:5.

C36.1. The composition of embodiment C36 comprising 5 polypeptides, comprising: a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:1 , a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:2, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:3, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:5.

D1. A pharmaceutical composition, comprising:

(a) one or more polypeptides each independently comprising an amino acid sequence consisting of, or 95% or more identical to, an amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5; and

(b) a pharmaceutically acceptable excipient.

D2. The pharmaceutical composition of embodiment D1, comprising 2, 3, 4 or 5 polypeptides whose sequences differ from each other and each of which independently comprises an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

D3. The pharmaceutical composition of embodiment D2, comprising 5 polypeptides each independently comprising an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

D4. The pharmaceutical composition of embodiment D3 comprising 5 polypeptides, comprising: a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:1, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:2, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:3, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:4, and a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:5. D4.1. The pharmaceutical composition of embodiment D4 comprising 5 polypeptides, comprising: a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 , a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:2, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:3, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:5.

D5. The pharmaceutical composition of any one of embodiments D1-D4.1 that is formulated for systemic, parenteral, topical, oral, mucosal, intranasal, subcutaneous, aerosolized, intravenous, bronchial, pulmonary, vaginal, vulvovaginal, esophageal, or oroesophageal administration.

D6. The pharmaceutical composition of embodiment D5 that is formulated for oral administration.

D7. The pharmaceutical composition of any one of embodiments D1-D6 that is formulated as a gel, ointment, liquid, suspension, aerosol, tablet, pill, powder or lyophile.

D8. The pharmaceutical composition of any one of embodiments D1-D7, wherein the composition comprises a microparticle.

D9. The pharmaceutical composition of embodiment D8, wherein the microparticle comprises a sustained-release polymeric matrix.

D10. The pharmaceutical composition of embodiment D8 or D9, wherein the microparticle comprises b-cyclodextrin, ethyl cellulose (EC) or b-cyclodextrin and ethyl cellulose (EC).

D11. The pharmaceutical composition of embodiment D10, wherein the microparticle comprises about or equal to 10% to about or equal to 20% w/w ethylcellulose, based on the weight of the microparticles.

D12. The pharmaceutical composition of embodiment D10 or D11, wherein the microparticle comprises about or equal to 15% w/w ethylcellulose, based on the weight of the microparticles.

D13. The pharmaceutical composition of any one of embodiments D10-D12, wherein the microparticle comprises about or equal to 50% to about or equal to 70% w/w b-cyclodextrin, based on the weight of the microparticles.

D14. The pharmaceutical composition of embodiment D13, wherein the microparticle comprises about or equal to 60% w/w b-cyclodextrin, based on the weight of the microparticles.

D15. The pharmaceutical composition of any one of embodiments D8-D14, wherein the microparticle is formulated for oral administration. D16. The pharmaceutical composition of any one of embodiments D1-D15, wherein the composition or microparticle comprises an agent that protects the components of the composition or microparticle against degradation in the acidic environment of the stomach.

D17. The pharmaceutical composition of embodiment D16, wherein the agent is hydroxypropyl- methyl cellulose acetate succinate (HPMCAS).

D18. The pharmaceutical composition of embodiment D17, wherein the composition is a microparticle and the microparticle comprises about or equal to 20% to about or equal to 40% w/w HPMCAS, based on the weight of the microparticles.

D19. The pharmaceutical composition of embodiment D18, wherein the microparticle comprises about or equal to 30% w/w HPMCAS, based on the weight of the microparticles.

D20. The pharmaceutical composition of any one of embodiments D8-D19, wherein the composition comprises a microparticle of a size between about or equal to 1 pm and about or equal to 5 pm in diameter, as determined by dynamic light scattering (DLS).

D21. The pharmaceutical composition of any one of embodiments D8-D20, wherein the composition comprises a microparticle and the microparticle comprises a lectin.

D22. The pharmaceutical composition of embodiment D21, wherein the lectin is selected from among Aleuria aurantia lectin (AAL), wheatgerm agglutinin and Ulex europaeus- 1.

D23. The pharmaceutical composition of embodiment D22, wherein the lectin is AAL.

D24. The pharmaceutical composition of any one of embodiments D21-D23, wherein the microparticle comprises about or equal to 0.1% to about or equal to 0.5% w/w lectin, based on the weight of the microparticles.

D25. The pharmaceutical composition of any one of embodiments D1-D24, comprising about or equal to 1% to about or equal to 10% w/w polypeptides.

D26. The pharmaceutical composition of any one of embodiments D1-D25, wherein the composition is formulated for prophylactic treatment.

D27. The pharmaceutical composition of embodiment D26, wherein the composition is formulated as a vaccine.

D28. The pharmaceutical composition of embodiment D27, wherein the composition comprises one or more suitable pharmaceutically acceptable adjuvants and/or one or more suitable pharmaceutically acceptable carriers. E1. A device, comprising the composition of any one of embodiments B1-B32, the isolated polypeptide, polynucleotide or composition of any one of embodiments C1-C36, or the pharmaceutical composition of any one of embodiments D1-D28.

E2. The device of embodiment D1 that is in unit dosage form, liquid dosage form, solid dosage form, oral dosage form, a tablet, a capsule, a topical patch, a syringe, an inhaler, a dosage cup, a dropper, a pump, a spray bottle, an aerosol container, a wound dressing, or an applicator for administering the pharmaceutical composition.

F1. A kit, comprising the composition of any one of embodiments B1-B32, the isolated polypeptide, polynucleotide or composition of any one of embodiments C1-C36, or the pharmaceutical composition of any one of embodiments D1-D28 and a device for administration of the composition.

F2. The kit of embodiment F1, wherein the composition of any one of embodiments B1-B32, the isolated polypeptide, polynucleotide or composition of any one of embodiments C1-C36, or the pharmaceutical composition of any one of embodiments D1-D28 is contained in the device for administration.

F3. The kit of embodiment F1, wherein the composition of any one of embodiments B1-B32, the isolated polypeptide, polynucleotide or composition of any one of embodiments C1-C36, or the pharmaceutical composition of any one of embodiments D1-D28 is present as a separate component that is distinct from the device.

F4. The kit of any one of embodiments F1-F3, wherein the device is a dressing, a topical patch, a pump, a spray bottle, an aerosol container, a syringe, an inhaler, a dosage cup, a dropper, or an applicator.

G1. A method for treating a disease or condition associated with a pathogen in a subject, comprising administering the composition of any one of embodiments B1-B32, the isolated polypeptide, polynucleotide or composition of any one of embodiments C1-C36, or the pharmaceutical composition of any one of embodiments D1-D28 to a subject in need thereof, in an amount sufficient to induce an immune response.

G2. A method for treating a disease or condition associated with a pathogen in a subject, comprising administering a therapeutically effective amount of the composition of any one of embodiments B1-B32, the isolated polypeptide, polynucleotide or composition of any one of embodiments C1-C36, or the pharmaceutical composition of any one of embodiments D1-D28 to a subject in need thereof.

G3. The method of embodiment G1 or G2, wherein the pathogen is a virus. G4. The method of embodiment G3, wherein the virus is a coronavirus.

G5. The method of embodiment G4, wherein the coronavirus is SARS-CoV-2.

G6. The method of any one of embodiments G1-G6, wherein the treatment is a prophylactic treatment.

G7. The method of any one of embodiments G1-G6, wherein the disease or condition is COVID-19.

H1. A method of inducing an immune response in a subject, comprising administering the composition of any one of embodiments B1-B32 or the isolated polypeptide, polynucleotide or composition of any one of embodiments C1-C36 in an amount sufficient to induce an immune response.

H2. The method of embodiment H1, further comprising obtaining polyclonal antibodies from the subject and/or antiserum that immunospecifically binds to a polypeptide, or a polypeptide encoded by, the composition of any one of embodiments B1-B32 or the isolated polypeptide, polynucleotide or composition of any one of embodiments C1-C36.

