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Cross-Reference to Related Applications

This Patent Appl ication claims priority from Italian Patent Application No . 102022000005633 filed on March 22 , 2022 , the entire disclosure of which is incorporated herein by reference .

Technical Field

The present invention relates to peptides for the preparation of vaccines against Sars-CoV-2 and for diagnosing a SARS-CoV-2 infection .

Coronavirus disease 2019 ( COVID- 19 ) is caused by a respiratory tract infection caused by the coronavirus , SARS- CoV-2 , first described in Wuhan, China, in December 2019 .

Background

Based on the data provided daily by the World Health Organi zation, there are now more than 230 million confirmed cases with almost 5 million deaths . Most people with COVID- 19 show only mild symptoms , however, about 15% develop a serious illness that requires hospitali zation and respiratory support and 5- 10% require admission to an intensive care unit . Older age and comorbidities constitute risk factors associated with an increased likelihood of lethality .

To date , vaccination remains the only ef fective protection against SARS-CoV-2 infection .

The study of the immune response to SARS-CoV-2 aimed at assessing the status of immunological memory in patients and convalescents and the ef fectiveness of vaccination in healthy subj ects has become of primary importance to identi fy the best prophylaxis and therapy strategies . Initially, attention was paid to assessing the serum concentration of neutrali zing antibodies against the virus and, for the vaccinated people , speci fically against the Spike protein . Subsequently, many studies have demonstrated the central importance of the cell-mediated immunity for an ef fective immune response against SARS-CoV-2 infection .

Two precise indications have emerged from the latter results : on the one hand, the need to develop immunological tests adapted to assess the consistency and the ef fector capacity of the T-lymphocyte mediated response , and on the other hand, attention, in the formulation of new vaccines , to their capacity to evoke an ef fective response by the T lymphocytes .

The current immunological tests proposed for the study of the T response are mainly based on the elispot or ELISA method for the evaluation of I FN-y production . The stimulus used is given by 2 or more pools of peptides belonging to the Spike protein necessary to cover the entire length and stimulate the T lymphocytes of all subj ects . Some research groups have developed cytof luorimetric tests that use the same stimulus and that also evaluate other cytokines in addition to I FN-y .

The most common vaccines currently on the market in Western countries are divided into mRNA vaccines and viral vector vaccines . The former above all have showed a more than good ef ficacy in inducing a protective immunity against SARS-CoV-2 infection, but a few months after the booster shot , there is an inevitable decline in the level of neutrali zing Ab against the Spike protein and the cell- mediated response therefore assumes a central importance .

The need is therefore felt in the art for alternative diagnostic approaches , in particular aimed at the development of as says that allow to evaluate the T-mediated response , in particular by CD8 ⁺ T lymphocytes . The need for new vaccines usable against SARS-CoV-2 is also felt .

Summary

Therefore , aim o f the present invention is to provide new peptides capable of being used for the preparation of new vaccines against SARS-CoV-2 and diagnostic tests to evaluate the T-mediated response , in particular by T lymphocytes CD8 ⁺ .

This aim is achieved by a peptide according to claim 1 , a nucleic acid according to claim 4 , a recombinant vector according to claim 5 , a host cell according to claim 6 , a composition according to claim 7 and their use according to claim 8 .

Brief Description of the Drawings

The present invention will now be described in detail with reference to the figures of the accompanying drawings , wherein :

Figure 1 shows A) gating strategy for the HLA-E molecule stabili zation assay . B ) Histograms representative of the mean fluorescence intensity of the HLA-E molecule .

Figure 2 shows the results of the stabili zation test of the HLA-E/peptide complex : The index was calculated based on mean fluorescence intensity (MFI ) values . The table reports the medians of the index for each of the peptides tested (N=3 ) the y-axis corresponds to the median values of the stabili zation tests .

Figure 3 shows the frequency of CD8 + T lymphocytes producing di f ferent cytokines after the stimulus with SARS peptide C0V2 : the first column represents the median frequency of CD8 ⁺ T lymphocytes , the second column represents the frequency of total CD8+ T lymphocytes , each row represents a di f ferent cytokine A: I FN-y B : TNF-a, C : IL- 10 D : IL-2 .

