FIELD OF INVENTION

[0001] This invention generally relates to adjuvants for use with vaccine formulations. More particularly, aspect of the invention relates to adjuvants for use with coronavirus vaccine formulations.

BACKGROUND

[0002] New coronaviruses appear from time to time in the last couple of decades, for example, SARS-CoV-1 (SARS), EMC/2012 (MERS) and SARS-CoV-2 (COVID-19). Takes SARS- CoV-2 as an example. COVID-19 has a mean incubation period of 5.2 days and causes fever, cough, and other flu-like symptoms. It can affect multiple tissues and organ systems and many affected patients develop pneumonia and progress into severe acute respiratory syndrome, which has a very poor prognosis and high mortality. Studies have shown that over 60% of patients died once they progressed into an advanced sever e/criti cal illness stage.

[0003] The outbreak of COVID-19 has already taken more than 40,000 lives and still counting. Not only that, SARS-CoV-2 is also extremely contagious and spreads easily from person to person through close contact. Some studies also suggested that asymptomatic transmission of SARS-CoV-2 is likely to occur.

[0004] In a world of denser cities and increased international travel, diseases can spread quickly and cause serious devastation. Vaccines can be an important tool to manage the diseases and save lives. However, effectiveness of vaccines varies.

[0005] Furthermore, the development of vaccines for each new emerging diseases caused by the same virus family is challenging. Vaccines are usually developed after the emergence of novel infectious diseases and the development of vaccines for novel diseases often takes years. Some diseases caused by some species in the same virus family may be deadlier and spread quicker than the others in the same family. By the time the vaccine for the novel disease becomes available, many lives may have been gone.

SUMMARY

[0006] Since new human infectious virus from the same virus family appears from time to time, development of cross-species immunity to species, which is not present in the vaccinating composition or unique protein(s) is not present in the vaccinating composition, meets the requirements of pandemic preparedness and is extremely important. Further, in order to better protect lives, it is also important to improve the effectiveness of vaccines through the enhancement of immune response. [0007] In the light of the foregoing background, an aspect of the present invention provides A method for improving an immune response to vaccination of a subject, comprising: applying a composition comprising a toll-like receptor 7 agonist and/or a toll-like receptor 9 agonist to the subject, and administrating a vaccine at a vaccination site of the subject, whereby the composition improves the immune response, wherein the improvement comprises the increase of antibody titer relative to that produced by administration of the vaccine in the absence of application of the composition.

[0008] Accordingly, in another embodiment of the present invention, a medical kit for improving an immune response to vaccination of a subject comprising: a composition comprising a toll-like receptor 7 agonist and/or a toll-like receptor 9 agonist adapted to apply to the subj ect and improve the subj ecf s immune response, wherein the improvement comprises the increase of antibody titer relative to that produced by administration of the vaccine in the absence of application of the composition; an optional vaccine adapted to be administrated to the subject; and a pre-printed instruction portion disposed on the kit adapted to include instructions for using the kit, wherein the administration is selected from a group consisting of sub-dermal injection, intradermal injection, intramuscular injection, subcutaneous injection, and microneedle vaccination, wherein the composition is adapted to apply to the subject between a predetermined period of time, wherein the instructions comprise the administration and application method.

BRIEF DESCRIPTION OF FIGURES

[0009] Persons of ordinary skill in the art may appreciate that elements in the figure are illustrated for simplicity and clarity so not all connections and options have been shown. For example, common but well-understood elements that are useful or necessary in a commercially feasible embodiment may often not be depicted in order to facilitate a less obstructed view of these various embodiments of the present disclosure. It may be further appreciated that certain actions and/or steps may be described or depicted in a particular order of occurrence while those skilled in the art may understand that such specificity with respect to sequence is not actually required. It may also be understood that the terms and expressions used herein may be defined with respect to their corresponding respective areas of inquiry and study except where specific meanings have otherwise been set forth herein.

[0010] FIG. 1 provides a schematic view of the SARS-CoV-2 spike protein (S-protein) according to one embodiment.

[0011] FIG. 2 provides a representative elution chromatograph of the recombinant RBD protein on a SUPERDEX 200 increase column according to one embodiment. [0012] FIG. 3 provides a chart showing Glycosylated peptides in RBD identified by mass spectrometry according to one embodiment.

[0013] FIG. 4 provides a chart showing the abundance of glycosylation according to one embodiment.

[0014] FIG. 5 is a schematic view of the glycosylation sites illustrated based on the complex structure of SARS-CoV-2 RBD bound to angiotensin-converting enzyme 2 (ACE2) according to one embodiment.

[0015] FIG. 6 provides a graph showing the real-time binding profile between our purified RBD protein and ACE2 characterized by surface plasmon resonance (SPR) BIACORE according to one embodiment.

[0016] FIG. 7 provides graphs showing (a) IgG and (b) IgM responses (against S protein RBD) of the sera of the mice according to one embodiment.

[0017] FIG. 8 provides charts showing the antibody reaction (a) IgG and (b) IgM (against S protein RBD) of the sera of the mice according to one embodiment.

[0018] FIG. 9 provides a graph showing the antibody reaction (against S protein RBD) of the sera of the mice according to one embodiment.

[0019] FIG. 10 provides a graph showing the antibody reaction (against S protein RBD) of the sera of the mice according to one embodiment.

[0020] FIG. 11 provides charts showing the antibody reaction (a) IgG and (b) IgM (against S protein RBD) of the sera samples according to one embodiment.

[0021] FIG. 12 provides charts showing the inhibition of the S protein RBD binding to cell- surface ACE2 according to one embodiment.

[0022] FIG. 13 provides charts showing the neutralization of the infection of SARS-CoV-2 pseudovirus by the sera according to one embodiment.

[0023] FIG. 14 provides a chart showing the virial load in the hACE2 mice which were intraperitoneally injected with 0.8 ml of the pooled sera from the mice immunized with the vaccine 7 days after the first vaccination, or normal mouse sera from the mice treated with PBS as a control according to one embodiment.

[0024] FIG. 15 provides images showing the histopathological changes, under the light microscopy, in the lung tissues from the hACE2 mice which were intraperitoneally injected with 0.8 ml of the pooled sera from the mice immunized with the vaccine 7 days after the first vaccination, or normal mouse sera from the mice treated with PBS as a control according to one embodiment. [0025] FIG. 16 provides a chart showing the body weight change (%) of the hACE2 mice which were intraperitoneally injected with 0.8 ml of the pooled sera from the mice immunized with the vaccine 7 days after the first vaccination, or normal mouse sera from the mice treated with PBS as a control according to one embodiment.

[0026] FIG. 17 provides a graph showing the measurement of the level of the circulating IgG against the recombinant RBD protein in the recipient mice by ELISA 1 day after the transfer of 0.8 ml sera from the donor mice 7 days after the first vaccination according to one embodiment. Similar results were repeated in two independent experiments. Also, the untreated mice were used as additional control.

[0027] FIG. 18 provides a chart showing antibody bindings of wild-type C57BL/6 mice and mice deficient in Cd4 ^/_ , Cd8a ^/_ , T1G2 ^' , T1G4 ^' , Sting l ^/_ , Caspl , Nlrp3 ^/_ , and I1-1b , which were immunized with the recombinant RBD protein (5pg per mice) and sera were collected on day 7 after the first vaccination and were tested for the antibody against RBD at the dilution of 1:25, according to one embodiment.

[0028] FIG. 19 provides charts showing the cytokines (a) IL-4 and (b) IFN-g produced by the spleen lymphocytes according to one embodiment.

[0029] FIG. 20 provides charts showing gating (a) CD4+ CD44high + B220- MHCII- PTM-g+, (b) CD4+ CD44high + IL-4+, (c) CD8+ CD44high + B220- MHCII- IFN-y+ and (d) CD8+ CD44high + IL-4+ of the lymphocytes in the spleen, which were collected from the mice with the recombinant RBD protein (5pg per mice) 7 days after the first vaccination and were incubated with RBD for three days according to one embodiment.

[0030] FIG. 21 provides charts showing cytokines in the sera collected from the mice 7 days after the first vaccination: (a) TNF-a, (b) IFN-g, (c) IFN-a, (d) IFN-b, (e) IL-6, (f) IL-4 were measured by ELISA. AL means AL(OH)3, RBD means recombinant RBD protein and RBD+AL means the addition of the recombinant RBD protein to AL(OH)3 according to one embodiment.

[0031] FIG. 22 provides charts showing IgG response against S protein RBD at different dilution of the sera of the rabbits, which were immunized with (a) AL, (b) 20pg RBD, (c) 1 pg RBD + AL, (d) 5pg RBD,+ AL, (e) 10pg RBD,+ AL, (f) 20pg RBD,+ AL and (g) 40pg RBD,+ AL. AL: AL(0H)3, RBD: recombinant RBD protein, RBD+AL: the addition of the recombinant RBD protein to AL(OH)3 according to one embodiment.

[0032] FIG. 23 provides a graph showing potent neutralization of SARS-CoV-2 pseudovirus infection by the sera from the rabbit immunized with recombinant RBD vaccine according to one embodiment. [0033] FIG. 24 schematically depicts a front view of an immunogenicity enhancement device according to one embodiment.

[0034] FIG. 25 schematically depicts a cross section of the device shown in FIG. 24, demonstrating laminar construction according to one embodiment.

[0035] FIG. 26 schematically depicts an exploded view of the device of FIG. 24. All the four layers are aligned to the center of the device according to one embodiment.

[0036] FIG. 27 depicts a procedure for using a device of the inventive concept in an immunization process according to one embodiment.

[0037] FIGS. 28A and FIG. 28B schematically depict an alternative embodiment of the device having an attached "wing" portion that can act as a dressing or bandage. FIG. 28A schematically depicts a front view of the device. FIG. 28B schematically depicts an exploded view of the device.

[0038] FIG. 29 depicts positioning of adhesive layers in a device of the inventive concept as shown in FIGS. 28 A and 28B according to one embodiment.

[0039] FIG. 30 depicts a procedure for using a device of the inventive concept with a dressing according to one embodiment.

[0040] FIGS. 31A to 3 ID: FIG. 31A shows an alternative embodiment of a device of the inventive concept that is configured for use with external bandage. FIG. 3 IB shows an exploded view of such a device. FIGS. 31C and 3 ID schematically depict steps of a method for using such a device with an external bandage.

[0041] FIG. 32 depicts an embodiment of a measuring and application tool for use with other devices of the inventive concept.

[0042] FIGS. 33A and 33B: FIG. 33A schematically depicts a method for using an applicator as shown in FIG. 32 according to one embodiment. FIG. 33B provides a stepwise series of photographs (I to VI) showing use of an applicator of the inventive concept with an occlusive film.

[0043] FIGS. 34A and 34B depict examples of kits of the inventive concept according to one embodiment. FIG. 34A depicts a kit that includes instructions for use of a simplified version of a device of the inventive concept. FIG. 34B depicts a kit that includes instructions for use of a topical medication measuring device and applicator and for use of a vaccine-enhancing device of the inventive concept.

DETAILED DESCRIPTION

[0044] Embodiments may now be described more fully with reference to the accompanying drawings, which form a part hereof, and which show, by way of illustration, specific exemplary embodiments which may be practiced. These illustrations and exemplary embodiments may be presented with the understanding that the present disclosure is an exemplification of the principles of one or more embodiments and may not be intended to limit any one of the embodiments illustrated. Embodiments may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure may be thorough and complete, and may fully convey the scope of embodiments to those skilled in the art. The following detailed description may, therefore, not to be taken in a limiting sense.

Immunogenicity Enhancement

[0045] The present invention provides compositions and methods that enhance an immune response of a treated subject to a vaccinating compositions. The composition of the present invention includes a toll-like receptor 7 agonist and/or toll-like receptor 9 agonist. Enhanced immune responses include: (1) improved antibody titer relative to that produced by administration of the vaccine in the absence of application of the composition, and (2) generation of an effective immune response to species, which is not present in the vaccinating composition or its unique proteins is not present in the vaccinating composition. The composition of the present invention may increase antibody titer due to the improved activation of B-cells and T-cells.

[0046] When the composition of the present invention is applied within a predetermined period of time before or after the administration of a vaccine, the immune responses of the subject are improved. In some examples, the predetermined period of time is four hours before to four hours after the administration of the vaccine. In yet other examples, the predetermined period of time is 30 minutes before to 15 minutes after the administration of the vaccine. In further examples, the predetermined period of time is 0-10 minutes before to 0-10 minutes after the administration of the vaccine. In some embodiments, the composition of the present invention is applied simultaneously during the administration of the vaccine.

[0047] The vaccine may be (i) recombinant vaccine; (ii) live-attenuated vaccine; (iii) inactivated vaccine; (iv) toxoid vaccine; (v) dendrite cell vaccine; (vi) DNA vaccine; (vii) recombinant vector vaccine; (viii) RNA vaccine; or (ix) peptide-based vaccine.

[0048] In some embodiments, the vaccine is recombinant vaccine comprising receptor binding domain (RBD) of SARS-CoV-2 generated by insect cells using baculovirus expression vector (which will be explained in greater detail below). In some embodiments, the vaccine is a recombinant vaccine comprising receptor binding domain (RBD) of a pathogen generated by Chinese hamster overy cells using a predetermined expression vector. [0049] The vaccines and compositions may also be administered to the subject intranasally, intratracheally, orally, sub-dermally, intradermally, intramuscularly, intraperitoneally, or subcutaneously. In one particular embodiment, the vaccine is administrated by intradermal injection. In yet another embodiment, the vaccine is administrated by microneedle vaccination. [0050] The following discussion provides example embodiments of the inventive subject matter. One should appreciate that the disclosed techniques provide many advantageous technical effects including improving the effectiveness and breadth of protection afforded by viral vaccination preparations without the need for reformulation of the vaccine.

[0051] In one embodiment, the present invention provides compositions and methods that enhance an immune response of a treated subject to a vaccinating compositions. This is accomplished by applying a topical preparation that includes a toll-like receptor 7 agonist to the skin surrounding an area where a transdermal vaccination is applied. Enhanced immune responses include: (1) improved antibody titer relative to that produced by administration of the vaccine in the absence of application of the topical preparation, and (2) generation of an effective immune response to species, which is not present in the vaccinating composition or its unique proteins is not present in the vaccinating composition.