H3. The method of embodiment H2, further comprising: isolating spleen cells from the subject, and combining the spleen cells with myeloma cells under conditions that produce monoclonal antibody generating hybridomas.

H4. The method of embodiment H3, further comprising screening the hybridomas for those that produce monoclonal antibodies that immunospecifically bind to a polypeptide, or a polypeptide encoded by, the composition of any one of embodiments B1-B32 or the isolated polypeptide, polynucleotide or composition of any one of embodiments C1-C36.

H5. A monoclonal antibody produced by the method of embodiment H4.

11. A method of treating a disease or condition associated with a pathogen comprising administering, to a subject in need thereof, the monoclonal antibody of embodiment H5.

12. The method of embodiment 11, wherein the pathogen is a virus.

13. The method of embodiment I2, wherein the virus is a coronavirus.

14. The method of embodiment I3, wherein the coronavirus is SARS-CoV-2.

15. The method of any one of embodiments 11-14, wherein the disease or condition is COVID-19. Examples

The examples set forth below illustrate certain implementations and do not limit the technology. Example 1: Selection of Immunogenic Polypeptide Epitopes of the SARS-CoV-2 Coronavirus

DIAMOND™ is a computational platform and a neural network that can identify immunologic targets for T- and B-lymphocytes, which then can be used for the development of prophylactic treatments, such as vaccines, or to generate antibodies (e.g., monoclonal antibodies) for the treatment of diseases or conditions caused by pathogens. SpliceDiff™ is a proprietary software module for the identification of immunogenic polypeptide epitopes and is part of an integrated bioinformatics and artificial intelligence (Al) system as described herein and as described in PCT Application No. PCT/US20/35183, filed on May 29, 2020, the contents of which are expressly incorporated by reference herein. This example describes the use of this platform to identify immunogenic polypeptide epitopes of the SARS-CoV-2 coronavirus, which causes COVID-19.

DIAMOND™ was deployed to focus on the S1 sub-unit of the SARS-CoV-2 surface glycoprotein, also known as Spike (S) protein, which contains the receptor binding-domain (RBD) for ACE2, as it is essential for the virus to enter host cells. It was expected that anti-S1 antibodies directed against epitopes located in the RBD could be protective by neutralizing virus entry. Combined B and T cell activation are required for an effective immune response, hence peptides predicted to bind to HLA class I and class II molecules were included. The prediction of peptide affinity for HLA molecules is currently performed using standard artificial neural networks (ANN), while Diamond is equipped with a state-of-the-art capsule neural network (CNN), which applies an iterative routing-by- agreement mechanism, and therefore when multiple capsules agree, the probability of correct prediction is expected to be higher than that obtained with other network architectures.

The DIAMOND™ platform was used to select immune-dominant peptides from the S1 subunit of the Spike protein that were predicted to bind to the most common HLA class I and class II alleles and would be most likely to elicit neutralizing antibodies. From the predicted immune-dominant peptides, those that are accessible on the S1 protein surface were selected using a proprietary 3D rendering tool. A Phase I clinical study was conducted to evaluate the tolerability and the seroconversion rates in healthy individuals. The platform analyzed the sequence of the S glycoprotein to identify peptides in immune hotspots. The immune hotspot regions were defined as follows:

1) Sequences predicted to bind with high affinity to the most common HLA alleles in the world’s population.

2) Regions of the protein where the sequences identified in 1) clustered and/or overlapped

3) Regions predicted to be antibody epitopes, containing at least 1 HLA class II binder peptide

4) Regions where the predicted antibody peptides were accessible in 3D space. The CNN returned a list of possible epitope candidates predicted to be recognized by B and T cells. This list was then refined to include epitopes for the most common HLA alleles in the world’s population, to ensure effectiveness of a polypeptide-based vaccine against SARS-CoV-2 against as large a population as possible. Within this shortlisted epitope panel, antibody epitope sequences were selected that overlapped with HLA Class I and Class II binding peptides, to enhance the chance of a B and T cell response against the same sequences. The positions of the overlapping peptides were determined and further selected based on whether they were accessible on the surface of the S protein, thereby maximizing the chance that antibodies elicited by the vaccine will specifically neutralize the virus, thus minimizing the risk of antibody-dependent enhancement (ADE), which is driven by non-neutralizing antibodies.

The peptide selection steps described above resulted in the identification of 5 peptides (SEQ ID NOS: 1-5): two for B cell overlapping, two for CD4+ T-cells (HLA-Class II restricted), and three for CD8+ T cells (HLA Class l-restricted). These sequences are predicted to bind with high affinity to the following common HLA alleles in the world’s population:

HLA-DRB1*01:01 (94.27% of the world population)

HLA-B*18:01 (95.23% of the world population)

HLA-A*11:01 (83.39% of the world population)

HLA-B*58:01 (95.00% of the world population)

In addition, the selected peptides might provide cross-protection against coronaviruses other than SARS-CoV-2. It was found that the SARS-CoV-2 sequence where three of the 5 epitopes are located (one HLA-A*11:01- and one HLA-B*18:01- restricted peptide, and one Ab epitope overlapping with the HLA-DRB1*01:01 restricted peptide) shares an 86% homology with that of the SARS-CoV virus, with only two amino acid substitutions. The remaining 2 epitopes (one Ab epitope overlapping with the HLA-DRB1*01:01 restricted peptide, and the HLA-B*58:01 restricted peptide) are located in a region which shares 88% homology to that of the SARS-CoV virus. When analyzed by the DIAMOND™ CNN platform, the differences in the sequence did not alter the predicted B cell epitope localization, nor did they significantly reduce the affinity for HLA-DRB1*01:01, and HLA:18:01, indicating that a vaccine formulation containing these peptides might afford for a level of cross-protection against other coronaviruses, especially those that use ACE2 as a cellular receptor due to the tendency to conserve the RBD sequence.

Mutations in proteins of many viruses are common and they can pose an obstacle to vaccine development due to amino acid substitutions that reduce the binding of neutralizing antibodies, hampering the neutralization effect and possibly triggering ADEs. A multi-sequence alignment tool (MSA) was used to investigate the possible presence of emerging non-synonymous mutational hotspots in the epitopes selected for the SARS-CoV-2 vaccine formulation. The selected epitopes were found not to be affected by any mutations across all the available sequenced deposits from COVID19 outbreaks worldwide (https://www.ncbi.nlm.nih.gov/genbank/sars-cov-2-seqs/).

Example 2: Preparation, Characterization and Cytotoxicity of Microparticles for Formulation of a SARS-CoV-2 Vaccine

The 5 viral peptide antigens (VPAs) identified as discussed in Example 1 above (SEQ ID NOS:1-5) can be incorporated into a microparticle formulation that can be administered orally, e.g., as an oral vaccine. Microparticles containing immunogenic polypeptides, such as the VPAs, can be prepared as described, for example in WO 2010/037142 and WO 2016/081783, the contents of which are incorporated in their entirety by reference herein. The polymer matrix of the microparticles is loaded with specific components to increase target specificity and immunogenicity. The benefits of using MP for immunotherapy include uptake by antigen presenting cells (APC) such as dendritic cells (DCs) and macrophages, targeting, shielded trafficking of cargo, co-delivery of immunomodulatory agents and antigen(s), and sustained release of antigen(s), eliminating the need for repeated doses of the vaccine (7).

The microparticles are composed of b-cyclodextrin, hydroxypropyl-methyl cellulose acetate succinate (HPMCAS), and ethyl cellulose (EC). The HPMCAS component is used for its enteric properties to protect proteins from the harsh pH of the stomach, while the b-cyclodextrin and ethyl cellulose function as a sustained-release polymeric matrix. This formulation has previously been shown as ideal to deliver immune-stimulatory agents to the Gut-Associated Lymphoid Organs (GALT), in a murine model of ovarian cancer treated with an analogue oral vaccine (5). The vaccine formulation also contains microfold (M)-cell targeting ligand Aleuria Aurantia lectin (AAL), which has been shown to improve the targeting of the particles to the Peyer's Patches (PP).