Description of Embodiments

The term "peptide" is used herein to designate a series of amino acid residues , typically linked together by peptide bonds between the alpha-amino and carbonyl groups of adjacent amino acids. The peptides are preferably between 7 and 14 amino acids in length, more preferably between 9 and 14 even more preferably between 11 and 14.

The term "peptide" also comprises "oligopeptide". The term "oligopeptide" is used herein to designate a series of amino acid residues, typically linked together by peptide bonds between the alpha-amino and carbonyl groups of adjacent amino acids. The length of the oligopeptide is critical to the invention, as long as the correct epitope or epitopes are maintained within it.

According to a first aspect of the invention, there is provided an epitope peptide of CD8 + cells from SARS-CoV-2, characterized in that said peptide has a maximum length of 14 amino acids and comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9.

Preferably, the epitope peptide of CD8 ⁺ cells from SARS- CoV-2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17 and SEQ ID NO. 18.

More preferably the epitope peptide of CD8 ⁺ cell from SARS-CoV-2 is an amino acid sequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17 and SEQ ID NO. 18.

Even more preferably the peptide is selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9.

The peptides of the invention with immunogenic potential derive from different SARS-CoV-2 virus proteins and are capable of binding the HLA-E molecule.

These peptides can be presented by the extremely poorly polymorphic MHO lb HLA-E molecule, as opposed to classical HLA class I molecules. To date, only two alleles expressed by the world population are known, the molecule HLA-E* 01:01 and the molecule HLA-E* 01:03, which differ by a single amino acid located outside the binding pocket for the antigenic peptides .

Therefore, since the HLA-E molecule has an invariant binding pocket, the immunogenic action of these peptides can induce the activation of CD8 lymphocytes of any individual, regardless of the expression of the classical HLA molecules.

By binding the HLA-E molecule and having an immunogenic potential, they then directly induce a cell-mediated immune response by activating the restricted CD8 ⁺ HLA-E T lymphocytes , according to a TCR-dependent mechanism . The CD8 ⁺ T lymphocytes thus activated perform a cytotoxic ef fector function and produce a cytokine pattern that can trigger a Thl-type response , which, as is known, is ef fective against viruses .

The capacity to directly activate the cellular compartment of the adaptive immunity makes these peptides very useful in inducing a protective immune response in immunodeficient , innate or acquired subj ects and are therefore usable as a subunit of a vaccine .

In addition, these peptides can also be used in the diagnostic field for the development of immunological tests aimed at evaluating the ef fectiveness of the cell-mediated immune response against SARS-CoV-2 infection .

Advantageously, moreover, compared to the current tests , the use of one or more peptides of the invention that bind the HLA-E molecule allows to reduce production costs without compromis ing the possibility of using the tests for the entire population thanks to the low polymorphism of HLA- E to which these peptides bind .

According to a second aspect of the present invention, there is provided a nucleic acid encoding an epitope peptide of CD8 + cells from SARS-CoV-2 of any one of SEQ ID NO . 1- 18 .

According to a third aspect of the present invention, there is provided a recombinant vector comprising a nucleic acid sequence as described above .

According to a fourth aspect of the present invention, there is provided a host cell comprising a nucleic acid sequence or into which a recombinant vector as described above has been introduced .

According to a fi fth aspect of the present invention, there is provided a vaccine composition or immunogenic composition comprising at least one peptide , at least one nucleic acid sequence or at least one recombinant vector as described above and at least one pharmacologically acceptable excipient .

According to a sixth aspect of the present invention, there are provided the peptides or the nucleic acid sequences as described above for use for immuni zing a subj ect against SARS-CoV-2 or for use for the diagnosis of SARS-CoV-2 infection .

Further characteristics of the present invention will become apparent from the following description of some merely illustrative and non-limiting examples .

Examples

EXAMPLE 1

Initially, a SARS-CoV-2 peptide binding prediction study was performed in order to identi fy peptides of variable length between 11 and 14 amino acids capable of binding HLA- E . By using a combination of di f ferent bioinf ormatic methods , 9 potential peptides from the full genome of the 2019- nCoV/USA-WAl-A12/2020 (MT020880) virus capable of binding the HLA-E molecule were identified.