[0052] In one embodiment, an immune response to vaccination of the present invention may be enhanced by applying a topical formulation to a vaccination site at or immediately prior to vaccination, where the topical formulation comprising a toll-like receptor 7 agonist and/or a toll-like receptor 9 agonist. A vaccine of the present invention is also applied at the vaccination site. A barrier may be applied to the treated area following application of the topical preparation and/or administration of the vaccine. In some embodiments the topical preparation is applied within 5 minutes of the administration of the vaccine. After a period of time (for example, 1 to 6 hours) following application of the vaccine the topical formulation is removed. The topical formulation is selected to provide a toll-like receptor 7 agonist and/or a toll-like receptor 9 agonist in a quantity sufficient (i) to provide effective protection against a non-vaccinating species that is not present in the vaccine or its unique protein is not present in the vaccine, and/or (ii) to an improved antibody titer to the vaccinating species, relative to an antibody titer generated on vaccination without the topical formulation. In some embodiments the non vaccinating species includes species in coronavirus family that are not represented in the vaccinating species. In a preferred embodiment the toll-like receptor 7 agonist comprises imiquimod. In some embodiments the topical formulation is formulated to provide between about 2 mg and about 20 mg of the toll-like receptor agonist to the skin. In some embodiments the topical formulation is provided as a nano-emulsion of the toll-like receptor 7 agonist and/or a toll-like receptor 9 agonist. In still other embodiments the topical formulation is supplied as part of an application device.

[0053] In another embodiment, an immune response to vaccination of the present invention may be enhanced by using a toll-like receptor 7 agonist and/or a toll-like receptor 9 agonist in the preparation of a topical formulation that provides an enhanced response to a vaccine. The enhanced response includes development of a protective immune response to a species that is not present in a vaccine or its unique protein is not present in the vaccine, when such vaccine is used to immunize a vaccinated subject treated with the topical formulation. Such a vaccine can include a coronavirus, in which can protective immune response is elicited to a different coronavirus (e.g. other than SARS-CoV-2, which will be explained in greater detail below). In some embodiments such an enhanced immune response includes development of an improved antibody titer to a species present in the vaccine, relative to vaccination response achieved in a vaccinated subject that has not been treated with the topical formulation. In a preferred embodiment the toll-like receptor 7 agonist is imiquimod. In some embodiments the topical formulation is formulated to provide between 2 mg and 20 mg of the toll-like receptor 7 agonist and/or a toll-like receptor 9 agonist on application. In still other embodiments the topical formulation is provided as part of an appliance.

[0054] Another embodiment, an immune response to vaccination of the present invention may be enhanced by the use of a toll-like receptor 7 agonist and/or a toll-like receptor 9 agonist in the preparation of a device that provides an enhanced response to a vaccine. The enhanced response includes development of a protective immune response to a species that is not present in a vaccine or its unique protein is not present in the vaccine, when such vaccine is used to immunize a vaccinated subject treated with the topical formulation. Such a vaccine can include a coronavirus, in which can protective immune response is elicited to a different coronavirus (e.g. other than SARS-CoV-2, which will be explained in greater detail below). In some embodiments such an enhanced immune response includes development of an improved antibody titer to a species present in the vaccine, relative to vaccination response achieved in a vaccinated subject that has not been treated with the topical formulation. In a preferred embodiment the toll-like receptor 7 agonist is imiquimod. In some embodiments the topical formulation is formulated to provide between 2 mg and 20 mg of the toll-like receptor 7 agonist and/or a toll-like receptor 9 agonist on application. In still other embodiments the topical formulation is provided as part of an appliance, for example an appliance that adheres to the skin. In some embodiment the kit may include a barrier, film and/or a microneedle device. [0055] The topical composition containing a toll-like receptor 7 agonist and/or a toll-like receptor 9 agonist is applied topically at or near an injection/application site of the vaccine of the present invention, either at or immediately prior to the time of vaccination. The sites may be any skin surface including but not limited to the arms, legs, face, nose or interior of the nasal channel. The resulting immune response to the vaccination is enhanced in providing both a higher titer immune response to vaccinating viral species (relative to a response observed in the absence of the topically applied composition) and in providing an effective immune response to viral strains not found in the immunizing composition.

[0056] In some embodiments, the composition containing a toll-like receptor 7 agonist and/or a toll-like receptor 9 agonist is applied topically at or near a virus vaccine injection site, either at or immediately prior to the time of vaccination. The resulting immune response to the vaccination is enhanced in providing both a higher titer immune response to vaccinating viral species (relative to a response observed in the absence of the topically applied composition) and in providing an effective immune response to viral strains not found in the immunizing composition. Suitable viral species include influenza and/or coronavirus species.

[0057] The inventors have found that toll-like receptor 7 agonists (such as imiquimod) and/or a toll-like receptor 9 agonist can be applied topically (for example, as an ointment) to provide enhanced immune response to viral vaccine preparations. In a preferred embodiment, the vaccine preparation includes viral antigens corresponding to coronaviruses (which will explain in greater detail below). For the topical formulation, the enhanced immune response can be observed on application of the toll-like receptor 7 agonist and/or a toll-like receptor 9 agonist at the time of vaccination, immediately prior to or following vaccination. For example, a toll-like receptor 7 agonist and/or a toll-like receptor 9 agonist composition of the inventive concept may be applied to the surface of the skin at and/or around a vaccination injection site less than or equal to 1 hour, less than or equal to 30 minutes, less than or equal to 20 minutes, less than or equal to 10 minutes, less than or equal to 5 minutes, less than or equal to 2 minutes, or less than or equal to 1 minute prior to vaccination. Similarly, in other embodiments the toll-like receptor agonist composition may be applied 1 hour, less than or equal to 30 minutes, less than or equal to 20 minutes, less than or equal to 10 minutes, less than or equal to 5 minutes, less than or equal to 2 minutes, or less than or equal to 1 minute following vaccination. In yet another embodiment, in other embodiments the toll-like receptor agonist composition may be applied up to 4 hours prior to or following vaccination. [0058] For the topical formulation, following application and/or vaccination, the toll-like receptor 7 agonist and/or a toll-like receptor 9 agonist composition may be left in place on the skin surface for a period of time. For example, a toll-like receptor 7 agonist and/or a toll-like receptor 9 agonist composition may be left in place for at least 15 minutes, at least 30 minutes, at least one hour, at least 2 hours, at least 3 hours, at least 6 hours, at least 12 hours, at least 16 hours, or 24 hours or more following vaccination. In some embodiments the area of skin treated with the toll-like receptor 7 agonist and/or a toll-like receptor 9 agonist containing composition may be protected by a barrier, such as a barrier film or bandage, during this post-vaccination period.

[0059] Vaccination can be performed by any suitable method. Suitable methods include sub- or intra-dermal injection, intramuscular injection, and microneedle vaccination. Such microneedle vaccination can be carried out using a microneedle device or through the use of a microneedle patch. Similarly, immunization can be carried out transcutaneously using methods that disrupt the stratum corneum layer of the skin, including tape stripping and disruption using laser and/or ultrasound energy.

[0060] A topical preparation that includes a toll-like receptor 7 agonist and/or a toll-like receptor 9 agonist may be applied to an area at or near that of the site of vaccination. In some embodiments of the inventive concept, such a topical preparation may be applied to an area of about 1 cm ² that surrounds the site of vaccination. In other embodiments the area treated using the topical preparation may be about 4 cm ² - 16 cm ² , or more surrounding the site of vaccination. In some embodiments, the area treated with the topical preparation at least partially overlaps the site of vaccination. In a preferred embodiment the site of vaccination may be approximately centered in the topically treated area.

[0061] Some embodiments of the inventive concept include an applicator or application device that can assist a healthcare provider with proper utilization of such a topical preparation. Such a device can be utilized following application of the topical preparation to the skin surface. In other embodiments such a device can be applied simultaneously with application of the topical preparation to the skin surface. In such embodiments the topical preparation can be included with and/or form part of the application device. Suitable application devices can include a barrier (such as a barrier film), which can prevent transfer of an applied topical preparation from the skin surface. Such a barrier film can be secured to a skin surface by any suitable means, for example an adhesive, elastic bandage, or pressure from a garment. Alternatively, in some embodiments the topical preparation can be formulated to provide adhesion of such a barrier film. For example, a component of the vehicle of the topical preparation can be selected to provide sufficient traction and/or adhesion to at least transiently fix a barrier film to a treated skin surface (for example, by providing a moist, tacky, and/or gelatinous surface texture). In some embodiments of the inventive concept such a kit can include a template or similar representation of an area over which the topical preparation is to be applied. Such a template can include an indication of the desired vaccination site, and in some embodiments can at least transiently adhere to the skin surface.

[0062] Another embodiment of the inventive concept is a kit for utilization of a topical preparation that includes a toll-like receptor 7 agonist and/or a toll-like receptor 9 agonist. Such a kit can be used to enhance a patient' s response to vaccination. Such a kit can include a topical preparation that includes a toll-like receptor 7 agonist and/or a toll-like receptor 9 agonist in a pharmaceutically acceptable medium and an application device as described above. In some embodiments such a kit can include instructions for use. Such instructions can include directions for timing of the application of the topical preparation relative to the delivery of the vaccine to the patient, time that the topical preparation is to be kept in place on the treated skin surface, instructions for removal of the topical preparation from the skin surface, and/or instructions for aftercare of the vaccination and/or treated site. In yet some embodiments, such kit may include pre-printed instruction portion disposed on the kit. Instructions on the pre-printed instruction portion can include the vaccine administration method and application method of the composition of the present invention discussed herein. [0063] Suitable toll-like receptor 7 agonists include imiquimod, CL075, CL097, CL264, CL307, gardiquimod™, loxoribine, resimiquimod, R848 and combination thereof. Suitable toll-like receptor 9 agonists include paromycin, miltefosine, paromoycin, CpG, agatolimod, lefitolimod, MGN1703, CPG 7909, PF-3512676, ISS 1018, IMO-2055, CpG-28 and combination thereof. A topically applicable composition containing such a toll-like receptor 7 agonist and/or a tolllike receptor 9 agonist may include a pharmaceutically acceptable vehicle, and may be formulated as a spray, lotion, ointment, gel, emulsion, micro-emulsion, nano emulsion, or other suitable solution and/or suspension. Such topical formulations may be formulated to provide from about 0.1 mg - 20 mg or more of the toll-like receptor 7 agonist and/or a toll-like receptor 9 agonist to an individual on application of the formulation. In some embodiment, the imiquimod (IMQ) may be carried by ultradeformable archaeosomes. Optionally, such a topical formulation may include an indicator, for example a dye, to provide a visible indication that an area of skin has been treated. In some embodiments, the toll-like receptor 7 and/or a toll-like receptor 9 agonist composition is provided as part of a patch that adheres to the skin surface, and which applies the toll-like receptor 7 agonist and/or a toll-like receptor 9 agonist to the skin while additionally providing a barrier film. Such a patch may be formulated to permit vaccination through the material of the patch following application. In some embodiments, the topical formulation includes a nano-emulsion of the toll-like receptor 7 agonist and/or a toll-like receptor 9 agonist, which may speed absorption of the toll-like receptor 7 agonist and/or a toll like receptor 9 agonist relative to conventional suspensions, solutions, and/or emulsions. In other embodiments, the topical formulation is applied with microneedles (for example, a microneedle array or patch).

[0064] It should be appreciated that two distinct and different forms of enhanced vaccination response may be generated through the use of a toll-like receptor 7 agonist and/or toll-like receptor 9 agonist. In one form of enhanced vaccination response, a quantifiable immune response (for example, antibody titer) to an immunizing species or viral strain is enhanced (i.e. improved) in an individual or population receiving treatment at the vaccination site with a toll like receptor 7 agonist and/or a toll-like receptor 9 agonist relative to an equivalently vaccinated control (e.g. receiving the same vaccination) that does not receive such treatment. For example, an individual or population receiving treatment with a toll-like receptor 7 agonist and/or a toll like receptor 9 agonist at or near a vaccination site may have a 1.5 fold - 10 fold or higher GMT (geometric mean titer) than that observed from a control individual or population receiving the same vaccine by the same route of administration, but lacking the treatment.

[0065] Another form of enhanced vaccination response may be the induction of a functional immunity to viral species or strains, that are not present in the vaccine formulation or unique protein is not present in the vaccine formulation, when such vaccine is used to vaccinate an individual that receives treatment with a toll-like receptor 7 agonist and/or a toll-like receptor 9 agonist at or near the site of vaccination (i.e. cross protection). For example, an individual or population receiving treatment with a toll-like receptor 7 agonist and/or a toll-like receptor 9 agonist at or near a vaccination site may develop an effective immune response (for example, antibody titer) to an coronavirus species or strain that does not form part of the vaccinating mixture, whereas such a protective response to the non-vaccinating species or strain is not observed from a control individual or population receiving the same vaccine by the same route of administration, but lacking the treatment. In some embodiments both types of enhanced vaccination response are produced.

[0066] Method

[0067] In a double-blind, randomized controlled trial, healthy volunteers aged 18-30 years in early 2014 received the 2013-14 northern hemisphere winter trivalent influenza vaccine (TIV). Eligible subjects were randomly allocated (1 : 1: 1: 1) into 4 groups: topical imiquimod ointment followed by intradermal TIV (IQ), topical aqueous-cream followed by intradermal TIV (ID), topical aqueous-cream followed by intramuscular TIV (IM), and topical imiquimod ointment followed by intradermal normal saline injection (NS). Volunteers and investigators were blinded to the type of topical treatment applied. Hemagglutination inhibition (HI) and neutralization antibody titers (NT) were measured. Primary outcome was day 7 seroconversion rate. Other outcomes included seroprotection rate and GMT-fold increase against the vaccine and non-vaccine strains (including the A/Switzerland/9715293/2013 -like virus which emerged in late 2014) at day 7 and 21.