The microparticles are formulated using a single-step spray drying method (8). OroVAX with VPAs will be formulated under cGMP constraints at Kiromic’s GMP facility located at 7707 Fannin St., Suite 140, Houston, TX 77054. In brief, a bio-degradable polymer matrix consisting of 20%

Hydroxyl propyl methyl cellulose acetate succinate (HPMCAS), 60% beta-cyclodextrin, and 20% ethyl cellulose is prepared in a sterile aqueous alkaline solution (1 mM NaOH in RNase-free H ₂ 0). The M cell-targeting ligand AAL (0.25% w/w loading relative to total mass of polymers) is added, followed by the peptide mix (1% w/w loading per each peptide). This feeding solution is sprayed, resulting in an aerosolized peptide/AAL/polymer matrix mixture. Optimal water removal from the aerosolized droplets results in polymeric microparticles containing the encapsulated peptide (VPAs)/AAL. Formulation details form part of a US and International Patent filed in 2009 entitled “A one-step method of formulating bioactive nano-particles with the use of a spray drying technique”, and a provisional patent filed in 2014 entitled “Method of Production and Use of a Novel Nanoparticle-Based Vaccine Targeting Cancer Testes Antigens in Solid and Hematologic Malignancies” (No. 62/081,825).

Physical characterization of the final spray-dried product is conducted for each OroVAX batch as described previously and incorporated by reference herein (21). Ideally, >90% of particles in any given batch have a diameter between 1 pm and 5 pm as determined by dynamic light scattering (DLS). The zeta potential of the product should be >0 mV when suspended in distilled water. Particle morphology can be determined by scanning electron microscopy, which also can be used for size analysis as an alternative for DLS (20, 22).

The encapsulation efficiency of the VPAs can be determined by ELISA and MS following their extraction from a particle sample dissolved in a slightly basic medium (Trizma®, pH 8.3), while the potency of the OroVAX batch can be determined by an immature dendritic cell (iDC) activation assay (21).

Biologic characterization of the final spray-dried product can be conducted for each OroVAX batch to ensure the desired properties and biological efficacy. The characterization includes induction of maturation markers in immature dendritic cells (iDC) generated from adherent peripheral blood monocytes in the presence of IL4 and GM-CSF. A passing result for the biological release assays is defined as DCs expressing ³60% DC80, ³65% CD83, ³50% CD86, and ³90% MHC class I (20).

The components of the microparticles are generally recognized as safe (GRAS) for human use by the FDA and have shown no toxicity, even at very high concentrations, in cultured cells and primates. As shown in Figure 1, OroVAX microparticles without VPAs (w/w composition, based on the weight of the microparticles, is 20% HPMCAS, 60% b-cyclodextrin, 15% EC and 0.25% AAL) were found not to be cytotoxic within the tested concentration range (0.0625-2 mg/mL). Percent cytotoxicity is relative to negative control of cells treated with complete DMEM media only. Positive control of benzalkonium chloride was found to be cytotoxic, with 21% cell viability (Kiromic unpublished results).

Example 3: M Cell Targeting, Uptake and Immune Adjuvant Properties of Microparticle Formulations Containing AAL

Previous reports have shown that AAL allows for microparticle anti-gravitational uptake by M cells in vitro in inverted orientation (11, 12). At the doses used, AAL optimizes M cell targeting, while not harming intestinal cells and it is therefore very likely that it will be safe and suitable for application also in humans (10, 11). When AAL was administered orally by Roth-Walter F. et al. (10-12), they reported no toxicity in Balb/C mice of oral microparticles carrying a 80-times higher dose of AAL than that of the amount of AAL in OroVAX. By testing escalating doses of AAL, it was found that normal intestinal epithelium cells were not affected until a 40-times higher dose than that of the amount of AAL in OroVAX was reached (13). Potency and efficacy studies of oral AAL-containing microparticles in several tumor models have demonstrated not only Microfold cells (M cells) targeting, but also activation of mucosal DCs with a cytotoxic T lymphocyte (CTL)-polarizing profile, resulting in antitumor effects (14-16). Data obtained in collaboration with Dr. Martin J D’Souza over the last decade demonstrate that immunization using AAL microparticles loaded with antigen- containing whole tumor cell lysate results in effective antibody titers and decreased tumor volumes in mice inoculated with melanoma (17), prostate (18), ovarian (19), and breast cancer cells (6).

In it known that enzymes called neuraminidases act as a virulence factors for several pathogens that invade the human body through Peyer's Patch M-cells. Because of the structural similarity of Aleuria Aurantia lectin (AAL) to neuraminidases, the hypothesis was made that AAL might also target human M-cells (11, 12), and it was shown that AAL is not only structurally similar to neuraminidases, but it also has similar immune cells activation abilities: upon incubation with AAL, PBMCs proliferated in response to AAL and secreted the cytokines, IL-2, IFN-gamma, IL-10 and IL- 5 in a concentration-dependent manner, without signs of toxicity.

Figure 2 demonstrates preliminary uptake studies using fluorescent-labeled (fluorescein isothiocyanate-labelled bovine serum albumin; FITC-BSA) and AAL blank microparticles in mice; previous studies with human M-cells have demonstrated excellent uptake into PPs within 60 minutes following oral administration. Mice were dosed with the microparticles, euthanized shortly thereafter and microparticle localization determined by fluorescence analysis. The results are presented in Figure 2. Particle distribution (green dots, represented in grayscale) in small intestine (Figure 2A) and uptake of labeled microparticles in Peyer’s patches (Figure 2B) microvilli are shown (Kiromic unpublished results).

Preliminary studies on immature dendritic cells (iDC) showed that AAL-containing OroVAX particles and free ALL, but not AAL-negative OroVAX particles, are capable of triggering a significant activation of iDC from healthy donors. iDC were obtained by exposing monocyte-enriched PBMCs cultures to 1,000 U/mL IL4 and 800 U/mL GM-CSF for 4 days in DC serum-free GMP medium (CellGenix). Half volume of medium was replaced on day 2 with fresh medium containing 2,000 U/mL IL4 and 1 ,600 U/mL GM-CSF. On day 4, microparticles with or without AAL, or free ALL, were added (1 mg microparticles / 2 x 10 ⁶ iDC in 2 mL medium, or the corresponding amount of AAL, i.e. 0.25% of 1 mg = 2.5 pg). Cells were incubated overnight before analysis. Consistently with previous results, cell viability was not affected (³98% in all conditions). Representative flow- cytometry results are depicted in Figure 3, which illustrates immunophenotype of human dendritic cells exposed to BSK02 microparticles or to control stimuli (Kiromic unpublished results).

The microparticle formulations described in a recent publication (16) and in WO 2016/081783 include, in certain aspects, the TLR9 agonist, CpG ODN, as immune adjuvant. To further assess the contribution of CpG ODN to the immunogenicity of the vaccine, OroVAX-CpG microparticles were manufactured and tested for their ability to activate HEK-Blue™ cells (InVivogen). HEK- Blue™-hTLR9 cells were obtained by co-transfection of the human TLR9 gene and an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene into HEK293 cells. The SEAP gene was placed under the control of the IFN-b minimal promoter fused to five NF-KB and AP-1- binding sites (16). Stimulation with a TLR9 ligand activates NF-KB and AP-1, which induces the production of SEAP. Cells were exposed to OroVAX, OroVAX-CpG, or free CpG ODN for 26 hours, before reading the optical density (OD) at 655 nm to assess reporter activity. As shown in Figure 4, which illustrates measurement of CpG ODN-dependent TLR9 activation before and after spray drying (16), CpG ODN does not show any residual activity after the spray-drying process, indicating that the immune stimulant function of OroVAX is exerted by AAL (16). The results demonstrate that AAL serves both as an M-cell targeting agent and as an immune adjuvant, collectively enhancing the immunogenicity of the peptides loaded into OroVAX.