In particular such peptides, the proteins from which they derive, as well as the binding affinity value with the HLA-E molecule are shown in Table 1.

Table 1

By using the Immune Epitope Database Analysis Resource (IEDB) and by setting the homology level at 80% (Blast-80%) , the sequence homology of the peptides identified with other epitopes not derived from SARS-CoV-2 was verified. The binding affinity of the identified peptides was then tested by bioinf ormatic studies. From the obtained binding prediction models, all generated peptides can be housed in the binding pocket of HLA-E*01:01 F.

Subsequently, the HLA-E/peptide binding affinity was verified in vitro. An RMA-S cell line (accession no. CVCL_2180) (TAP2 mutant Rauscher Murine Leukemiavirus induced T cell lymphoma) transfected to express the molecule HLA-E*01:01 F was used for this test. The cells, cultured at 37 °C with 5% of CO2, were subsequently incubated with the test peptides at 26°C and 37°C for 3 and 24 hours in Iscove medium supplemented with 1% glutamine, 20 mM HEPES, 100 U/mL penicillin, 100 pg/mL streptomycin. The bond of the peptides to the HLA-E molecule stabilizes the expression of the same on the surface of the cell; therefore, by using an antihuman HLA-E mAbPE it was possible, by cytof luorimetry, to verify the bond between the peptides and the HLA-E molecule. The cells were acquired using a BD FACSAria™ cytof luorimeter and analysed using FlowJo software (BD bioscence) (Fig 1) . The results were expressed as geometric mean of the fluorescent intensity (MFI) and the binding index was calculated as follows:

Expression Index = MFI (37°C with peptide- 37°C without peptide) / MFI (26°C with peptide- 26°C without peptide)

Based on the data obtained from the in silico affinity tests, the cut-off was set to 0.1 EMI.

As positive and negative controls, use was made of a nonamer peptide (VMAPRTLIL - SEQ ID NO. 19) derived from the HLA-C polypeptide chain leader sequence and a WNSNNLDSKVGG peptide (SEQ ID NO. 20) that does not bind to the HLA-E binding pocket. The tests performed at 24 hours yielded an expression of the HLA-E molecule calculated as mean HLA-E expression index ± SD for the positive and negative control of 1.323 ± 0.574 and 0.02 ± 0.01, respectively. With regard to the values obtained for the peptides of the invention, the data on the stabilization of the HLA-E molecule on the surface of the RMAS, these are summarized in Tables 2 and 3.

Table 2

Table 3

From the results obtained it can be observed that the peptides of the invention are able to stabili ze the molecule HLA-E E* 01 : 01 on the surface of RMAS cells .

EXAMPLE 2 Functional tests were performed to assess the immunogenicity of SARS-CoV-2 peptides and thus their capacity to induce an ex vivo response in the CD8 ⁺ T lymphocytes . PBMC cells , obtained by ficoll gradient separation, from donors cured of COVID 19 (negative for the COVID- 19 PCR test ) and hyperimmune plasma donors were used for these experiments . The CD8 ⁺ T cells from these samples were tested to assess their capacity to respond to the stimulus given by the cells of the TAP2-def icient RMA-S-HLA- E +/+ line (HLA-E cannot be loaded with self peptides) incubated with the 9 different SARS-CoV-2 peptides (ID 10 to 18) , in terms of cytokine production such as IFN-y TNF-a, IL-10 and IL-2.

The preliminary results obtained show the presence of a CD8 ⁺ T population IFN-y+ and significant, although lower than IFN-y, TNF-a production was observed. Although at a lower frequency CD8 T lymphocytes producing both IL2 and IL10 were also observed (Fig. 3) .

Finally, based on the combined results of the carried out test of HLA-E/peptide bond stabilization and of the test of immunogenicity understood as the capacity to produce cytokines upon specific stimulation, five peptides of the invention (PP02, PP07, SP09, SA01 and 8A01) having the best immunogenicity characteristics were identified. These peptides are able to activate HLA-E-restricted CD8 ⁺ T lymphocytes through TCR-mediated specific recognition, the activation of these cells involves the production of cytokines, mainly of the Thl type, useful to induce an efficient immune response for the clearance of SARS COV2.