[0068] Study Design and Interventions

[0069] At the time of inclusion, demographic data of the participants were obtained. Simple randomization with no stratification was performed. Participants were randomly assigned into 4 groups, the experimental group (IQ) and three control groups (ID, IM and NS respectively). A square of 16 cm ² was marked on the deltoid region of the right arm of all participants by the study nurse. In the IQ and NS groups, the study nurse would apply the content of one sachet of Aldara™ (5% , 12.5 mg of imiquimod in 250 mg cream) to the marked surface on the skin 5 minutes before vaccination. In the ID and IM groups, aqueous cream BP™ (AFT pharmaceuticals, New Zealand) with no effect was applied instead of the Aldara™ by the study nurse. Participants in the IQ and ID groups received a single dose of 0.1 mL intradermal influenza trivalent vaccine (15 pg of hemagglutinin [HA] per strain). In the IM group, participants received a single dose of 0.5 mL intramuscular influenza vaccine (15 pg of hemagglutinin per strain). In the NS group, participants received a single dose of 0.1 mL of normal saline as sham vaccine. The vaccine was injected in the center of the marked area after the cream was absorbed and skin disinfection by 70% alcoholic swab. The ointment (Aldara™ or aqueous cream BP) was removed by the participant by washing with tap water 6 hours after vaccination.

[0070] To maintain blinding, each participant was assigned to a serial number, and the randomization list linked each serial number with the 4 study groups, differed in the route of delivery and the type of pre-treatment ointment applied. Only the study nurse had knowledge of the type of topical treatment applied. Both participants and investigators remained blinded to the type of topical treatment applied until the completion of the study. The route of delivery was unblinded to the participants during vaccination.

[0071] Influenza Vaccine

[0072] Both the intradermal Intanza™15 (Sanofi-Pasteur, Lyon, France) and the intramuscular Vaxigrip™ (Sanofi-Pasteur, Lyon, France) influenza vaccines used were manufactured by Sanofi-Pasteur MSD™. Both vaccines were inactivated, non-adjuvanted vaccines formulated to contain 15pg of HA of influenza A/Califomia/07/2009 (HlNl)-like virus, influenza A/Victoria/361/2011 (H3N2)-like virus and influenza B/Massachusetts/2/2012-like virus (B/Yamagata lineage). The intradermal injection device, the BD Soluvia™ microinjection system consists of a prefilled trivalent influenza vaccine, with a single 1.5 mm needle penetrating perpendicularly to the skin. The BD Soluvia™ is currently the only prefilled intradermal device licensed for influenza vaccine.

[0073] Safety was evaluated by first asking the subjects to remain in the clinic premise for 30 minutes for post-immunization observation. An immediate adverse event checklist was filled before discharge, covering the period for severe anaphylactic reaction. In addition, a diary was given to the subjects to document symptoms of local and systemic adverse events presented within the first 7 days post-vaccination. Systemic symptoms included fever (body temperature >37.5°C), headache, malaise, myalgia, arthralgia and severe adverse events, and local symptoms included redness, swelling, induration, ecchymosis and pain were documented as solicited events. Redness, swelling, induration, and ecchymosis were graded based on size: grade 1 <20mm and grade 2 >20mm. Pain was graded accordingly: grade 1 was pain on touch and grade 2 was pain when arm was moved. The diaries were collected upon follow-up on day 21 -post vaccination.

[0074] Immunogenicity Measurements

[0075] Blood was taken from participants at baseline, 7 and 21 days after vaccination for antibody assay. Serum antibody titer was measured using a hemagglutination-inhibition (HI) assay for the vaccine strains, and by both HI and neutralization antibody (NT) assays for the non- vaccine strains, according to standard methods. The Committee for Proprietary Medicinal Products (CPMP) guidelines of the European Medicines Evaluation Agency was adopted for immunogenicity measurements of the HI assay. A satisfactory (i.e. effective) antibody response in adult subjects, aged between 18 and 60 is based on at least one of the following indicated requirements: 1) >70% achieving a HI titer of >40 (seroprotection rate) or 2) a geometric mean titer (GMT) fold increase >2.5-fold or 3) >40% achieving a 4-fold rise in antibody titer (seroconversion rate). For the NT assay, the GMT of the four non vaccine strains was compared among the 4 groups.

[0076] A primary outcome measure is the seroconversion rate by HI assay on day 7. Secondary outcome measures included GMT, GMT fold increases and the seroprotection rate by HI assay and the GMT by NT assay from day 7 and 21 post vaccination. The seroconversion rate by HI assay from day 7 and 21 and adverse events post-vaccination were also compared among the 4 groups. [0077] In addition, in order to assess the cross-protection effect to the four non-vaccine influenza strains: A/HK/485197/14 (H3N2 Switzerland lineage), A/HK/408027/09 (prepandemic seasonal H1N1), A/WSN/33 (H1N1), B/HK 418078/11 (Victoria lineage) by imiquimod pretreatment before TIV vaccination, the seroprotection, seroconversion and GMT fold increase by HI and NT assay against these virus strains were measured on day 7 and 21 after vaccination.

[0078] Hemagglutination-inhibition Assay

[0079] Paired serum samples (pre- and post- vaccination) were tested for hemagglutination- inhibiting (HI) antibody using reference antigens including the three vaccine strains: A/California/07/2009 (HlNl)-like virus, influenza A/Victoria/361/2011 (H3N2)-like virus and influenza B/Massachusetts/2/2012-like virus (B/ Yamagata lineage), and the four non vaccine strains as stated above. HI antibody assays were performed by standard microliter techniques after removal of non-specific inhibitors in serum by pre-absorption with turkey erythrocytes for A(H1N1) antibody testing or guinea pig erythrocytes for A(H3N2) & B antibody testing, and followed by receptor destroying enzyme (RDE) (1 :3) after incubation overnight at 37 °C before heat-inactivation at 56 °C for 30 minutes. All serum samples from each subject were tested in parallel for each of the test antigens. Serial two-fold dilutions of RDE-treated serum from 1: 10 were titrated against 4 hemagglutinin units of reference antigens using 0.5% turkey or 0.75% guinea pig erythrocytes.

[0080] Neutralization Antibody Assay

[0081] The Neutralizing Antibody assay (NT) was performed in 96-well microwell plates seeded with Madin Darby canine kidney cells. Two fold serial dilutions of paired serum (pre- and post- vaccination) were tested in duplicate by inoculation with 100 TCID50 of A HK/485197/14 (H3N2 Switzerland lineage), A HK 408027/09 (pre-pandemic seasonal H1N1), A WSN/33 (H1N1), B/HK 418078/11 (Victoria lineage) viruses. A corresponding set of cell controls with sera but without virus inoculation was used as controls. The cells were scored for inhibition of the cytopathic effect (CPE) at 72 hours after inoculation. The titer of a neutralization antibody is defined as the maximum dilution of serum at which the percentage of CPE is less than or equal to 50%.

[0082] Statistical Analysis

[0083] The sample size of this study was determined based on a previous intradermal influenza vaccination studies on elderly patients with chronic illness (12). The seroconversion rate of the IQ group was assumed to be superior to the control IM group, and the seroconversion rate for the A(H1N1) strain by the intradermal and intramuscular seasonal influenza vaccination to be 35% and 20% respectively. With a power of 80% and a two-sided type 1 error of 5%, 40 participants would be needed for each treatment arm that would also allow for a 5% loss to follow-up rate. ANOVA was used to compare the demographic parameters and the immunogenicity among the four different groups. IBM SPSS Statistics 20.0™ was used for statistical computation. A P value <0.05 was considered to represent significant difference.

[0084] Results

[0085] A total of 160 subjects were enrolled in and completed the study. Forty subjects were randomized equally among the 4 groups. All recruited subjects were healthy volunteers without any past medical history and were not on any regular medications. None of the recruited subjects received influenza vaccination in the previous 5 years. The median age was 20 years (interquartile range 19-21 years) and 50% of the recruited subjects were male. There were no difference in age (p=0.875) and sex (p=0.5) among the four groups.

[0086] Safety

[0087] No serious adverse events related to vaccination were reported (see Table 1). Incidence of local or systemic adverse events was infrequent and self-limiting. Although grade 1 redness or swelling was more commonly found in IQ and ID groups, there were no differences amonj the four groups. None of the subjects had visible vaccine leakage from the injection site.

Table 1 IQ: imiquimod ointment + intradermal vaccine; ID: aqueous cream + intradermal vaccine;

IM: aqueous cream + intramuscular vaccine;

NS: imiquimod ointment + intradermal normal saline vaccine

Fever: body temperature >37.5°C Redness, swelling, induration and ecchymosis were graded based on size: grade 1, <20mm; grade 2, 20-50mm

Pain was graded as follows: grade 1, pain on touch; grade 2, pain when arm is moved. NA: not applicable

[0088] Immunogenicity by HI and NT Assays for the Vaccine Strains [0089] The day 7 and 21 immunogenicity measurement in all 3 parameters (seroprotection, seroconversion and GMT fold increase) for the A/California/HINI strain was determined to be significantly higher in the IQ group than for the three control groups (p<0.0001). Surprisingly, 97.5% and 100% achieved seroconversion and seroprotection respectively against the A/California/HINI strain on day 7 and 21 in the IQ group; with a GMT 631 [95% confidence interval (C.I.): 441.4-902] and GMT fold increase of 18 [95% C.I.: 9.9-26.2] on day 7 and a GMT 687.9 [95% C.I.: 476-994] and GMT fold increase of 19.8 [95% C.I.: 11.4- 28.3] on day 14 (see Table 2). The day 7 and 21 seroconversion rate and GMT fold increase for both the A/Victoria/H3N2 (which has relatively low immunogenicity) and B/Massachusetts strains were also significantly higher in the IQ group than the three controls (p<0.0001). Similar results were found for the NT assay (see Table 4) achieving a significantly higher GMT (p<0.0001) on day 7 and 21 for all 3 vaccines' strains when compared to the 3 controls: A/Califomia/HINI strain [248.3 (95% Cl: 132-465.6); 322.1 (95% Cl: 176.6-568.1)], A/Victoria/H3N2 strain [140.6 (95% Cl: 81.8-241); 201.8 (95% Cl: 119.7-340.4)] and B/ Massachusetts strain [198.6 (95% Cl: 133.7-294.4); 285.1 (95% Cl: 193.2-420.7)].

Table 2

IQ: imiquimod ointment + intradermal vaccine; ID: aqueous cream + intradermal vaccine;

IM: aqueous cream + intramuscular vaccine; NS: imiquimod ointment + intradermal normal saline vaccine; GMT: geometric mean titer; CPMP: Committee for Proprietary Medicinal Products; CPMP guideline: at least one of the following criteria must be met for the viral strain in the vaccine: GMT fold increase >2.5, seroconversion rate >40% and seroprotection rate >70%. [Significant P -values in bold]

[0090] Cross-protection

[0091] Surprisingly, effective cross-protection was demonstrated for all four non- vaccine strains by HI (see Table 3) and NT (see Table 5) assays in the IQ group for A/HK 4851970/14 (H3N2 Switzerland lineage), A/HK/408027/09 (seasonal H1N1), A/WSN/33 (H1N1), and B/HK/418078/11 (Victoria lineage). By HI assay, 70% and 97.5% achieved seroconversion and seroprotection respectively against the A/HK/485197/14 (H3N2 Switzerland lineage) on day 7 in the IQ group, with a GMT 86.7 (95% C.I. 70.8-105.9) and a GMT fold increase of 4.8 [95% C.I.: 3.7-5.9] on day 7. Similar results were demonstrated by the NT assay with GMT 40 (95% C.I. 28.6-55.5) and GMT 42 (95% C.I. 30.1-58.3) on day 7 and 21 respectively, with the IQ group as the only group achieving the satisfactory antibody response according to the CPMP guideline. Immunogenicity measurement in all 3 parameters (seroconversion, seroprotection and GMT fold increase) for all 4 non- vaccine strains was significantly higher in the IQ group than the three controls (p<0.0001).

Table 3

IQ: imiquimod ointment + intradermal vaccine; ID: aqueous cream + intradermal vaccine;

IM: aqueous cream + intramuscular vaccine; NS: imiquimod ointment + intradermal normal saline vaccine GMT: geometric mean titer; CPMP: Committee for Proprietary Medicinal Products; CPMP guideline: at least one of the following criteria must be met for the viral strain in the vaccine: GMT fold increase >2.5, seroconversion rate >40% and seroprotection rate >70%. [Significant P-values in bold]

Table 4

IQ: imiquimod ointment + intradermal vaccine; ID: aqueous cream + intradermal vaccine;

IM: aqueous cream + intramuscular vaccine; NS: imiquimod ointment + intradermal normal saline vaccine.

Table 5

IM: aqueous cream + intramuscular vaccine; NS: imiquimod ointment + intradermal normal saline vaccine [0092] Overall, topical imiquimod pretreatment before intradermal influenza vaccination significantly expedited and augmented the immunogenicity of the vaccine strains with at least 10 fold increase in antibody against vaccine strains on day 7. The Inventors found that such treatment can augment the effective breadth of otherwise conventionally formulated seasonal influenza vaccination by providing important and significant cross-protection against non vaccine influenza strains, with at least about a 4-fold increase in antibody titer. This is especially notable for the hitherto non-included antigenically drifted influenza strain that only emerged after the WHO recommendation on the components of the seasonal influenza vaccine for the forthcoming season. Surprisingly, this novel approach can also induced good immunity against an archived prototype A(H1N1) virus isolated 83 years ago, for which considerable genetic drift would be expected. Compositions and methods of the inventive concept can form part of a vaccination strategy that can have important global healthcare implications and should translate into better clinical protection by reducing the seasonal influenza burden of sick leave, outpatient visits, hospitalization and mortality in both young and elderly subjects. The ability of this approach to generate sufficiently high GMT and seroconversion rate within 7 days against a hitherto non-included influenza virus due to antigenic drift, can provide a highly flexible, safe and inexpensive way to induce rapid and sustained immunity against such newly emerged virus in serologically naive individuals. [0093] HI antibody assay results also demonstrate that the vaccine strain A/Victoria/H3N2 virus appeared less immunogenic than the A(H1N1) and the influenza B vaccine virus. Nevertheless, the GMT by HI assay for the four non-vaccine strains were similar. NT assay was also performed to confirm the finding of cross-protection in the study group. According to the CPMP guideline, the intradermal vaccination with topical imiquimod pretreatment was the only group achieving the satisfactory antibody response (>40) with the NT assay. None of the control groups were able to achieve the sufficiently high seroprotection GMT. A third control group with topical imiquimod ointment followed by sham intradermal normal saline injection allowed us to exclude the possible immunogenic effect of the imiquimod pretreatment.