Example 3: Potency of the Microparticle Formulations

The potency of the OroVAX particles can be inferred based on potency studies that were performed using BSK-02 microparticle formulations (Composition, as w/w based on the weight of the microparticles, is 20% HPMCAS, 60% b-cyclodextrin, 15% EC, 0.25% AAL and 5% SP17 polypeptide (SEC ID NO:7)).

Figure 10 illustrates a potency assay for BSK-02 microparticles incubated at 37 °C for 7 days or 14 days, as measured by activation of iDC. iDC were generated and tested as indicated in Example 2 above. There was no significant difference between BSK-02 stored at -20 °C for 7 days or 14 days and thawed prior to use and BSK-02 stored at 37 °C for up to 14 days (Kiromic unpublished data).

In Figure 10, BSK02-1 is microparticles stored for 7 days at -20 °C, BSK02-2 is microparticles stored for 7 days at 37 °C, BSK02-3 is microparticles stored for 7 days at -20 °C, and BSK02-4 is microparticles stored for 7 days at 37 °C. The control is freshly prepared BSK-02 microparticles.

Example 4: Immunogenicity of the VP As, and Ability to Elicit T-cell Mediated Killing

Monocytes were used as antigen presenting cells (APCs) to present the immunogenic polypeptides identified as described in Example 1 (SEC ID NOS:1-5). As shown in Figure 11, in vitro immunogenicity testing indicated that these peptides are presented by APC in selected HLA type donors and elicit cytotoxic T cell responses and Interferon g release. Figure 11 depicts light micrographs of APCs co-cultured with autologous peripheral blood lymphocytes (PBL), and IFNy and Turbofectin release in the medium, as measured by ELISA. T- cell mediated cytotoxicity is visible in APCs pulsed with the peptides of SEQ ID NOS: 1-5. As shown in the Table, the experiment was conducted on cells isolated from the peripheral blood of a donor presenting HLA-A*11 :01 and HLA- DRB1 ^* 01 :01 , which bind three of the 5 predicted peptides (CPFGEVFNATRFASV _aa 336-35o and VGGNYNYLYRLFRKS _aa445 -459 bind to HLA-DRB1 ^* 01 :01, ASVYAWNRK _aa348 -356 binds to HLA- A*11:01). A) Vehicle B) APC pulsed with 10 ug/mL equimolar solution of the 5 peptides C) APCs pulsed with 2 ug/mL immunogenic CEFT pool (JPT Peptide Technologies).

Furthermore, in vitro killing assays were performed to test the killing ability of cytotoxic T cells (CTLs) activated by APCs pulsed with the 5 SARS-CoV-2 peptides (SEQ ID NOS: 1-5). Cells from two donors bearing the HLA Types HLA Class I A* 11:01 , HLA Class II DRB1 *01:01 (A), and HLA- B*58:01 (B) were used in this assay. Autologous monocytes were used as targets (APCs), with an effector to target ratio of 1 :1. Apoptosis was detected as increase of fluorescence following the activation of Caps3/7 Green Reagent (Incucyte®).

Figures 12 A and 12B depict T-cell mediated cytotoxicity of target cells pulsed with SARS-CoV-2 peptides. Cells from two donors bearing the HLA Types HLA Class I A* 11 :01 , HLA Class II DRB1 *01 :01 (A), and HLA-B*58:01 (B) were utilized in this assay. Autologous monocytes were utilized as targets with an effectors to targets ratio of 1 :1. Apoptosis was detected as increase of fluorescence following the activation of Caps3/7 Green Reagent (Incucyte®). The kinetics of apoptosis of monocytes enriched with 10 ug/mL equimolar solution of the indicated peptides after addition of activated PBL is shown in squares in Figure 12A. In circles in Figure 12A is shown the killing of CEFT enriched monocytes by CEFT -APC activated PBL.

As shown in Figure 12, the results showed the selective killing of target cells, specifically autologous monocytes, “decorated” with the SARS-CoV-2 peptides, by the activated CTLs. Conversely, PBL activation by APCs pulsed with SARS-Cov-2 peptides appeared to have no significant cytotoxic effects on only monocytes. As a positive control, the cytotoxicity of PBLs activated after interaction with CEFT pulsed APCs was measured utilizing CEFT enriched monocytes (Figure 12A). Figure 12B depicts the kinetics of apoptosis of monocytes enriched with 10 ug/mL equimolar solution of each of the SARS-CoV-2 peptides as indicated, as well as a cocktail of all 5 peptides. These results demonstrate that APCs decorated with each of the the 5 SARS-CoV-2 immunogenic polypeptides, and with a cocktail containing all 5 polypeptides, selectively induce T-cell mediated killing of the APCs, thereby demonstrating the promise of these peptides as a vaccine. Example 5: Criteria, Objectives and Guidelines for Phase I Pilot Study (Clinical Trial)

This phase I pilot study is an open-label, multi-site trial in 10 healthy male and non-pregnant female subjects, ages 18-55 years of age, who are in good health and meet all inclusion and exclusion eligibility criteria. Each subject will receive one 5mg dose and will be followed as per the study calendar (Appendix 1) for one year after the vaccination. This study is designed to rapidly evaluate the safety and efficacy of a novel oral vaccination delivery mechanism (OroVAX) designed to prevent the development of Covid-19 without generating cytokine mediated toxicity such as ARDS. The trial is designed to meet 2 key objectives. First, it will evaluate the safety and tolerability of this novel oral vaccine delivery platform. Second, it will assess the ability of OroVAX to generate an immune response, by quantifying the IgG and IgM antibody titer values (ATV) to the selected coronavirus peptide antigens. The ATV generated from OroVAX will also be used to determine the percentage of those subjects who seroconvert (a surrogate endpoint for protection against the coronavirus).

I. Hypotheses and Objectives Hypotheses

We hypothesize that the treatment of patients with the OroVAX formulation incorporating AAL and VPAs, will result in minimal adverse events. We further hypothesize that OroVAX will elicit an immune response as measured by peptide-specific IgG/lgM in the serum of treated subjects. Seroconversion is defined as a 4X increase in the ATV above baseline.

Primary Objective

To determine the feasibility, tolerability, reactogenicity, and adverse effects of solicited and unsolicited adverse effects (AE) of a single dose of an orally-administered OroVAX formulation incorporating VPAs and AAL.

Secondary Objective

To determine the immunogenicity of OroVAX incorporating VPAs and AAL, by measuring anti-S protein IgG/lgM Antibody Titer Values (ATV) before and after vaccination. Seroconversion will be defined as a 4X increase in the ATV compared to baseline. 2. Study Design

A. Study Population

Number and Characteristics of Patients Enrolled

1. The study population will be drawn from patients at various clinical institutions following contractual agreements.

2. The study population will consist of 10 patients.

3. There will be no patient restrictions based on race or gender.

4. Healthy adults ages 18-55 years of age.

3. Eligibility Criteria

Patients must have baseline evaluations performed prior to administration of the first dose of OroVAX and must meet all inclusion and exclusion criteria. In addition, the patient must be thoroughly informed about all aspects of the study, including the study visit schedule, required evaluations, and all regulatory requirements for informed consent. The written informed consent must be obtained from the patient prior to enrollment.

Each clinical trial site will have the option to enroll patients for a particular indication and/or stage as allowed by the protocol, if that indication and/or stage is more prevalent, accruable, or clinically relevant to that particular institution, as long as the patient meets all the inclusion and exclusion criteria.