[0094] Conventional seasonal influenza vaccination relies on the induction of neutralizing antibodies against the globular head of the viral hemagglutinin. The antibody is short-lived and annual revaccination is necessary to maintain the neutralizing antibody level. The inventors speculate, without wishing to be bound by theory, that the breadth of the influenza vaccine against seasonal antigenic drift or pandemic antigenic shift could be achieved by novel vaccine development targeting the highly conserved protein epitopes or by the addition of an adjuvant. Both approaches would induce cross-protective humoral responses.

[0095] Baculovector generated vaccine including the receptor-binding domain (RBD) of SARS-CoV-2

[0096] In some embodiments, the immunogenicity enhancement compositions of the present invention may be used in conjunction with recombinant vaccine comprising receptor binding domain (RBD) of SARS-CoV-2 generated by insect cells using baculovirus expression vector to enhance its immunogenicity and provide cross-protection.

[0097] SARS-CoV-2 uses its spike protein receptor-binding domain (RBD) to engage with the host cell receptor angiotensin-converting enzyme 2 (ACE2).

[0098] The following data shows a recombinant protein comprising residues 319-545 of the SARS-CoV-2 spike protein (S-protein) receptor-binding domain (RBD) may a potent functional antibody response after single dose injection. The recombinant protein may be recombinant SARS-CoV-2 Spike RBD protein (SARS-CoV-2 S RBD protein) it is found that the sera from the immunized subject contained the elevated level of antibodies against the recombinant RBD protein, blocked RBD binding to ACE2 expressed on the cell surface, neutralized SARS-CoV-2 pseudovirus in vitro and prevented the subject from infection of live SARS-CoV-2 in vivo. Moreover, the elevated RBD-specific antibodies were found in the sera from immunized subject with COVID-19. Therefore, a protective vaccine through the induction of antibody against RBD binding of S-protein of SARS-CoV-2 may be developed. [0099] The SARS-CoV-2 S RBD protein may be generated by insect cells using baculovirus expression vector. The details of which are discussed below.

[0100] Characterization of the SARS-CoV-2 S RBD protein prepared from insect cells [0101] The recombinant receptor-binding domain (RBD) protein of SARS-CoV-2 spike protein from insect cells was prepared using the Bac-to-Bac baculovirus expression system. To ensure effective protein secretion, a GP67 signal peptide sequence was added to the N-terminus of RBD (FIG. 1). In FIG. 1, the indicated domains and elements in the SARS-CoV-2 S protein includes signal peptide (SP), N-terminal domain (NTD), receptor binding domain (RBD), heptad repeat 1 and 2 (HR1 and HR2), transmembrane domain (TM) and cytoplasmic domain (CP), are marked. For S RBD preparation, the RBD region was engineered to include an N- terminal GP67 signal peptide before expression in insect cells. Following protein expression, recombinant RBD was harvested from the cell culture supernatant via Ni-NTA affinity chromatography.

[0102] Further purification of the protein using gel filtration revealed a symmetric elution peak (FIG. 2), indicating its high homogeneity in solution. In FIG. 2, the inset figures showed the SDS-PAGE and western-blotting analyses of the eluted RBD samples. The pooled samples were further analyzed by both SDS-PAGE and western blotting, which revealed a purity of over 98% (FIG. 2). It is found that the apparent molecular weight of the purified RBD protein is about 34 kDa, which is approximately 1/4 larger than that calculated from the molecular weight of the RBD amino acid sequence (about 27 kDa). This result highlighted post- translational glycosylation of the protein when expressed in insect cells. Thereafter identification of the glycosylated sites in the SARS-CoV-2 S protein RBD was set out. The purified protein was digested with trypsin and analyzed by mass spectrometry (MS). The intact N-glycopeptides and glycans were analyzed using the GPSeeker software. In total, three N- glycosylation sites on asparagine were identified (FIG. 3). The N-glycosylation and O- glycosylation sites are marked with the residue numbers.

[0103] Among them, 17 glycan moieties on N331, 12 glycans on N334 and 19 glycans on N343 were characterized. The O-glycosylation sites were also investigated by analyzing the MS results using SEQUEST in Proteome Discoverer (version 2.3). Some well-known O-linked glycans such as HexNAc and Hex(l)HexNAc(l) were searched as variable modifications. In total, ten O-glycosylation sites, including seven serine (S366, S371, S373, S375, S438, S443 and S514) and three threonine residues (T333, T376 and T523), were found (FIG. 3). To determine the abundance of glycosylation, the number of MS/MS spectra of glycosylated peptides and their corresponding unmodified peptides were counted, respectively. Overall, a much higher degree of N-glycosylation than O-glycosylation was observed (FIG. 4). In one embodiment, the number of mass spectrometry (MS)/MS spectra of each glycosylated peptides listed in FIG. 3 and their corresponding unmodified peptides were counted. Nine sample preparation and MS acquisition methods are applied. Each spot means the results from one method.

[0104] These identified glycosylation sites were mapped on the recently solved complex structure of the SARS-CoV-2 RBD bound to its receptor ACE2. On the whole, a majority of the sites were located on the core subdomain of the RBD molecule (FIG. 5). In one embodiment, the identified sites are shown as spheres and labeled. The approximate boundary between the core and external subdomains in RBD are marked with a dashed line. The right panel (surface representation) was generated by rotating the structure in the Left panel (cartoon representation) around a vertical axis for about 90°. In addition, all the sites were found to be distant from the bound ACE2 molecule (FIG. 5), indicating that the decorated glycans on these sites may not play a significant role in receptor recognition.

[0105] The functional of our prepared RBD protein to interact with the ACE2 receptor was further confirmed via surface plasmon resonance biacore. In line with a recent study, potent interactions with a typical si ow-on/slow-off binding kinetics between our protein and ACE2 were observed. The binding affinity was calculated to be about 1.54 nM in dissociation constant (KD), with a ka of 1.33xl07M-lS-l and kd of 2.05xl0-2S-l (FIG. 6). This finding indicates that our recombinant RBD can bind with ACE2 with a high affinity, suggesting close approximation of our recombinant protein and the native confirmation.

[0106] Identification of serum antibody against S protein RBD in patients and in mice [0107] Since Alum-precipitated protein vaccines elicit long-lasting neutralizing antibody responses that prevent bacterial or viral infection and Alum adjuvant is a commonly used in vaccine products, we therefore prepared the vaccine by addition of the recombinant RBD protein to aluminum hydroxide gel, resulting in Alum-precipitated protein vaccine (if not indicated otherwise). Mice were immunized at the different dose (0.1 to 20pg) and intervals. For example, mice were immunized with a single injection on Day 0 and collected sera on day 7; or with two vaccinations on day 0, day 7 and collected sera on day 21, with two doses on day 0, day 14 and collected sera on day 21. Also, we investigated a third vaccine on day 21 or longer. In order to detect the humoral immune responses induced by recombinant protein RBD, the sera from vaccinated mice were measured by enzyme linked immunosorbent assay (ELISA) for RBD-specific antibodies. In particular, the early antibody reaction and its ability to neutralize SAR-CoV-2 were determined as an early protective antibody response will enable better protection against infection by SARS-CoV-2. Sera at day 7 after the first vaccinations showed elevated IgG and IgM responses to the recombinant RBD protein (FIG. 7). In one embodiment, the mice were immunized with 5 pg recombinant RBD protein per mouse in 50 pi in the presence of aluminum hydroxide, compared with the control groups including recombinant RBD protein alone, aluminum hydroxide, pre-immune or Phosphate-buffered saline (PBS) alone. Sera were collected from the mice 7 days after the first vaccination and were tested at the different dilution for IgG and IgM against recombinant RBD protein using ELISA was as described in Methods. Data are presented as the mean ± SEM of five mouse sera in each group. P values were determined by two-way ANOVA. P values indicated RBD+AL vs AL or PBS or pre-immune groups. RBD+AL vs RBD alone in IgG level: P<0.0001 and P<0.0020 at dilution of 1:25 and 1:50 respectively. AL: AL(OH)3, RBD: recombinant 415 RBD protein, RBD+AL: the addition of the recombinant RBD protein to AL(OH)3. Similar results were repeated in three independent experiments.

[0108] In contrast, the sera from pre-immunization and those from PBS controls had only background-level antibody responses. Furthermore, the antibody reaction was dose-dependent, and could be induced at a very low dose by the vaccinated protein (O.lpg/mice) 7 days after the after only one dose of the candidate vaccine (FIG. 8), where the mice were immunized with 0.1-10 pg recombinant RBD protein per mouse in 50 pi in the presence of aluminum hydroxide, or aluminum hydroxide or PBS alone. Sera were collected from the mice 7 days after the first vaccination and were tested at 1:50 dilution for IgG and IgM against S protein RBD using ELISA. Data were presented as the mean ± SEM of six mouse serum samples in each group. P values were determined by one-way ANOVA.

[0109] Although the recombinant protein alone can already induce antibody response, the addition of aluminum hydroxide adjuvant was found to significantly increase the induction of the antibodies by day 7 (FIG. 7) and even more by day 21 (FIG. 9). In one example, the reaction were collected from 14 days after two vaccinations on day 0 and day 7 with 5 pg recombinant RBD protein per mouse in 50 mΐ in the presence of aluminum hydroxide, or recombinant RBD protein alone, and the level of the IgG of the sera was tested at the different dilution by ELISA. Similar results were repeated in three independent experiments.

[0110] The sera from 7 days after the second vaccination on day 14 showed a stronger antibody response (FIG. 10), compared with 14 days after the second vaccination on day 7 (FIG. 9). The reactions from FIG. 10 were collected from 7 days after two vaccinations on day 0 and day 14 with 5 pg recombinant RBD protein per mouse in 50 mΐ in the presence of aluminum hydroxide, or recombinant RBD protein alone.

[0111] To verify the degree of immunogenicity of RBD protein, the antibody responses against recombinant RBD protein by ELISA in sera from the patients with COVID-19 were measured. Sera from 20 healthy donors and 16 patients with COVID-19 (which were confirmed by RT- PCR using a nucleic acid detection kit) were tested for their IgG and IgM responses to the recombinant RBD protein. All 16 COVID19 patients showed elevated IgG and IgM levels against the recombinant RBD protein, compared with those in healthy donors (FIG. 11). The reaction data was collected from 16 patients infected with SARS-CoV-2 and 20 healthy donors and detected with ELISA as described in Methods. Data are presented as the mean ± SEM. P values were determined by one-way ANOVA.

[0112] There was no overlap between the two groups, indicating that it is likely that a cut-off can be defined that can differentiate between subjects with or without previous exposure to the virus with high degree of sensitivity and specificity, and this will have significant diagnostic implications. The finding suggests that S-protein RBD of SARS-CoV-2 could be recognized and triggered an antibody response, thus providing clinical evidence that support the development of a vaccine based on the RBD. In addition, the strong separation between patients with exposure and healthy individual without previous exposure may allow the development of a diagnostic assay that differentiate these two groups based on this protein antigen.

[0113] Functional characterization of the sera from the immunized mice and the prevention of the mice from SARS-CoV-2 infection

[0114] We select ACE2-positive Huh7 cells (a human hepatoma cell line) for detecting the RBD-binding activity in a flow-cytometric assay. RBD-Fc protein was added to the ACE2- positive Huh7 cells in the absence of the sera, followed by incubation with a secondary anti- Human IgG-FITC conjugate. As a result, 90.24% Huh7 cells were RBG-ACE2-positive (FIG. 12), in contrast, only 14.34% Huh7 cells were RBD-ACE2 positive in the presence of the immunized sera collected on day 7 after the first vaccination with the recombinant RBD protein. Sera from the mice treated with PBS at the same dilution had nearly no inhibitory activity with 87.40% ACE2 positive cells (FIG. 12). These findings indicated that the sera from the early vaccination after a single dose could block RBD binding to AEC2 receptor on the cells.

[0115] In one embodiment, referring to FIG. 12, recombinant SARS-CoV-2 RBD-Fc fusion protein was added to ACE2-positive Huh7 cells to a final concentration of lpg/ml in the presence or absence of the sera at a dilution of 1 :5 and followed by incubating with anti-human IgG-FITC conjugate. The binding assay of RBD-Fc with ACE2 was performed by flow cytometry as described in details in Methods. Sera/RBD (stained with sera pooled from 5 mice immunized with RBD vaccine day 7 after the first vaccination), sera/PBS (stained with sera from the mice treated by PBS as a control), positive control (without the presence of Sera/RBD or Sera/PBS), Negative control (the cells stained with anti-human IgG-FITC conjugate alone). Similar results were repeated in three independent experiments.

[0116] A neutralization assay using pseudovirus is regarded as a sensitive and quantitative method for SARS-CoV and MERS-CoV19. In the present study, we constructed SARS-CoV- 2 pseudovirus expressing EGFP, produced by co-transfecting with a plasmid encoding codon- optimized SARS-CoV-2 S protein, a pLenti-EGFP vector and a gag/pol expression plasmid. sera from the mice immunized with recombinant RBD protein was tested for the neutralizing activity against SARS-CoV-2 pseudovirus in 293T cells expressing ACE2. The sera from the mice immunized 7 days after the first vaccination were effective in blocking the infection by SARS-CoV-2 pseudovirus (FIG. 13). In one example, referring to FIG. 13, supernatants containing SARS-CoV-2 pseudovirus were preincubated with mouse sera which was serially diluted 2-fold. After incubated for 1 hour at 37 °C, the mixture was added to ACE2 -transfected 293T (293T/ACE2) cells to detect viral infectivity. The number of green fluorescent protein (GFP) expression in the infected cell was determined by fluorescent microscopy and flow cytometry. Both Sera/RBD and Sera/PBS (in the same as indicated in FIG.12 above), Untreated (infection with SARS-CoV-2 pseudovirus without sera).

[0117] The pre-immune sera or those from the mice treated with PBS at the same dilution had no inhibitory activity on SARS-CoV-2 pseudovirus infection. These findings suggest that the vaccine targeting S Protein RBD can induce the neutralizing activities against the pseudovirus. [0118] More importantly, we tested whether the humoral immunity alone at an early stage of the vaccination could block the infection in mice inoculated with SARS-CoV-2. Human ACE-2 transgenic mice were challenged with SARS CoV-2 viruses and mouse lung tissues were collected 5 days post-viral challenge to measure the virus replication status after the mice were given the day 7 immune sera. We used a quantitative real-time reverse transcription-PCR (qRT-PCR) to measure the levels of viral replication. No detectable viral replication was observed in the mice treated by the immune sera from the vaccinated mice (FIG. 14), while high levels of virus replication were detected in the lung tissues of the control mice. Also, referring to FIG. 14, the untreated mice were used as additional control. 1 day later after the transfer of the sera, the mice were challenged with SARS-CoV-2 stock virus at a dosage of 105 TCID50 intranasally. RNA copies in the lung tissues of mice challenged with SARS-CoV-2 were measured by a quantitative real-time reverse transcription-PCR reaction and expressed as RNA copies/ml of lung tissues, as described in Methods. Three mice in each group. Sera/RBD: sera pooled from the mice immunized with RBD vaccine 7 days after the first vaccination. Sera/PBS: sera from the mice treated by PBS as a control. Untreated: mice also infected with virus without the treatment with sera.

[0119] Consequently, the lung tissues from the control mice revealed typical histopathological changes of interstitial pneumonia, a defining feature of COVID-19. These findings included apparent thickened alveolar walls, heavy interstitial infiltrates by mononuclear cells and lymphocytes, congestion, as well as serosanguineous exudates in the alveolar spaces. In contrast, the mice treated with the sera from the mice immunized with the recombinant RBD protein vaccine exhibited either no histopathological changes or slight infiltrates (FIG. 15, also the untreated mice were used as additional control.). In addition, the mice treated with the sera from the vaccinated mice had an increase (about 8%) in body weight at 5 days post infection, as compared to no body weight gain in a same treated group or an 8% body weight decrease in untreated mice (FIG. 16, the untreated mice were used as additional control). To measure the levels of the IgG against the recombinant RBD protein in the recipient mice and not irritate the mice for live SARS-CoV-2 infection 1 day after the injection of the sera, we performed a separate experiment for the detection of the level of the IgG in parallel. The level of the circulating IgG against the recombinant RBD protein in the recipient mice by an ELISA assay one day after transferring 0.8 ml sera from the donor mice were measured and found that the level of the IgG response to RBD protein in the recipient mice was reduced to about 60% of the IgG level of the donor sera (collected from the donor mice at day 7 after the first vaccination) (FIG. 17). The fact that the live SARS-CoV-2 infection was completely inhibited in the recipient mice with the donor IgG from day 7 sera could provide the protective immunity. [0120] Pathways involved in the Elicit of the Cellular Immune Response by the RBD Protein

[0121] Potential cellular immune pathways involved as elicited by the recombinant RBD protein was investigated. Recombinant RBD protein was injected into Cd4 ^/_ , Cd8a ^/_ , Stingl , Tlr2 ^/_ , Tlr4 ^/_ , Caspl , NlrpS , and I1-1b mice. As shown in FIG. 18 (four to six mice per group), the mice deficient in Cd4 ^/_ , Sting l ^/_ , Caspl , Nlrp3 ^/_ , and I1-1b , Tlr2 ^/_ , Tlr4 ^/_ showed a reduction in the level of IgG against RBD protein, as compared to wild-type mice, while others (Cd8a ^/_ mice) showed no effect on the level of IgG. These findings implied that the NLRP3 inflammasome, Sting as well as TLR-4 and TLR-2 pathways and CD4 T lymphocytes may be involved in the induction of IgG against RBD protein by our vaccine. Similar results were repeated in two independent experiments. In one embodiment, the data are expressed as mean ± SEM. P values were determined by two-way ANOVA.

[0122] Since cellular immune responses may play a role in the clearance of SARS-CoV infection in which both CD4 and CD8 T cells are involved in the immune response against the SARS virus infection, the potential cellular immune responses in the vaccine of the present invention were also examined. The lymphocytes were collected 7 days after the first vaccination, and the cytokines including IFN-g and IL-4 produced by the lymphocytes were assayed by ELISA. The lymphocytes isolated from the vaccine immunized mice induced elevated IFN-g and IL-4 when stimulated with recombinant RBD protein (FIG. 19), while only a background level was detected in the lymphocytes stimulated by the recombinant RBD protein from the mice treated by a PBS control. In one embodiment, the lymphocytes were detected by ELISA under the stimulation of the recombinant RBD protein. Mice immunized with RBD protein vaccine or treated with PBS were sacrificed 7 days after the first vaccination to isolate lymphocytes which were then stimulated with recombinant RBD protein for three days, and the supernatants were collected for detecting IL-4 and IFN-g by ELISA assay, as described in Methods. The data are expressed as mean ±SD. P values were determined by unpaired Student t tests. Five mice per group. Similar results were repeated in three independent experiments. Furthermore, we identified the potential memory lymphocytes against the recombinant RBD protein by analyzing the phenotypes of these cultured lymphocytes. Using flow cytometry, we found that the number of the memory lymphocytes, CD4+CD44 ^hi§h +IL-4+, CD4+CD44 ^hi§h +IFN-Y, CD8+CD44 ^hi§h +IFN-Y,were increased in the mice after the vaccination (FIG. 20). In one embodiment, the RBD-reactive memory CD4 or CD8 was analyzed by flow cytometry by gating CD4+ or CD8+ CD44high + B220- MHCII- IFN-Y+ or IL-4+, as described in Method. Similar results were repeated in three independent experiments.

[0123] These findings indicated that the vaccination with the recombinant RBD protein vaccine could trigger T cell responses and induced increased numbers of memory T lymphocytes.

[0124] Since an elevated level of the INF-g and IL-4 in the lymphocytes stimulated by RBD protein in vitro were found, the level of the cytokines in blood was measured, and did not found the similar changes in the blood of the immunized mice when compared to that of the control groups (FIG. 21). This finding indicates that the vaccination may not induce a systemic inflammatory reaction.

[0125] As the safety of vaccine is a major concern, potential toxicity of our vaccine in animals was investigated. No adverse consequences were observed in gross measures such as weight loss, ruffling of fur, lifespan, behavior, or feeding. No pathologic changes in liver, lung, kidney, spleen, brain, heart, or other tissues were found on microscopic examination. No changes in serum biochemistry, the peripheral blood counts and differentials were found (data not shown). [0126] Identification of serum antibody against S protein RBD in rabbits using ELISA. [0127] The rabbits were immunized with 1-40 pg recombinant RBD protein per rabbit in 500 pi in the presence of aluminum hydroxide, or aluminum hydroxide or PBS alone. In particular, The rabbits were immunized with (a) AL, (b) 20 pg RBD, (c) 1 pg RBD + AL, (d) 5pg RBD,+ AL, (e) lOpg RBD,+ AL, (f) 20pg RBD,+ AL and (g) 40pg RBD,+ AL. AL means AL(OH) ₃ , RBD means recombinant RBD protein, RBD+AL means the addition of the recombinant RBD protein to AL(OH)3. Rabbit were immunized with three vaccinations on day 0, day 14 and day 21, collected sera 7 days after each boost. Sera were collected from the rabbits 7 days after the third vaccination and were tested at different dilution for IgG against S protein RBD using ELISA. Data are presented as the mean±SEM of six rabbits sera in each group. As shown in FIG. 23, sera at day 7 after the third vaccinations showed apparent elevated IgG responses to the recombinant RBD protein. In contrast, the sera from pre-immunization and those from to AL(0H)3 controls had only background-level antibody responses. Furthermore, the antibody reaction was dose-dependent, and could be induced at a very low dose by the vaccinated protein (lpg/rabbit). The recombinant protein RBD alone can induce increased antibodies, but the addition of aluminum hydroxide adjuvant could significantly enhance the induction of the antibodies. It has been known that the dose of the vaccine for human use is about equal to the dose of the rabbit that has a response.

[0128] As the safety of vaccine is a major concern, the potential toxicity of our vaccine in rabbits was investigated. No adverse consequences were observed in gross measures such as weight loss, ruffling of fur, lifespan, behavior, or feeding.

[0129] FIG. 24 shows neutralization of SARS-CoV-2 pseudovirus infection by the sera from the rabbit immunized with recombinant RBD vaccine. Infection of HEK293 cells expressing human ACE2 by SARS-CoV-2 pseudovirus was determined in the presence of rabbit sera at a series of 3-fold dilutions. Percentage of neutralization was presented as mean±SEM. In regard to SARS-CoV-2 pseudovirus that express spike protein, its backbone was provided by VSV G pseudotyped virus (G*AG-VSV) that packages expression cassettes for firefly luciferase instead of VSV-G in the VSV genome. Results showed that the sera from the rabbits immunized 7 days after the third vaccination with the dose of 20pg recombinant RBD protein per rabbit in 500pl in the presence of aluminum hydroxide had the very potent neutralization of SARS-CoV-2 pseudovirus infection and indicate that the antibodies induced by the vaccine can function.

Summary

[0130] The immune response against SARS-CoV-2 may be provoked by immunization of the vaccine, and an antibody targeting RBD protein alone is able to provide the protective immunity as early as 7 days after the first vaccination. These claims are supported by the data above, including 1). the elevated level of the antibodies against RBD protein in the immunized mice and COVID19 patients, 2). blocking of the binding of RBD with its host cell surface receptor ACE2, 3). inhibition of the infection of the pseudovirus in vitro and by live virus in vivo in a mouse model, 4). The identification of the involvement of pathways like NLRP3 inflammasome, Sting, TLR-4 and TLR-2 and CD4 T lymphocytes. The data and conclusions are also supported by the previous findings that the vaccines based on S Protein and its domains can induce protective immunity and prevent infection with SARS-CoV and MERS-CoV in animal models.

[0131] One dose in some embodiments may comprise 0.1-40pg recombinant RBD protein. In one embodiment, the dose may further comprise [0.1-1000m1] aluminum hydroxide, a predetermined amount of physiologically acceptable carrier, or a combination thereof. In other embodiments, one dose may comprise over 40pg recombinant RBD protein. In yet other embodiments, the dose may further comprise over IOOOmI aluminum hydroxide.

[0132] Examples [0133] Methods

[0134] Bacterial strains and cell lines

[0135] All E. coli strains were cultured in Lysogeny broth (LB) medium (1% w/v Tryptone, 0.5% w/v yeast extract and 1% w/v NaCl) using a non-humidified shaker at 37 °C. Spodoptera frugiperda (Sf9) cells and Trichoplusia ni (Hi5) cells were individually maintained in the SIM SF medium and the SIM HF medium (Sino Biological, Beijing, China) using a non-humidified shaker at 27°C. HEK293T and Huh-7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplementedwith 10% fetal bovine serum (FBS), 100 units of Penicillin and 0.1 mg/ml of Streptomycin with 5% C02 at 37 °C.

[0136] Gene cloning, expression and protein purification

[0137] The spike RBD protein of SARS-CoV-2 used in this study for antigenicity evaluation was expressed using the Bac-to-Bac baculovirus expression system (Invitrogen). The coding sequence (codon optimized for insect cells) for the RBD region, which spans residues 319-545 of the spike of the SARS-CoV-2 Wuhan-Hu-1 isolate (accession number MN908947), were synthesized by Convenience Biology Corporation (Changzhou, Zhejiang Province, China). For gene cloning, a previously described gp67 signal peptide sequence was first incorporated into the pFastBacl vector via the BamH-I and EcoR-I restriction sites. The RBD gene was then sub-cloned into the modified vector via the EcoR-I and Hind-III sites. In addition, an 8><His tag was further added to the protein C terminus to facilitate protein purification. The sequencing-verified plasmid was subsequently transformed into E. coli DHlOb cells to generate recombinant bacmids.

[0138] For protein expression, the bacmid was first transfected into Sf9 insect cells using Lipolnsect Transfection Regent (Beyotime Biotechnology, Shanghai, China) according to the manufacturer's instructions. The cell culture supernatants, which contain the packaged recombinant baculoviruses, were harvested about 72 hours post transfection. The baculovirus was then passaged in Sf9 cells for 2-3 times before used for protein production in Hi5 cells. [0139] For protein purification, the culture supernatants from the Hi5 cells were collected about 72 hours after infection and passed through a 5-ml HisTrap excel column (GE Healthcare) for primary purification. The recovered proteins were further purified on a SUPERDEX 200 Increase 10/300 GL column (GE Healthcare). Finally, the proteins were exchanged into a buffer consisting of 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl for further use. The purity of the protein was determined by SDS-PAGE and visualized by staining with Coomassie blue and by western blotting using the mouse anti-His monoclonal antibody (Zen BioScience, Chengdu, Sichuan Province, China).

[0140] LC-MS/MS analysis to identify glycosylation sites

[0141] For MS test, the protein was first trypsin-digested as previously described. In brief, the purified RBD protein was precipitated with 4 volumes of pre-cooled acetone at -20 °C overnight. The protein pellets were collected with centrifugation at 20,000g for 10 min. After dried on ice, the protein pellets were dissolved in a denaturing buffer (5% 8 M urea in 50 mM NH4HCO3, v/v). The proteins were reduced with 20 mM DTT at 55 °C for 60 min, and then alkylated with 55 mM iodoacetamide in the dark at room temperature for additional 30 min. After carbamidomethylation, the proteins were digested with trypsin (1:50 w/w) at 37 °C overnight.

[0142] After being desalted with Cl 8 ZipTip (Millipore) according to the manufacturer's instructions, the digested peptides were analyzed by LC-MS/MS using an EASY-nano-LC 1200 coupled to a Q-Exactive HF-X (Thermo Scientific) without the trap column. Specifically, the peptide samples were loaded onto an in-house packed reversed-phase Cl 8 analytical column (30 cm length><360 pm OD x75 pm ID, 3 pm particle, DIKMA) and were separated at a flow rate of 330 nL/min. The column oven temperature was 60 °C. Buffer A was 0.2% formic acid in water, and Buffer B was 80% ACN with 0.2% formic acid. A two hours gradient was applied with 3% B for 6 min, 3-48% B for 100 min, 48-100% B for 6 min, 100% B for 6 min, and 2% B for the last 2 min. MS spectra were acquired with m/z 700-2000 and a resolution of 60,000 at m/z 200. The automatic gain control (AGC) was set at 2e5 with maximum fill time of 50 ms. For MS/MS scans, the top 20 most intense parent ions were selected with a 2.0 m/z isolation window and fragmented using HCD with normalized collision energies of 20%/30%/30%. The AGC value for MS/MS was set to a target value of 5e5, with a resolution of 3,000 and a maximum fill time of 250 ms. Parent ions with a charge state of z = 1, 8 or unassigned were excluded and the intensity threshold was 3.3e4. The dynamic exclusion period was 20s. The temperature of the ion transfer capillary was set to 280 °C. The spray voltage was set to 2.8 kV.