5. Inclusion Criteria

The following criteria apply to all patients enrolled on the study, unless otherwise specified:

1. Ability to provide informed consent for healthy adult patients 18-55 years of age.

2. CBC with differential, serum creatinine (Scr), alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), total bilirubin (TB), lipase, prothrombin time (PT), and partial thromboplastin time (PTT) are within normal reference range limits of the laboratory processing the results 7 days

(+/- 3 days) or less prior to enrollment.

3. Negative urine or serum pregnancy test 7 days (+/- 3 days) or less prior to enrollment. In women of child-bearing age, and must agree to use accepted contraception methods for at least 3 months after the final vaccination. 4. Either a female or male of reproductive capacity (females not of childbearing potential are post-menopausal as defined by having a history of amenorrhea for at least one year, or are surgically sterile via hysterectomy, bilateral oophorectomy, or tubal ligation/salpingectomy) wishing to participate in this study must be using, or agree to use, one or more types of birth control during the entire study and for

3 months after the final vaccination. Birth control methods may include condoms, diaphragms, birth control pills, anti-gonadotropin injections/implants/inserts, intrauterine devices (IUD), or surgical sterilization. All post-pubertal males are considered to be of reproductive capacity unless vasectomy for 180 days or more prior to first vaccination or bilateral orchiectomy. Women of reproductive capacity will be required to undergo a urine pregnancy test before completion of the post screening informed consent process. Accordingly, males agree to decline sperm donations until greater than 3 months after the final vaccination.

5. Temperature less than 100.0 degrees Fahrenheit at enrollment and within 24 hours prior to first vaccination

6. Subject agrees to have samples stored for additional and/or other research projects

7. Negative for SARS-Cov-2 RT-PCR (or other CDC-approved screening methods)

7 days (+/- 3 days) or less prior to enrollment.

8. Negative for anti-SARS-Cov-2 IgG 7 days (+/- 3 days) or less prior to enrollment.

6. Exclusion Criteria

1. Positive pregnancy test.

2. Positive for SARS-Cov-2 RT-PCR (or other CDC-approved screening methods)

7 days (+/- 3 days) or less prior to enrollment.

3. Positive for anti-SARS-Cov-2 IgG 7 days (+/- 3 days) or less prior to enrollment.

4. Subject is breast feeding or plans to breast feed within 3 months after the final vaccination.

5. Patients with geographic conditions that prevent adequate follow-up or compliance with the study protocol, or other extenuating circumstances regarding overall medical condition and/or potential compliance issues.

6. Immunodeficiency of any cause. 7. COPD or severe asthma requiring daily medication support or hospitalizations for respiratory distress within 3 years of enrollment.

8. History of significant neurological diseases including stroke, seizures, encephalomyelitis or transverse myelitis within 3 years prior to enrollment.

9. Malignancy treatment within 5 years of enrollment except for basal cell or squamous cell carcinoma of the skin.

10. Development of an acute illness during the vaccination protocol not felt to be potentially related to the vaccination protocol

11. History of CHF, CAD, myocarditis, or pericarditis.

12. History of autoimmune disease.

13. History of ulcerative colitis, peptic ulcer disease including prescription or OTC medications for alleviation of gastritis, celiac disease, and/or any other type of intestinal malabsorption syndrome related disease such as pancreatic insufficiency

14. History of HIV, HCV, or HBV.

15. History of prior SARS-CoV-2 investigational study participation or plans to do so within the reporting follow-up period of this study.

16. History of use of any medications that may suppress the immune system such as systemic corticosteroids greater than 10mg/day of a prednisone equivalent, or any other medication, cytotoxic drug, or any immunomodulatory agent that the PI decides may impair the immune response. Topicals, ophthalmics, inhaled, or intranasal steroids will be allowed.

17. Any blood products within 3 months of enrollment.

18. History of alcohol or substance abuse within 6 months of enrollment.

19. Any live, attenuated, or inactive vaccine administered within 3 months before or after the vaccination protocol has been completed

20. Plan to travel outside the U.S. prior to Day 42 of the study.

21. Plan to donate blood or blood products during the reporting follow-up period of the study.

22. Patient on chronic antibiotics for any reason per PI discretion. Withdrawal Criteria

A. Reasons for Withdrawal of Patients from Study

1. If, at any time, a patient is found to be ineligible for the protocol according to patient inclusion and exclusion criteria, he or she will be removed from the study.

2. Patients will be withdrawn from the study in the event of limiting toxicity.

3. Patients will be withdrawn from study in the event of protocol non-compliance.

4. Patients may voluntarily withdraw from study for any reason.

5. Patients will be withdrawn from the study in the event they test positive for a CDC- approved SARS-Cov-2 detection test (except for IgG/lgM) at any point during the study follow-up.

B. Follow-up of Withdrawn Patients

1. A subject withdrawn from the study may be followed indefinitely, or until resolution of the event(s) which caused the withdrawal.

2. All subjects will be requested to adhere to the protocol schedule for clinic visits.

3. Blood draws will be requested according to the protocol schedule.

As far as it is reasonable, data will continue to be collected at the indicated protocol time-points. Subject Replacement

A. Reasons for Replacement

1. Subjects will be replaced in the event of screening failures post-enrollment.

2. Voluntary withdrawal of subjects prior to initiation of treatment.

3. If the subject is lost to follow-up after at least 3 phone call attempts to reach the subject, then a certified letter will be sent to the subject asking them to call to update us on their status, otherwise they will be withdrawn from the trial 7 days from the date of the letter. If the subject is categorized as lost to follow-up they may be replaced within that subject’s dose cohort. Study Schedule

A. Summary of Pre-treatment Evaluations

1. Completed written informed consent for screening assessment and subsequent enrollment. Once all screening parameters are met, including all inclusion and exclusion criteria, then the patient will be scheduled for vaccination administration as per the study calendar.

10. Safety Endpoints

The primary endpoints for this Phase I pilot trial are feasibility, safety, and toxicity of a single 5mg OroVAX vaccination.

A. Definition of Adverse Event and Adverse Reaction

1. An adverse event is defined as any untoward medical occurrence associated with the use of a drug in humans, whether considered drug related. More specifically, an adverse event can be any unfavorable and unintended sign (e.g., an abnormal laboratory finding), symptom, or disease temporally associated with the use of a drug, without any judgment about causality. An adverse event can arise from any use of the drug (e.g., off-label use, use in combination with another drug) and from any route of administration, formulation, or dose, including an overdose.

2. An adverse reaction is defined as any adverse event caused by a drug. Adverse reactions are a subset of all suspected adverse reactions for which there is reason to conclude that the drug caused the event.

B. Causality

1. Suspected

A suspected adverse reaction is defined as any adverse event for which there is a reasonable possibility that the drug caused the adverse event. For the purposes of IND safety reporting, “reasonable possibility” indicates that there is evidence to suggest a causal relationship between the drug and the adverse event. A suspected adverse reaction implies less certainty about causality than an adverse reaction.

2. Unexpected

An adverse event or suspected adverse reaction is considered unexpected if it is not listed in the investigator brochure or package insert(s) or is not listed at the specificity or severity that has been observed, or, if an investigator brochure is not required or available, is not consistent with the risk information described in the general investigational plan or elsewhere in the current application. Adverse events that would be anticipated to occur as part of the disease process are considered unexpected for the purposes of reporting because they would not be listed in the investigator brochure. For example, a certain number of non-acute deaths in a cancer trial would be anticipated as an outcome of the underlying disease, but such deaths would generally not be listed as a suspected adverse reaction in the investigator brochure. Some adverse events are listed in the Investigator Brochure as occurring with the same class of drugs, or as anticipated from the pharmacological properties of the drug, even though they have not been observed with the drug under investigation. Such events would be considered unexpected until they have been observed with the drug under investigation. For example, although angioedema is anticipated to occur in some patients exposed to drugs in the ACE inhibitor class and angioedema would be described in the investigator brochure as a class effect, the first case of angioedema observed with the drug under investigation should be considered unexpected for reporting purposes.