[0143] For the identification of intact N-glycopepyides, all the raw files were searched with GPSeeker, developed by Tian’s groupl5. In order to search matching precursor and fragment ions, the isotope peak abundance cutoff (IPACO), isotope peak mass-to-charge ratio (m/z) deviation (IPMD), and isotope peak abundance deviation

[0144] (IPAD) were respectively set to 40%, 20 ppm and 50%. The search of intact N- glycopeptide spectrum matches (GPSMs) included the following parameters: Y1 ion, Top4; the minimum percentage of matched fragment ions of the peptide backbone, > 10%; the minimum matched fragment ion of the N-glycan moiety, >1; TopN hits, N = 2 with Topi hit(s) having the lowest P score; G-bracket, >1; and GF Score, >1. G-bracket for a given N-gly cosite is defined as the number of peptide backbone b/y fragment ion pairs each of which can independently localize the N-glycosite. GF score for a given N-glycan sequence structure is defined as the number of structure-diagnostic fragment ions each of which can independently distinguish the structure from all the other putative structures with the same monosaccharide composition. After DB search of all the raw datasets, GPSMs were combined and intact N- glycopeptides with the lowest P score were chosen as the final IDs.

[0145] The MS Raw files were further searched against the RBD sequence with SEQUEST in Proteome Discoverer (version 2.3; Thermo Fisher Scientific). The precursor peptide mass tolerance was 10 ppm and a fragment ion mass tolerance was 0.02 Da. Two missing cleavages were allowed. Cysteine carbamidomethylation was set as a fixed modification. HexNAc (S/T), Hex(l)HexNAc(l)(S/T) and other potential O-glycosylation modifications with conventional oxidation of methionine and protein N-terminal acetylation were set as variable modifications. Percolator was generated with false discovery rate (FDR) of 1%. All potential O-glycosylation sites were further manually confirmed by the b ions and y ions.

[0146] Surface plasmon resonance (SPR) analysis

[0147] Surface plasmon resonance (SPR)-based measurements were performed by Biacore 8K (GE Healthcare, Uppsala, Sweden), as described previously. Human ACE2-Fc was captured to -lOORU on Sensor Chip Protein A. For kinetic analysis, RBD protein was run across the chip in a 2-fold dilution series (1, 2, 4, 6, 8, 16, 32nM), with another channel set as control. Each sample bound across the antigen surface was dissociated by HBS-EP+ running buffer for 300 s at a flow rate of 30 pL/min. Regeneration of the sensor chips was performed for 60 s using regeneration buffer (Glycine pH 1.5). The association and dissociation rate constants ka and kd were monitored respectively and the affinity value KD was determined. [0148] Vaccine formulation and mice vaccinations

[0149] Alum-precipitated protein (alum protein) vaccines were prepared as described previously. Briefly, the purified recombinant RBD protein at the different concentrations was added and incubated with mixing with aluminum hydroxide gel for one hour at 5°C. The different formulations were prepared with the concentrations of l-100pg/ml for protein and 1.21mg/ml for aluminum hydroxide gel.

[0150] B ALB/c and C57BL/6 mice at 6 to 8 weeks of age were injected intramuscularly with different doses (0.1-20 pg per mouse) of recombinant RBD protein and different intervals. For example, the mice were immunized with a single injection on Day 0 and collected sera on day 7, or with two vaccinations on day 0, day 7 and collected sera on day 21, compared with two doses on day 0, day 14 and collected sera on day 21. Also, we are investigating the third vaccine on day 21 or longer. Additional control animals were injected with aluminum hydroxide adjuvant [Al(OH)3], recombinant RBD protein or PBS alone. Pre-immune sera also were collected before starting the immunization and the sera were collected 7 days after each boost. Sera were kept at 4°C before use. Also, we attempted to find the pathways through which our recombinant RBD protein may function. We injected recombinant RBD protein vaccine into genetic deficient mice, Cd4 ^/_ , Cd8a ^/_ , Caspl , Sting 1 , Tlr2 ^/_ , Tlr4 ^/_ (all from Jackson Laboratory), NIhH (from Genentech), and I1-1b (Tokyo University of Science). All studies involving mice were approved by the Animal Care and Use Committee of Sichuan University. [0151] Identification of serum antibody against S protein RBD in patients and in mice using an ELISA assay

Blood samples were collected from the retro-orbital plexus of mice after each antigen boost. After coagulation at room temperature for 1-2 h, blood samples were spun in a centrifuge, 3000 rpm/min for 10 min at 4 °C. The upper serum layer was collected and stored at -20 °C . Recombinant protein RBD or S2 protein as a control was used to coat the flat-bottom 96-well plates (Thermo Scientific NUNC-MaxiSorp) at a final concentration of 1 pg/ml in 50 mM carbonate coating buffer (pH 9.6) at 4°C overnight. The following day, plates were washed 3 times with phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBST), and blocking solution containing 1% BSA in PBST was added, followed by 1 h incubation at room temperature. Serially diluted mouse sera was added and incubated at 37°C for 1 h, and then washed the plates 3 times with PBST. Antibodies including goat anti-mouse IgG horseradish peroxidase (HRP)-conjugated antibody, or anti-mouse IgGl/IgM HRP-conjugated antibody were diluted 1/5000 in blocking solution, and added to wells (100 mΐ/well). After incubation for 1 h at room temperature, the plates were washed 5 times with PBST and developed with 3, 3', 5, 5'- tetramethylbiphenyldiamine (TMB) for 10 min. The reactions were stopped with 50 mΐ/well of 1.0 M H2S04 stop solution. The absorbance was measured on a microplate reader at 450 nm (A450). In order to measure the titer of RBD-specific antibodies induced by recombinant proteins, serum samples were serially diluted and measured by titration.

[0152] In order to investigate the potential immunogenicity of S protein RBD as vaccines in human, serum samples were collected from 16 patients infected with SARS-CoV-2 and 20 healthy donors detected with ELISA in similar way mentioned above. Briefly, the recombinant protein was used to coat 96-well microtiter plates, After blocking with 1% BSA, 1: 5 diluted sera were added and incubated, followed by four washes Bound Abs were detected with HRP- conjugated antibody (anti-human IgG/IgM antibody) at 1/2000 dilution. For the detection of IgM, serum samples were added to IgG sorbents and collect the supernatant for further detection centrifugation. All the patients with COVID-19 were confirmed by RT-PCR using a 2019-nCoV nucleic acid detection kit. This case series and healthy donors were approved by the institutional ethics board of Sichuan Provincial People's Hospital. The informed consent was obtained from all participants.

[0153] In order to investigate cell-mediated immune response, mice immunized with S protein RBD or PBS were sacrificed to isolate lymphocytes which were applied for IL-4, and IFNy ELISA assay. Briefly, the lymphocytes isolated from the spleens of the immunized mice or mice treated by PBS alone were cultured in RPMI medium 1640 supplied with 10% (vol/vol) FBS, 100 U/ml penicillin, 100 pg/ml streptomycin, 1 mM pyruvate (all from Gibco), 50 mM b- mercaptoethanol, and 20 U/ml IL-2 (all from Sigma-Aldrich). Simultaneously, 1 pg /ml RBD protein was added to activate cells. These cells (1x106 per well) were incubated for 72 h at 37 °C. Cells cultured without RBD protein were used as negative control. The supernatants were collected for ELISA. Also, we identified the potential memory lymphocytes against the recombinant RBD protein by analyzing the phenotypes of these cultured lymphocytes by flow cytometry, as mentioned below in flow cytometry.

[0154] Measurement of inhibition of the S protein RBD binding to ACE2 on the cell surface [0155] Binding assay of RBD-Fc with ACE2 was performed by flow cytometry as previously described. Briefly, ACE2-positive Huh7 cells (a human hepatoma cell line) were collected and washed with Hanks’ balanced salt solution. Recombinant SARS-CoV-2 RBD-Fc fusion protein was added to the cells to a final concentration of 1 pg/ml in the presence or absence of the sera at a different dilution. The cells were incubated further at room temperature for 30 min. Cells were washed three times with HBSS and then stained with anti-human IgG-FITC conjugate (Sigma-Aldrich, St. Louis, MO, U.S.) at 1:50 dilution for an additional 30 min. After washing, the cells were fixed with 1% formaldehyde in PBS and processed by the NovoCyte Flow Cytometer (ACEA Biosciences, Inc.), and the results were analyzed with FlowJo VI 0 software. [0156] Neutralization of pseudovirus infection

A neutralization assay based on the pseudovirus was performed by measuring infection of ACE2 -transfected 293T (293T/ACE2) cells as previously described, briefly, EGFP-expressing pseudotyped viruses were produced by co-transfecting with a plasmid enconding codon- optimized SARS-CoV-2 S protein, a pLenti-EGFP vector and a gag/pol expression plasmid. Supernatants containing pseudovirus were harvest 48h posttransfection and preincubated with the sera from the immunized mouse at various dilutions and control sera. After incubated for 1 hour at 37°C, the mixture was added to ACE2 -transfected 293T (293T/ACE2) cells to detect viral infectivity. Media was changed the following day and 48h after infection, EGFP expression in infected cell was determined by fluorescent microscopy and flow cytometry. [0157] Challenge of the mice with live SARS-CoV-2

All procedures involved in the animal study were reviewed and approved by the Institutional Animal Use and Care Committee of the Institute of Laboratory Animal Science, Peking Union Medical College. Murine studies were performed in an animal bio-safety level 3 (ABSL3) facility using HEPA-filtered isolators. The animal experiments of the infection of SARS-CoV- 2 were performed by specific pathogen-free transgenic hACE2 mice established by the Institute of Laboratory Animal Science, Peking Union Medical College, China. Transgenic mice were generated by microinjecting a transgene carrying a mouse ACE2 promoter driving the human ACE2 coding sequence into the pronuclei of fertilized ova from ICR mice, as described in detail (bioRxiv preprint doi: Linlin Bao et al. 2020. The Pathogenicity of SARS-CoV-2 in hACE2 Transgenic Mice. bioRxiv dokO.l 101/2020.02.07.939389v3). The adoptive therapy of the sera was described previously. The hACE2 mice were injected 0.8 ml of the pooled sera from the mice immunized with the vaccine 7 days after the first vaccination, or normal mouse serum from the mice treated with PBS as a control intraperitoneally 1 day before the challenge with SARS-CoV-2 stock virus at a dosage of 10 ⁵ TCID50 intranasally. Also, the mice infected with the virus were not treated with sera as a control. The mice were sacrificed 5 days after the challenge and lungs and other organs of the mice were removed. The lung tissues were used for detecting virus replication or were fixed with 10% buffered formalin solution for histopathological analysis. RNA copies in the lung tissues of mice challenged with SARS- CoV-2 were measured by a quantitative real-time reverse transcription-PCR (qRT-PCR) reactions using the PowerUp SYBG Green Master Mix Kit (Applied Biosystems, USA) and expressed as RNA copies/ml of lung tissues. The primer sequences used for qRT-PCR are targeted against the envelope (E) gene of SARS-CoV-2 and are as follows: Forward: 5’- TCGTTTCGGAAGAGACAGGT-3 ’ , Reverse: 5 ’ -GCGC AGT AAGGAT GGCT AGT -3 ’ . Sections were stained by Hematoxylin and Eosin (H&E) and histopathological changes were observed by light microscopy.

[0158] Flow cytometry

[0159] T cells was evaluated with flow cytometry. Mice immunized with S protein RBD or PBS were sacrificed to collect lymphocytes. The lymphocytes were cultured in RPMI medium 1640 supplied with 10% (vol/vol) FBS, 100 El/ml penicillin, 100 pg/ml streptomycin, 1 mM pyruvate (all from Gibco), 50 mM b-mercaptoethanol, 20 U/ml IL-2 (all from Sigma-Aldrich) for 72 h. At the same time, 1 pg/ml S protein RBD was added to activate cells. Brefeldin A (BD Biosciences) was administrated 4-6 h before staining to block intracellular cytokine secretion. Cells were then washed in PBS (Gibco) and stained for 30 min at 4°C with anti-CD8, anti-CD4, anti-CD44, anti-B220, anti-MHCII (all from BioLegend). Afterwards, cells were fixed and permeabilized to facilitate intracellular staining with anti-IFNy, and anti-IL-4 (all from BioLegend). Flow cytometry data were acquired on a NovoCyte Flow Cytometer (ACEA Biosciences, Inc.) and analyzed using FlowJo VI 0 software.

[0160] Statistical analysis

[0161] Statistical analyses were performed using the Prism 8.0 (GraphPad Software). Comparisons among multiple groups across multiple time points were performed using a two- way ANOVA test with Tukey’s multiple comparison post test. Comparisons among multiple groups were performed using a one-way ANOVA test followed by Tukey’s multiple comparison post test. Comparisons between two groups were performed using an unpaired Student’s t tests. P values of < 0.05 were considered a significant difference. *P < 0.05, **P < 0.01, ***p < 0.001, ****p < 0.0001. NS, no significance.

[0162] In yet another embodiment, the recombinant RBD protein of SARS-CoV-2 spike protein is prepared from insect cells using the Bac-to-Bac baculovirus expression system, which baculovirus expression vector comprises at least a portion of a genetically modified influenza viral genome comprising an insertion of a gene encoding for a RBD protein of SARS- CoV-2 spike protein. The at least a portion of a genetically modified influenza viral genome may further comprises a disruption in the non- structural (NS1) coding segment, one or more base substitutions in the matrix membrane protein coding segment, or any combination thereof. The deletion may be a deletion of at least part of an NS1 gene extending beyond nucleotides 57 to 528 of an NSl segment of the influenza viral genome. One or more base substitutions may lie outside of the M region of the genetically modified influenza viral genome, for example, (i) a G346A mutation in the H1N1 influenza virus genome or (ii) set of one or more mutation(s) at T261G or a A310G mutation in the H1N1 influenza virus genome. In another embodiment, One or more base substitutions may lie inside the M segment of the genetically modified influenza viral genome, for example, A14U and/or G917A mutations.

[0163] Immunogenicity Enhancement Device

[0164] The composition of the present invention may be used in conjunction with an Immunogenicity enhancement device.