3. Serious

An adverse event or suspected adverse reaction is considered serious if, in the view of either the investigator or sponsor, it results in any of the following outcomes: a. Death b. Life-threatening adverse event c. Inpatient hospitalization or prolongation of existing hospitalization d. A persistent or significant incapacity or substantial disruption of the ability to conduct normal life function e. Congenital anomaly/birth defect f. Important medical events that may not result in death, or require hospitalization yet may be considered serious when, based upon appropriate medical judgment, they may jeopardize the patient or subject and may require medical or surgical intervention to prevent one of the outcomes listed in this definition. Examples of such medical events include allergic bronchospasm requiring intensive treatment in an emergency room or at home, blood dyscrasias or convulsions that do not result in inpatient hospitalization, or the development of drug dependency or drug abuse.

4. Life-threatening

An adverse event or suspected adverse reaction is considered life-threatening if, in the view of either the investigator or sponsor, its occurrence places the patient or subject at immediate risk of death. It does not include an adverse event or suspected adverse reaction that, had it occurred in a more severe form, might have caused death. C. Recording of Adverse Events (AE)

1. The Investigator will assign attribution of the possible association of any AE with use of the investigational drug, and this information will be recorded using the classification system listed below: a. Related to investigational drug/intervention:

• Unrelated: The AE is clearly not related to the vaccination

• Doubtful: The AE is doubtfully related to the vaccination

• Possibly: The AE is possibly related to the vaccination

• Probable: The AE is likely related to the vaccination

• Definite: The AE is clearly related to the vaccination

2. Signs or symptoms reported as AE will be graded and recorded by the Investigator. When AEs are unsolicited/unspecified then they will be recorded as to the type/description of the AE, and graded by the Investigator as none, mild, moderate, severe, or life threatening according to the following grades and definitions:

All specified AE signs or symptoms that are seen within 28 days of the final vaccination will be graded and recorded by the Investigator according to the following:

* Subject should be at rest for all vital sign measurements.

** Oral temperature; no recent hot or cold beverages or smoking.

*** When resting heart rate is between 60 - 100 beats per minute. Use clinical judgement when characterizing bradycardia among some healthy subject populations, for example, conditioned athletes.

D. Follow-up of Adverse Events

All adverse events will be followed through 28 days after the final vaccination with appropriate standard of care medical management until resolved, (including patients removed or withdrawn from the study) as per the following:

E. Adverse Event Monitoring

1. All AE, whether unexpected, and whether considered to be associated with the use of the study drug, will be recorded, as noted above.

2. The Investigator will assess all AE and determine reportability requirements to the Food and Drug Administration (FDA), if it meets the FDA reporting criteria.

3. All AE recorded will be reviewed by the study Principal Investigator (PI) on a weekly basis and the Institutional Protocol Review Committee (I PRC) and/or IRB monthly, or as deemed necessary by the study PI. The I PRC will review and discuss at each meeting the selected toxicity, the toxicity grade, and the attribution of the relationship of the AE to study drug/treatment.

F. Definition of Dose-Limiting Toxicity (DLT)

1. Grade III or higher allergic reaction. Grade III is defined as symptomatic bronchospasm requiring medication, edema or angioedema, and Grade IV is defined as anaphylaxis. 2. Grade II or higher autoimmune reaction. Grade II is defined as an autoimmune reaction involving a non-essential organ or function requiring treatment.

3. Grade III or higher hematologic or non-hematologic toxicity including fever (> 40°C for > 24 hours).

11. Immune Response Endpoint

Immune Response (IR) Rules: The main secondary endpoint of this study is immune response (IR), defined as “Detection of a peptide-specific immune response in patients receiving oral OroVAX formulation incorporating viral peptides and Aleuria Aurantia Lectin (ALL)”.

B-cell (humoral) response

Immune Response Endpoint will be determined as follows: Peptide-specific IgG and IgM in the serum of patients will be analyzed by ELISA using 5-fold dilutions of serum and a standard colorimetric measurement. Immune response tests will be performed at Kiromic laboratories or by third-party contractors with ISO certification, if available.

12. Reasons for Discontinuation of Study

1. Dose limiting toxicities (DLTs): if more than 1 DLT is reported in either cohort then the study may be put on hold until the situation can be assessed, to determine if the events were possibly related to OroVAX vaccination. If the DLTs are considered to be clinically significant and possibly related to OroVAX administration, then the study will be closed. If not, it may proceed after IRB review and approval, and after the sponsor communicates to the FDA the outcome of the investigation.

2. Known Toxicities Associated with tumor peptides Immunotherapy: because peptides used in the present study are generated using a cell-free, in vitro GMP- grade synthesis system, and AAL is obtained at GMP grade, their administration can be safely controlled in terms of absence of toxic, infectious and pyrogenic material.

3. Known Toxicities Associated with AAL as Adjuvant: pre-clinical studies in human and mouse cells in vitro (10-12), and in vivo studies in mice (8, 11, 12, 17, 19, 20).

13. Methods and Assessments

1 Contraception and Pregnancy Testing

Either a female or male of reproductive capacity wishing to participate in this study must be using, or agree to use, one or more types of birth control during the entire_study and for 3 months after completing the study. These may include condoms, diaphragms, birth control pills, anti- gonadotropin injections, intrauterine devices (IUD), surgical sterilization, or subcutaneous implants. Women of reproductive capacity will be requested to undergo a urine or serum pregnancy test before completion of the screening process.

2 Screening, Study Visits, and Procedures

(A) Screening Visits and Procedures

1. Informed written consent will be obtained for screening and subsequent enrollment.

2. Once consent is obtained, all inclusion and exclusion criteria will be assessed including the following: a. History and physical examination. b. Pregnancy test (urine or serum). c. Fever less than 100 degrees Fahrenheit at enrollment on day -7 and on each day of vaccine administration. d. Evaluation of concomitant medications. e. CBC and differential leukocyte count. f. Hepatic and renal function tests. g. Negative history of HIV, HCV, and HBV. h. Negative SARS-Cov-2 RT-PCR (or equivalent CDC-approved test for the detection of the virus) i. Negative anti-SARS-Cov-2 IgG

3. Once the subject is determined to meet all inclusion and exclusion criteria, they will be scheduled for corresponding OroVAX administration.

(B) Study Visits and Procedures

1. Study visits for clinical toxicity evaluations will occur on each day of the first vaccination, and then on days 7, 14, and 28 (+/- 3 days).

2. ATV test will be obtained as a baseline (+/- 3 days), and 7, 14, and 28 days after the vaccination (+/- 3 days).

3. SARS-Cov-2 RT-PCR will be obtained 7 days or less (+/- 3 days) prior to the first vaccination and again on days 12, 42, 194 and 379 to confirm the patient does not develop Covid-19 during the study. 4. Anti-SARS-CoV-2 IgG/lgM 7 days or less (+/- 3 days) prior to the first vaccination to confirm the patient was not previously positive for Covid-19 prior to trial enrollment.

5. Procedures: Patients will receive one OroVAX vaccination on Day 0.

Patient enrollment: Six (6) patients will be treated to evaluate for potential toxicity and reactogenicity and if no more than 1 DLT is noted in either disease cohort, then the study may continue to accrue patients until accrual is complete.

6. Vaccination Schedule:

Day 0

Vaccination

(5mg)

7. All clinical laboratory tests will be performed in a CLIA and/or ISO-certified facility. Non-clinical tests (i.e. , immune response assays) may be performed at Kiromic’s laboratories.

(C) Final Study Visit

1. The final study visit will take place 28 days after the vaccination (+/- 3 days).

Laboratory tests at the final study visit will include hepatic and renal profiles, and CBC with differential. See Appendix 2 for laboratory adverse effect (AE) monitoring criteria.

(D) Post-Study Follow-up

Once the protocol is completed, patients will be followed off protocol as per standard of care by the patient’s physician(s).

(E) Discontinuation Visit

1. Will be identical to final study laboratories/procedures.

14. Study Materials and Procedures 1. Product

The micro particulate vaccine will contain a maximum of 1% weight ratio of VPAs mix. Peptides will be synthesized with ³95% purity, TFA-free, and prior to the spray-drying process they will be resuspended at 10-20 mg/mL in sterile water for injection (WFI), or DMSO, or sterile DPBS, depending on their solubility. 2. Detailed peptides list (VPAs)

Using a proprietary Artificial Intelligence-driven method of selection, we identified peptides from the subunit 1 of the SARS-Cov-2 S glycoprotein (YP_009724390.1 surface glycoprotein, Severe acute respiratory syndrome coronavirus 2) that are predicted to generate a protective immune response in the majority of the world’s population, basing on in silico binding predictions to HLA-DRB1-*01:01, HLA-A*01:01, HLA-B*18:01, HLA-B*58:01, and to antibodies. We used a 3D rendering engine to select target peptides that are accessible on the surface of the S protein, thus maximizing the probability of protective antibody binding to the virus.

The following figures show the mapping of the selected epitopes in the 3D space highlighted in red. The S protein is shown in gray in a vertical orientation, with the selected epitope in red at the top outer edge of the S-protein, where it is most likely to interact with the ACE2 cellular receptor.

The following are the selected peptide sequences:

Sequence HLA binding Ab epitope % population coverage ^†

CPFGEVFNATRFASVaa336-350 HLA-DRB1*01 :01 yes 94.72 GEVFNATRFaa339-347 HLA-B*18:01 yes 95.23

AS VY AWN R Kaa348-356 HLA-A*11 :01 yes 83.39

VGGNYNYLYRLFRKSaa445-459 HLA-DRB1*01 :01 yes 94.72 VGG N YN YI_Yaa445-453 HLA-B*58:01 yes 95.99

†) The estimated population coverage represents the percentage of individuals within the population that are likely to elicit an immune response to the indicated epitope. Population coverages were computed using the tool provided by the Immune Epitope Database (IEDB) (http://tools.iedb.org/population/). This tool uses the distribution of MHC alleles (with at least 4-digit resolution, e.g., A*02:01) within a defined population (obtained from http://www.allelefrequencies.net/) to estimate the population coverage for a given epitope.

The following figures (Figure 5A & 5B through Figure 9A & 9B) illustrate the mapping of the selected epitopes in the 3D space highlighted in red. The S protein is shown in gray in a vertical orientation, with the selected epitope in red at the top outer edge of the S-protein, where it is most likely to interact with the ACE2 cellular receptor. 15. Reagents

1. Aleuria Aurantia Lectin

AAL is obtained from Medicago (catalogue number 05-0134-2): Purity >= 95% by SDS-PAGE 2. Microparticle polymers a. b-cyclodextrin: International Chemical Identifier: 1 S/C42H70O35/c43- 1-8-29- 15(50)22(57)36(64-8)72-30-9(2-44)66-38(24(59)17(30)52)74-32- 11(4-46)68- 40(26(61)19(32)54)76-34-13(6-48)70-42(28(63)21(34)56)77-35-1 4(7-49)69- 41(27(62)20(35)55)75-33-12(5-47)67-39(25(60)18(33)53)73-31-1 0(3-45)65- 37 (71 -29)23(58) 16(31 )51/h8-63H , 1 -7H2/t8-,9-, 10-, 11-,12-,13-,14-,15-,16-

, 17-, 18-, 19-,20-,21 -,22-,23-,24-,25-,26-,27-,28-,29-,30-,31 -,32-,33-,34-,35- ,36-,37-,38-,39-,40-,41-,42-/m1/s1

Food-grade b-cyclodextrin is obtained from Sigma-Aldrich (catalogue number W402826). b- cyclodextrin is “generally recognized as safe” (GRAS) by the FDA. b. Hydroxypropyl-methyl cellulose acetate succinate: International Chemical

Identifier: 1S/C4H604.C3H802.C2H402.CH40/c5-3(6)1-2-4(7)8;1-3(5)2-

4;1-2(3)4;1-2/h1-2H2,(H,5,6)(H,7,8);3-5H,2H2,1 H3;1H3,(H,3,4);2H,1 H3 HPMC-AS (AquaSolve™) is obtained from Ashland (catalogue number 834123).

HPMCAS is “generally recognized as safe” (GRAS) by the FDA. c. Ethyl cellulose: International Chemical Identifier: 1S/C20H38O11/c1-6-26-10- 12-16(17(27-7-2) 18(28-8-3)20(25-5)30- 12)31-19-14(23) 13(22) 15(24-4) 11 (9- 21)29-19/h11-23H,6-10H2,1-5H3

Ethyl cellulose Aqueous Dispersion (Aquacoat® ECD, NF, JPE) is obtained from DuPont Nutrition USA, Inc. (catalogue number 422002-15).

Ethyl cellulose is “generally recognized as safe” (GRAS) by the FDA.

16. General Outline of OroVAX Manufacturing

OroVAX will be formulated under cGMP constraints at Kiromic’s GMP facility located at 7707 Fannin St., Suite 140, Houston, TX 77054. In Brief, a bio-degradable polymer matrix consisting of 20% Hydroxyl propyl methyl cellulose acetate succinate (HPMCAS), 60% beta- cyclodextrin, and 20% ethyl cellulose will be prepared in a sterile aqueous alkaline solution (1 mM NaOH in RNase-free H ₂ 0). The M cell targeting ligand AAL (0.25% w/w loading relative to total mass of polymers) will be added, followed by the peptide mix (1% w/w loading per each peptide). This feeding solution will be sprayed resulting in an aerosolized peptide/AAL/polymer matrix mixture. Optimal water removal from the aerosolized droplets will result in polymeric microparticles containing the encapsulated peptide/AAL. Formulation details form part of a US and International Patent filed in 2009 entitled “A one-step method of formulating bioactive nano particles with the use of a spray drying technique”, and a provisional patent filed in 2014 entitled “Method of Production and Use of a Novel Nanoparticle-Based Vaccine Targeting Cancer Testes Antigens in Solid and Hematologic Malignancies” (No. 62/081,825).

17. Storage and Drug Accountability

A. Spray Dried Product

OroVAX microparticles will be stored refrigerated (+4 °C) in a dry form, for up to -600 days, or at room temperature (+24 °C), for up to -90 days.

B. Rationale for the drug storage guidelines

We performed an accelerated stability test as follows (http://lso-inc.com/medical-package- testi ng/accelerated-ag i ng . htm I)

Two (2) sample aliquots were analyzed for each time point, in two separate incubators (+37°C). Acceleration is calculated with real storage at +4 °C and RT (24 °C). The temperature difference is 33°C and 13°C, respectively.

With Q10 value = 1.8, the acceleration factors are:

1.8exp33/10 = 1.8exp3.3 = 48

1.8exp13/10 = 1.8exp1.3 = 6.6

C. Schedule for accelerated stability study

Storage time (days) Calculated storage time Calculated storage time at 37°C (days) at +4 °C (days) at +24 °C

14 683 92

18. Treatment

18.1 Selection of Oro VAX dose level

A dose of 5mg of OroVAX has been previously shown to provide an efficient adjuvant effect in oral vaccine formulations tested in murine models (17, 19, 20), with a very low risk of adverse effects. 18.2 Study Drug Quality and Safety Measures

18.2a OroVAX Formulation

1. OroVAX will be administered for one dose on Day 0.

2. Peptides incorporated into the formulation will be at least 95% pure, and they will not contain TFA.

3. Plan of action in the event of OroVAX production batch fails:

If a bioburden test is positive, the OroVAX batch will be discarded. Treatment may proceed if it is possible to prepare a new batch. Otherwise, the patient will be withdrawn from the study.