[0165] In one embodiment, the device is an immunogenicity enhancement device comprises an occlusive dressing includes a barrier that covers the applied topical medication and a central opening through which an immunizing injection is administered. The topical medication/preparation may be applied to the surface of the skin prior to application of the occlusive dressing or applied to a skin-facing surface of the occlusive dressing. The occlusive dressing may be constructed in layers, and include a frame layer and a barrier layer. In some embodiments the occlusive dressing may include a portion that covers the central opening following administration of the injection. The occlusive dressing may include a wound dressing positioned to cover the injection site following immunization. In some embodiments the occlusive dressing may be held in place by a separate wound dressing. The device may include a measuring device for aliquoting a desired amount of the topical medication/preparation. Such a measuring device may be utilized to apply or spread the topical medication/preparation. [0166] The inventive subject matter provides apparatus, systems and methods in which an occlusive device or dressing that includes an access feature is provided that applies a desired amount of an immunization-enhancing formulation (for example, a toll-like receptor 7 agonist, and/or toll-like receptor 9) to an area of skin that is to receive an intradermal vaccination. After a suitable period of time the access feature is utilized (for example, by opening or removal) to permit access to an area of the skin surface for delivery of the intradermal vaccination. In some embodiments a portion of the occlusive device (for example, a "wing") may be deflected to provide a dressing over this access feature following immunization. In other embodiments a dressing may be provided as a separate item. After another suitable period of time (for example, a period of time suitable for providing an enhanced post-immunization enhancement) the device may be removed. The occlusive device may be configured to provide an immunization enhancing formulation to the immunization site, to a region surrounding the immunization site, or both. In some embodiments the device is self-adhesive to the skin surface. In other embodiments the occlusive device may be applied and/or affixed to the skin surface using a second device (for example, a bandage or dressing). [0167] The present invention provides occlusive devices and methods that enhance the effects of immunization. This is accomplished by providing an occlusive device that simplifies safe and effective topical application of a composition containing an immunization enhancing pharmaceutical at or near (e.g. surrounding) a vaccine injection site. Such an immunization enhancer may be an aluminum-based salt, such as alum, aluminum phosphate, and/or aluminum hydroxide. In some embodiments an immunization enhancer may be an organic compound, such as a squalene. In some embodiments a suitable immunization enhancer may be a toll like receptor agonist, such as a toll like receptor 7 agonist and/or a toll like receptor 9 agonist. In a preferred embodiment the immunization enhancer may be imiquimod. To avoid the potential infection during injection and permit time for tissue penetration, the topical composition is preferably applied on and/or around the vaccination site for a period of time (for example, 1 to 2 hours) prior to the vaccine injection, and may require protection from clothing, accidental contact, and other environmental factors during this period.

[0168] In practice this process is time-consuming and error-prone. Proper application may require the additional step of cleaning the residual topical composition from the site before injection, and may suffer from lack of compliance (even from medical professionals). For example, early removal of the topical composition containing the immunization enhancer (for example, due to limited personnel in a clinical setting) may compromise the immune-boosting effects on immunogenicity.

[0169] In some embodiments of the inventive concept, an applicator that may deliver a fixed or selectable volume of a topical formulation (for example a cream, powder, paste, ointment, lotion, liquid, suspension, foam, and/or gel) onto at least a portion of a device of the inventive concept that is subsequently brought into contact with a skin surface that is intended for use in immunization. Alternatively, skin at or around the intended immunization site of the individual to be immunized may supplied with such a volume of topical formulation. The volume of topical formulation may be selected to provide adequate coverage at and/or around an immunization site so as to enhance the immune response to the supplied immunogen. It should be appreciated that the volume of the topical formulation may be varied to accommodate the nature of the vaccine and/or characteristics of the individual to be immunized (for example, age, gender, size, body composition, underlying health conditions, previous disease status, etc.). In some embodiments the topical formulation may be supplied as part of the device, for example as a pre-applied layer that is brought into contact with the skin on application. In such embodiments dosing of the topical formulation may be controlled or adjusted by selection of a device of appropriate size. Alternatively, in some embodiments the topical formulation may be supplied as a pre-filled dose or bolus that is expressed onto an applicator and/or onto an area of the skin of the subject to be immunized. In some embodiments such an applicator may be sized to provide a desired amount of topical formulation, and/or may include indicia that permit a user to select a desired amount of topical formulation.

[0170] Devices and methods of the inventive concept may utilize or support the use of a formulation that includes toll like receptor 7 agonists and/or toll like receptor 9 agonists as immune enhancers. Suitable toll like receptor agonists include imidazoquinoline derivatives (e.g. imiquimod and/or resiquimod), guanosine analogs (e.g. loxoribine), pyrimidine analogs (e.g. bropirimine), phosphonic acid derivatives, and 8-oxoadenine derivatives and their carboxylate esters. In a preferred embodiment the immune enhancer is imiquimod, which may be provided as a topically applicable lotion, cream, or gel. Such an immune enhancer may be supplied in an applicator that is used in conjunction of a device of the inventive concept, or may be incorporated into one or more skin-contacting portions of the device as supplied. [0171] Topical composition suitable for use with occlusive devices of the inventive concept may include an immunization enhancing pharmaceutical (such as imiquimod or the composition of the present invention) that may be applied to an area at and/or near the sited of vaccination. In some embodiments of the present invention, the occlusive device provides or is supplied with dispensing or measuring device that includes a reservoir sized to include a suitable amount of the topical composition (for example, from 1cm to 10 cm x 1cm to 10 cm) that surrounds the intended site of vaccination following application to an individual to be vaccinated. The reservoir area for the topical composition may have a shape that covers an area greater that 1cm ² , for example up to 8 cm ² or more. Such a reservoir may have any suitable configuration. For example, such a reservoir may be circular, ovoid, square, rectangular, polygonal, and/or irregular. Such a reservoir may have a minimum dimension of about 1 cm and a maximum dimension of up to 8 cm. The reservoir may be made from any suitable material that is not reactive with the pharmaceutical stored therein, for example a polymer such as polyethylene, polypropylene, or silicone. In some embodiments the reservoir may include additional features to enhance the stability of the immunization enhancing pharmaceutical, for example impermeability to oxygen, ability to block UV and/or visible light, etc.

[0172] Embodiments of the inventive concept may include an adhesive-bearing portion that permits the occlusive device to cling to the skin during use. In some embodiments the adhesive bearing portion is coupled to the reservoir. In other embodiments the adhesive-bearing portion is provided as a separate piece, for example as a tape or bandage that is applied to the reservoir on use. An adhesive-bearing portion may be made of any suitably flexible and supportive material, such as a polymer sheet, a polymer mesh, a woven fabric (natural or synthetic), or a nonwoven fabric (natural or synthetic). In a preferred embodiment at least a part of the adhesive-bearing portion permits the free passage of air (i.e. is breathable) in order to promote comfort and skin integrity when the device is in use. The adhesive-bearing portion may include a surface that is applied to the skin when in use. At least a portion of this skin-facing surface may include a pharmaceutically compatible adhesive that provides a transient bond to skin surface. Such a transient bond should be stable for from 5 minutes to up to 48 hours following application to the skin. In some embodiments the occlusive device may be provided with a removable (e.g. peelable) cover that protects this adhesive prior to application.

[0173] Some embodiments of the inventive concept incorporate a bandage or dressing, which may be used to cover the puncture wound resulting from vaccination. Such a bandage or dressing may be coupled to or provided by a portion of the device. For example, a device of the inventive concept may include a tab or wing that may be folded over to act as a bandage or dressing. In other embodiments a bandage or dressing may be provided as a separate item that is applied following immunization. Such a bandage or dressing may include an adhesive portion, and absorbent portion, and a backing. The backing may be made from any suitably flexible material, for example a polymer sheet, polymer mesh, woven fabric, unwoven fabric, etc. The absorbent portion may be coupled to the backing and include a material suitable for absorbing and/or containing blood and other body fluids. Suitable absorbent materials include a fabric, wool, and/or gel made of natural or synthetic materials. In some embodiments the absorbent material may include an agent that provides pain control (such as a topical analgesic or anesthetic) and/or promotes healing (such as an antibiotic). The adhesive portion may be an adhesive layer that is coupled to the backing. Such an adhesive layer may be provided as an adhesive compound applied to at least a portion of the backing or as a layer of material that incorporates such an adhesive compound and is coupled to the backing.

[0174] Some embodiments of the inventive concept may include a dispensing or measuring device suitable for storing and dispensing the topical composition. Such a device may be provided as reservoir dimensioned to accommodate at least a volume of the topical composition suitable for a single use. Such a reservoir may be reversibly sealed, for example using a polymer film or sheet that is reversibly affixed to an opening of the reservoir. Such a dispensing device may include an applicator, which facilitates removal of the topical composition from the reservoir and application to the skin of a subj ect in need of immunization or to a skin-contacting surface of a device of the inventive concept. In some embodiments such an applicator may include indicia (for example, visible lines or similar symbols along its length) that permit metering or measurement of the amount of topical composition present on the applicator. [0175] An example of an occlusive device of the inventive concept is shown in FIG. 24. As shown, the occlusive device provides an occlusive dressing that protects and maintains a layer on the skin of a medicament that enhances immune response to an injected vaccine. The occlusive device provides a through-hole through which the vaccination may be administered. The example shown in FIG. 24 provides a frame (120) that supports a film (140) which serves to protect the layer of medicament. The frame (120) may be constructed of any suitable paper (for example, cardboard, fiberboard, etc.) or polymer (for example, polyethylene, polypropylene, nylon, silicone, etc.), and have sufficient thickness to support the film (140) and other elements of the occlusive device while being sufficiently pliant for application to a skin surface. The film may act as a barrier layer during use of the occlusive device. In some embodiments the frame (120) may include or support one or more tabs (130), which may serve to improve adhesion to the skin surface. The film may be made from a suitable polymeric material (e.g. polyethylene, polypropylene, nylon, silicone, etc.) that provides an environmental barrier (e.g. resistance to moisture, etc.), and in preferred embodiments is transparent or translucent. The frame (120) may support a through hole cover (110), which serves to cover a through-hole that extends through the occlusive device. Such a through-hole may be produced by the alignment of apertures in different portions of the occlusive device. [0176] In some embodiments occlusive devices of the inventive concept are constructed in a laminar or layered fashion. This may be seen in FIG. 25, which depicts a cross section of an occlusive device such as that shown in FIG. 24. As shown the occlusive device is constructed of a number of layers, including a through-hole cover (110), a frame (120), a film (140), and a backing (150). Each of these layers may be constructed of a different material. Layers of the occlusive device may be joined by any suitable means. For example, layers may be joined using an adhesive, by melting, by welding (e.g. ultrasonic welding), by crimping or folding, and/or by using joiners (e.g. stitching, stables, etc.). In some embodiments the connection between two or more layers may be readily disrupted without the use of tools (e.g. a "peelable" layer), permitting removal of or disconnection or a portion of one or more layers from the bulk of the occlusive device. Such laminar construction facilitates manufacture of the occlusive device. [0177] FIG. 26 provides an expanded view of an occlusive device as shown in FIG. 24. As shown, the through-hole cover (110) is centered over frame aperture (125) that is at least approximately centrally located on the frame (120). The frame aperture (125) may be defined by a ring or similarly shaped portion that extends from an interior edge of the frame (120). Remaining open space between the edges of the frame (120) define a frame cutout (127) through which the film (140) may be seen. As shown the film (140) includes a film aperture (145) that is in central alignment with the frame aperture (125). The backing (150) similarly includes a backing aperture (155) that is centrally aligned with the frame aperture (125) and the film aperture (145). This alignment of apertures in the assembled occlusive device provides a through-hole through which the vaccination is administered.

[0178] In some embodiments the backing (150) may include an adhesive on the side facing away from the film (140), which may aid in adhering the occlusive device to the skin during use. In such embodiments this adhesive may be covered by a removable or peelable film. In other embodiments the backing (150) may be removed prior to use to expose the surface of the film (140) facing away from the frame (120). In such embodiments the film (140) may include an adhesive interposed between the film (140) and the backing (150) that aids in affixing the occlusive device to the skin of a patient to be immunized. Alternatively, the film (120) may be constructed of a material having sufficient taction to cling to the skin without the need of an adhesive.

[0179] In some embodiments of the inventive concept the occlusive device may be provided with a medicament that enhances immune response to a vaccination already incorporated into the occlusive device. For example, such a occlusive device may include a layer of the medicament interposed between the film (140) and the backing (150), such that removal of the backing exposes the medicament. Alternatively, the backing (150) may include a layer of medicament applied to the surface facing away from the film (140). Such a layer may be covered with a removable barrier during storage, which is removed upon application to a patient. In other embodiments the medicament is provided in a separate container or applicator. [0180] FIG. 27 illustrates a method for using an occlusive device such as that shown in FIGS. 24 to 26. From left to right, a backing is removed from the underside of the occlusive device (1), which is then applied to a suitable immunization area with the through-hole cover oriented away from the skin (2). In this instance the upper arm is shown, however other sites are suitable. Occlusive devices of the inventive concept may be supplied in various sizes (e.g. from 4cm x 4cm to 10cm x 10cm) and configurations (e.g. square, rectangular, circular, ovoid, etc.) adapted to different immunization sites and/or different patient sizes. In some embodiments a medicament that enhances immune response to a vaccinating composition is applied to the skin prior to application of the occlusive device. In other embodiments such a medicament is incorporated into the occlusive device and is applied to the skin on application. The occlusive device may be left in place on the skin for a period of time sufficient for the medicament to provide the desired enhancement on administration of the vaccine (e.g. from 30 seconds to 48 hours) or, alternatively, the vaccine may be given immediately.

[0181] In order to administer the vaccine the through-hole cover is removed (3) to expose the area of skin where the immunization is to be administered (4). At this point a topical analgesic may be applied to this exposed area, if desired. In some embodiments the lower surface of the through-hole cover may include a topical anesthetic, such that the anesthetic is applied to the immunization area on application of the device. Finally, the immunization is administered (for example, by microneedle, intradermal, subdermal, or intramuscular injection). Following administration of the vaccine the occlusive device may be kept in place for a period of time suitable to enhance the immune response to the injection (e.g. from 5 minutes to 48 hours). In some embodiments a portion of the occlusive device may be removed. For example, the frame may be removed while leaving the film layer in place on the patient. Alternatively, if the occlusive device has been applied for a period of time prior to administration of the vaccine sufficient to enhance its effect, the occlusive device may be removed essentially immediately following administration.