4. Physical characterization of the final spray dried product will be conducted for each OroVAX batch prior encapsulation. Ideally, 90% of particles in any given batch have a diameter between 1 pm and 5 pm as determined by dynamic light scattering (DLS). The zeta potential of the product should be >0 mV when suspended in distilled water. Particle morphology will be determined by scanning electron microscopy, which can also be used for size analysis as an alternative for DLS.

5. The sterility of the final spray dried product will be assessed by performing bacteria/fungi culture prior to batch release.

18.3 Specific Restrictions and Requirements

18.3a Concomitant Therapy

1. All medications (prescription and over the counter), vitamin and mineral supplements, and/or herbs taken by the study subjects will be documented.

18.3b Compliance with Therapy

1. All treatments and study procedures will be administered in the clinic. Subjects will be given a checklist for daily recording of health status, treatment side effects, and documentation of all other medications and dietary supplements that may be taken.

19. Sample Size and Statistical Methods

19.1 Safety and Toxicity

Based on pre-clinical efficacy and toxicity evaluations, the tolerability of OroVAX will be tested. 19.2 Immune Response (IR)

For the IR part of the study, a Simon 2-stage design with a null hypothesis of 5% IR (pO = 0.05) and alternative hypothesis of 35% IR (p1 = 0.35), with desired significance level (a) and desired power (1-b) of 0.05 and 0.9, respectively, will be utilized. If three (3) or more responses out of a total of 6 patients are observed per cohort, OroVAX immunotherapy may move to a formal Phase I/ll clinical trial.

20. Ethical Considerations

20.1 Protection of Patients from Unnecessary Harm

Safety of subjects involved in human experimentation is protected by both the participating clinical investigators and independently by the Institutional Protocol Review Committee (IPRC) and/or the Institutional Review Board (IRB). The IRB reviews all proposed studies involving human experimentation and ensures that the subject’s rights and welfare are protected and that the potential benefits and/or the importance of the knowledge to be gained outweigh the risks to the individual. The IRB also reviews the informed consent document associated with each study to ensure that the consent document accurately and clearly communicates the nature of the research to be done and its associated risks and benefits.

20.2 Privacy and Confidentiality

Patients will be informed of the extent to which their confidential health information generated from this study may be used for research purposes. Following this discussion, they will be asked to sign the HIPAA form and informed consent documents. The original signed document will become part of the patient’s medical records, and each patient will receive a copy of the signed document. The use and disclosure of protected health information will be limited to the individuals described in the informed consent document.

To protect patient’s privacy, all study data will be identified only by initials and a specifically assigned study number. However, in compliance with Good Clinical Practices, the PI will allow the independent study monitor(s) to review that portion of the patient’s medical record that is directly related to the study. This shall include all relevant study documentation, including patient medical histories to verify eligibility, laboratory test results, x-ray reports, and admission/discharge summaries while the patient is on-study, and autopsy reports for deaths occurring during the study (if available). 20.3 Informed Consent

In obtaining and documenting informed consent, the investigators will comply with the IRB guidelines and the ethical principles originating in the Declaration of Helsinki.

20.4 IRB Approval

This protocol and the associated informed consent documents must be submitted to the IRB for review and approval.

21. Safety Considerations

21.1 Microparticulate formulation

Because the tumor specific antigens-derived peptides are generated using a cell-free system and administered orally using biodegradable M cell-targeted microparticles composed of generally recognized as safe (GRAS) materials, we do not expect any significant adverse reactions to the therapy.

21.2 Adverse Immune Responses

The available data indicates that these VPAs are selectively expressed by SARS-CoV-2. There is no significant similarity with any known human protein. It is therefore expected that the immune responses will not be directed against non-infected cells.

21.3 Regulatory A spects

This study will be conducted as part of an Investigational New Drug (IND) Application. The study will not commence until full approval is obtained from either the central and/or local IRB.

22. Financial Considerations

The expenses and costs incurred by this study will be provided by financial support entirely or partially from Kiromic, and/or external funding sources. It is expected that the follow-up monitoring (as part of the standard of care) will be paid either by the patient or their insurance companies. This arrangement will be clarified in the Patient Consent Form.

23. Potential Conflict of Interest

It is possible that in the future Kiromic may apply for a patent covering the potential use of some peptide antigens in cancer therapeutics and diagnostics. The investigators will declare this potential conflict of interest in the Patient Consent Form so that all patients recruited to this study will be aware that some or all data obtained may be used to support future application that may result in commercial value of the microparticle immunotherapy. A panel of independent physicians/investigators will serve as an external Independent Clinical and Scientific Review Board monitor team (ICSRB) that will review the clinical and immunologic data generated in the study to guarantee the un-biased evaluation of study results.

24. Quality Control and Quality Assurance The quality of the microparticle product will be determined and guaranteed by strict adherence to the Standard Operating Procedure (SOP) following Good Manufacturing Processing (GMP).

25. Publication Policy

Results of this clinical study may be presented and/or published in National Meetings and/or peer-reviewed medical or scientific journals, partially or completely. Patient’s personal data will remain anonymous during such presentations/publications and integrity of data preserved and guaranteed during the dissemination process.

26. Direct Access to Source Data and Documents. Data Handling, and Record Keeping and Retention of Study Documents Only the Study Chair, PI, and Kiromic’s Clinical Research personnel may have access to the study source data and documents. Data will be handled exclusively by the Study Chair, PI, designated Clinical Research personnel, and/or an officially designated person or persons such as one of the study sub-investigators and members of the ICSRB. Data will be handled under strict confidentiality and kept in a secure place at Kiromic. Only the Study Chair, PI, designated Clinical Research personnel, and members of the ICSRB may have access to the study data, including patient information and study outcomes. Study documents will be retained at Kiromic for at least 5 years following completion and closure of the study.

APPENDIX 1: STUDY CALENDAR APPENDIX 2:

* The laboratory values provided in the tables serve as guidelines and are dependent upon institutional normal parameters APPENDIX 2 (Continued):

* The laboratory values provided in the tables serve as guidelines and are dependent upon institutional normal parameters.

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The entirety of each patent, patent application, publication and document referenced herein is incorporated by reference. Citation of patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. Their citation is not an indication of a search for relevant disclosures. All statements regarding the date(s) or contents of the documents is based on available information and is not an admission as to their accuracy or correctness.

The technology has been described with reference to specific implementations. The terms and expressions that have been utilized herein to describe the technology are descriptive and not necessarily limiting. Certain modifications made to the disclosed implementations can be considered within the scope of the technology. Certain aspects of the disclosed implementations suitably may be practiced in the presence or absence of certain elements not specifically disclosed herein.

Each of the terms “comprising,” “consisting essentially of,” and “consisting of” may be replaced with either of the other two terms. The term “a” or “an” can refer to one of or a plurality of the elements it modifies (e.g., “a reagent” can mean one or more reagents) unless it is contextually clear either one of the elements or more than one of the elements is described. The term “about” as used herein refers to a value within 10% of the underlying parameter (i.e., plus or minus 10%; e.g., a weight of “about 100 grams” can include a weight between 90 grams and 110 grams). Use of the term “about” at the beginning of a listing of values modifies each of the values (e.g., “about 1, 2 and 3” refers to "about 1, about 2 and about 3"). When a listing of values is described the listing includes all intermediate values and all fractional values thereof (e.g., the listing of values "80%, 85% or 90%" includes the intermediate value 86% and the fractional value 86.4%). When a listing of values is followed by the term "or more," the term "or more" applies to each of the values listed (e.g., the listing of "80%, 90%, 95%, or more" or "80%, 90%, 95% or more" or "80%, 90%, or 95% or more" refers to "80% or more, 90% or more, or 95% or more"). When a listing of values is described, the listing includes all ranges between any two of the values listed (e.g., the listing of "80%, 90% or 95%" includes ranges of "80% to 90%, " "80% to 95%" and "90% to 95%").

Certain implementations of the technology are set forth in the claims that follow.