[0182] Vaccination by injection necessarily leaves a skin wound, which may bleed or become infected. Accordingly, some embodiments of the inventive concept incorporate or support the use of a wound dressing. FIG. 28A depicts an embodiment of the inventive concept in which the occlusive device includes a wound dressing (560). Other portions of the occlusive device may be similar to those of the occlusive device in FIG. 24, including a frame (520), a through- hole cover (510), and a film (540). In some embodiments one or more tabs (530) aid in holding the occlusive device in place while in use. The film (540) may act as a barrier layer during use of the occlusive device. The wound dressing (560) may be attached to or extend from an outside edge of the frame (520), film (540), or any portion of the occlusive device that is retained on the skin during use. FIG. 28B provides an expanded view of the device of FIG. 28 A. As shown the wound dressing (560) may include a dressing protective film (565) that is removed prior to application of the dressing. The wound dressing (560) may include an adhesive layer that is covered by the dressing protective film (565) during storage. As shown, the through-hole cover (510) is positioned to occlude a through-hole produced by the superimposition of apertures in the frame (520), film (540), and backing (550) during storage. In the embodiment shown the wound dressing (560) is attached along one edge to an outer edge of the frame (520), which permits it to be deflected over the through-hole following immunization.

[0183] As noted above, occlusive devices of the inventive concept may include one or more adhesive layers, strips, or patches that aid in positioning and use of the device. Such adhesive layers, strips, or patches may be covered by removable films that provide protection during storage and prior to use. FIG. 29 shows and example of the positioning of such adhesive portions in an occlusive device as shown in FIGs. 28A and 28B. The upper panel shows a view of the "front" (i.e. oriented away from the skin during use) of the occlusive device, where removal of a peelable film exposes a layer of adhesive used to secure the wound dressing (610). The lower panel shows a view of the "back" (i.e. oriented towards the skin during use) of the occlusive device, where removal of a peelable film exposes a layer of adhesive (620) that aids in fixing the occlusive device to the skin surface.

[0184] FIG. 30 depicts a method for using an occlusive device of the inventive concept that incorporates a wound dressing (such as the occlusive device shown in FIGs. 28A and 28B). From left to right, a peelable film is removed from the back of the occlusive device (1), which is then affixed to the skin such that the through-hole is oriented over the desired immunization site (2). As noted above, a medicament that enhances the immune response to the vaccine to be administered may be placed on the skin prior to application of the occlusive device. Alternatively such a medicament may be incorporated into the occlusive device such that it is applied to the skin on placement. In some embodiments the occlusive device may be left in place for a period of time in order for the medicament to take effect, as noted above. In order to administer the vaccine the through-hole cover is removed (3), and a topical anesthetic may optionally be applied to the vaccination site (4). As noted above, in some embodiments a topical anesthetic may be provided on the skin-facing surface of the through-hole cover. Following removal of the through-hole cover the vaccine may be injected (5). Following injection the wound dressing may be prepared for use by removal of a film protecting the wound-facing surface (6). The wound dressing may then be placed over the wound produced by vaccination, for example by folding over at or near an area where it is joined to the bulk of the device (7, 8). An adhesive layer applied to at least a portion of the skin-facing surface of the wound dressing may serve to keep it in place until the device is removed (for example after a period of time sufficient for the medicament to have the vaccination-enhancing effect, as described above).

[0185] FIG. 31A depicts an alternative, simplified occlusive device of the inventive concept. Such a simplified occlusive device is suitable for use with a separate wound dressing or bandage, and may be manufactured at low cost. As shown the occlusive device includes a frame (820) that may support one or more tabs (830), and also includes a film (840) and backing (not shown in this view). The film (840) may act as a barrier layer during use of the occlusive device. Alignment of apertures in these layers provides a through-hole (860). FIG. 3 IB provides an expanded view of the simplified occlusive device, and shows the arrangement of the frame (820), film (840), and backing (850) portions. As noted above, such an occlusive device may incorporate a medicament that enhances immune response to a vaccine composition that is positioned so that it is brought into contact with the skin during use (for example, on a skin facing surface of the backing or film). In other embodiments such a medicament is provided from a separate source prior to application of the occlusive device.

[0186] FIG. 31C illustrates a method for using a simplified occlusive device such as that depicted in FIGs. 31 A and 3 IB. From left to right, a protective film is removed from the device (1) and the occlusive device is oriented over and affixed to the skin such that the through-hole lies over the desired vaccination site (2). A topical anesthetic may be applied to at least a portion of the area of skin exposed by the through-hole prior to vaccination (3). As noted above, a medicament that enhances the immune response to vaccination is applied to the skin either on application of the occlusive device (e.g. where the medicament is provided with the occlusive device) or prior to application of the occlusive device. The occlusive device may then be kept in place for a period of time that permits the medicament to enter tissues at or near the vaccination site. Alternatively, vaccination may be performed essentially immediately and the occlusive device maintained in place afterwards (as described above). Vaccination may be accomplished by injection (4).

[0187] Additional optional steps are shown in FIG. 3 ID, where following injection (4) a wound dressing (870) is applied to the wound created by injection. Such a wound dressing may, advantageously, aid in holding the occlusive device in place following vaccination. As noted above, in some embodiments a portion of the occlusive device (e.g. the frame) may be removed during use while maintaining a remaining portion (e.g. the film) in contact with the skin. [0188] As noted above, in some embodiments a topical medicament is provided as part of the occlusive device that acts to enhance the immune reaction to the vaccine composition. In other embodiments the medicament is provided as a separate portion. In such embodiments it is advantageous to be able to dispense a predetermined or desired portion or dose of the medicament rather than rely on estimation of the amount applied by a health care professional. FIG. 32 depicts an example of such a measuring or portioning device. Such a measuring device may have a laminar construction. As shown the measuring device has a base (930) with a reservoir (940), which may be produced by indenting a portion of the base. Alternatively the reservoir (940) may be a distinct feature that is joined to the base (930), for example by gluing, melting, or welding. In some embodiments the reservoir may serve as part of a repository for a portion of topical medicament (950), which may reside in the measuring device until use. In other embodiments the reservoir provides a defined volume into which a portion of medicament is introduced prior to use. The base (930) may be made of any suitable material, such as a metal foil or polymer sheet. The base (930) may be joined to a plate (920), which provides structural support. The plate (920) includes a plate aperture (925) that is aligned with the reservoir (940). The reservoir (940) is sealed by a removable film (910), which may be removably attached to the plate (920). The removable film (910), plate (920), and base (930) may be joined by any suitable method, including the use of an adhesive, melting, welding, and fixing devices.

[0189] FIG. 33 depicts a method of using a measuring device as shown in FIG. 32 with a separate supply of a topical medicament. From left to right, the removable film is peeled away to expose the reservoir (1). A dispenser (960) of the topical medicament is used to place a portion of the medicament in the reservoir (2), which provides a defined volume that represents a desired unit dose of the topical medicament. It should be appreciated that the reservoir may have any suitable shape, including a hemisphere, ovoid, or polygonal volume. The volumes of such shapes are readily calculable from known geometric formulae. To dispense the topical medicament a healthcare professional may exert pressure on a protruding portion of the reservoir until it everts (3). The exposed portion of topical medicament may then be applied to the skin surface (4). Flat surfaces of the measuring device may be utilized to spread the topical medicament and avoid contamination of the healthcare professional's hand.

[0190] In some embodiments, a measuring device may be used independently of an occlusive device of the inventive concept. As shown in FIG. 33B, the reservoir portion of a measuring device may be filled with a topical medicament (I) in order to provide a desired unit dose. This unit dose may be exposed for application by everting the reservoir portion of the measuring device (II). The exposed topical medicament is then applied to the skin surface at or near the intended vaccination site (III) and distributed over the skin (IV). This provides a treated vaccination site (V), which may be protected by the application of a dressing or film (VI) to maintain the topical medicament at the vaccination site for the desired period of time (for example, up to 48 hours). In some vaccination protocols this period of time may be prior to, following, or both prior to and following application of the vaccine to the patient.

[0191] In some embodiments an occlusive device of the inventive concept as described above may be provided as part of a kit. Such a kit may include instructions for use, and may also include a measuring device and/or a supply of topical medicament. In some embodiments a kit may include two or more devices and/or measuring devices. Kit packaging may include indicia suitable for product identification and/or tracking (for example, 1 and 2 dimensional bar codes). Such packaging may also include a memory device (such as an RFID chip) that may store and transmit information related to the package and/or its contents, such as package contents, date of manufacture, expiration date, and so on. In some embodiments packaging for devices of the inventive concept may include indicators that provide indications of exposure to water or moisture, extreme temperatures, and other conditions that may adversely impact contents of the package. FIG. 34A shows a photograph of such a kit, with instructions for use of a simple version of the occlusive device. FIG. 34B shows a photograph of a kit with instructions for use of an occlusive device and of a measuring device.

[0192] In a typical vaccination method of the inventive concept, when vaccination is performed using an occlusive device as described above a protective layer is initially remove from the skin- facing surface of the occlusive device. In some embodiments the exposed surface may include a topical preparation of an immunization enhancing pharmaceutical that is deposited during manufacturing, and is exposed by removal of then protective layer. In other embodiments a clinician may apply a topical preparation of an immunization enhancing pharmaceutical following removal of the protective layer. For example, a suitable amount of 5% imiquimod cream (e.g. one containing 12.5 mg of imiquimod in 250 mg of cream) may be evenly spread on the skin facing surface of the occlusive device. Afterwards the occlusive device is placed on the skin of the patient where a vaccine will be administered. In another embodiment, imiquimod cream may be applied directly to the skin of a person to be vaccinated. After a suitable period of time (for example, about 5 minutes) the through-hole cover of the occlusive device is removed. The skin thus exposed may be disinfected (for example, using 75% alcohol) and then the influenza vaccine may administered to the patient intradermally through the exposed opening. In some embodiments a skin facing surface of through-hole cover may include a topical preparation of a local anesthetic, such as lidocaine. Alternatively, as an optional step before intradermal injection, local anesthetic may be applied to the skin at the vaccination site either by injection or pressure gun to reduce pain.

[0193] The skin facing surface area of the occlusive device may provide a consistent amount of immunization enhancing pharmaceutical compound, which is evenly and consistently provided to patient skin in each treatment. Use of an occlusive device of the inventive concept simplifies disinfection of the skin at the injection site; as a result the time required for vaccine administration may be shortened, thereby improving the efficiency of the vaccination program. This method may also allow the vaccination enhancing pharmaceuticals to remain on the skin for a defined period of time. Shown below are examples in how a vaccination enhancing pharmaceutical such as imiquimod may be used with the device to enhance the effectiveness of influenza and other vaccinations. [0194] In vaccination using a 2 cm x 2 cm sized occlusive device a protective layer on the skin facing surface of the occlusive device is first removed. Approximately 0.2 mL of 5% imiquimod cream (containing 12.5 mg of imiquimod in 250 mg cream or gel) is applied evenly on to the exposed surface of the occlusive device. The occlusive device is then placed on the arm of the patient, with the vertical axis being aligned with the arm. After a suitable period of time (for example, about 5 minutes), the through-hole cover is removed. The skin exposed is then disinfected and the influenza vaccine is administered intradermally via the exposed through-hole.

[0195] As shown above, in some embodiments the occlusive device includes a 'wing' protrusion or attachment that incorporates an elastoplast-like wound dressing or bandage to facilitate the vaccination process. The vaccination procedure as described above may be used to perform vaccination. After the intradermal injection is completed and a 'balloon' or bleb of inoculant is formed under skin has been absorbed, a protective layer may be removed to expose a bandage portion that extends from the main body of the device. This bandage 'wing' may be folded over the main body of the device to cover and protect the injection site.

[0196] The example above is for illustrative only and is not restrictive. Many variations of embodiments may become apparent to those skilled in the art upon review of the disclosure. The scope embodiments should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the pending claims along with their full scope or equivalents.

[0197] As used herein, the term “vaccine” or “vaccine formulation” refers to a composition that stimulates an immune response to a particular antigen or antigens (i.e., influenza virus). Thus, a vaccine refers to a composition that is administered to a subject with the goal of establishing an immune response and/or immune memory to a particular influenza virus. It is also contemplated that the vaccine compositions may comprise other substances designed to increase the ability of the vaccine to generate an immune response.

[0198] The term “pharmaceutically acceptable,” as used herein with regard to compositions and formulations, means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals and/or in humans.

[0199] The term “carrier” refers to a diluent, excipient, and/or vehicle with which the compositions described herein are administered. Such pharmaceutical carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions may also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include, but are not limited to, starch, glucose, sucrose, gelatin, lactose, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, glycerol, propylene, glycol, water, ethanol and the like. The compositions and formulations described herein may also contain wetting or emulsifying agents or suspending/diluting agents, or pH buffering agents, or agents for modifying or maintaining the rate of release of the composition. Formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, sodium saccharine, starch, magnesium stearate, cellulose, magnesium carbonate, etc. Such compositions and vaccines will contain an effective amount of the protein together with a suitable amount of carrier so as to provide the proper form to the patient based on the mode of administration to be used.

[0200] If for intravenous administration, the vaccines and compositions may be packaged in solutions of sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent. The components of the composition are supplied either separately or mixed together in unit dosage form. If the composition is to be administered by infusion, it may 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 or saline may be provided so that the ingredients may be mixed prior to injection. The vaccines and compositions may also be administered to the subject intranasally, intratracheally, orally, intradermally, intramuscularly, intraperitoneally, or subcutaneously.

[0201] One or more features from any embodiment may be combined with one or more features of any other embodiment without departing from the scope embodiments. A recitation of "a", "an" or "the" is intended to mean "one or more" unless specifically indicated to the contrary. Recitation of "and/or" is intended to represent the most inclusive sense of the term unless specifically indicated to the contrary.

[0202] While the present disclosure may be embodied in many different forms, the drawings and discussion are presented with the understanding that the present disclosure is an exemplification of the principles of one or more inventions and is not intended to limit any one embodiments to the embodiments illustrated.

[0203] Further advantages and modifications of the above described system and method may readily occur to those skilled in the art.

[0204] The disclosure, in its broader aspects, is therefore not limited to the specific details, representative system and methods, and illustrative examples shown and described above. Various modifications and variations may be made to the above specification without departing from the scope or spirit of the present disclosure, and it is intended that the present disclosure covers all such modifications and variations provided they come within the scope of the following claims and their equivalents.