Novel peptide conjugate vaccines

Abstract

The present invention provides novel fusion polypeptides and compositions comprising the same. The fusion polypeptides are capable of triggering and / or enhancing an immune response against the peptide sequence of interest contained therein and are thus useful in vaccinating or treating a mammalian subject. Kits comprising the fusion polypeptides are also provided.

Description

Technical Field

[0001] The present invention provides a novel fusion polypeptide and a composition comprising the fusion polypeptide. The fusion polypeptide can trigger and / or enhance an immune response to a peptide sequence of interest contained therein, and can therefore be used for vaccination or treatment of mammalian subjects. A kit comprising the fusion polypeptide is also provided. Background Art

[0002] The rapid spread of infectious diseases and the increasing number of cancer diseases around the world indicate that the immune system of each individual is facing increasing challenges. In the case of infectious diseases, the immune system of mammals is triggered to fight non-mammalian (i.e. non-self) antigens. In contrast, in the case of cancer, a specific immune response against certain self-antigens needs to be triggered.

[0003] The poor immunogenicity of key disease-associated epitopes is one of the major drawbacks of protein-based vaccine development and efficacy against infectious diseases. Even for highly immunogenic disease-associated epitopes, boosting the immune response may be necessary to obtain a stronger humoral and / or cellular immune response. In both cases, boosting the immune response to non-self antigens is desirable.

[0004] In contrast, when protein-based therapeutics are used to treat cancer, they must elicit an immune response against tumor-specific self-antigens. Historically, it has been extremely difficult to generate humoral and / or cellular immune responses against such self-antigens. This difficulty stems from immune tolerance mechanisms that prevent antigen-driven expansion of B cells and / or T cells with self-specificity. For these reasons, the development of protein-based vaccines for the treatment of cancer is challenging. In this setting, an enhanced immune response to self-antigens is required.

[0005] There is a need for new compositions that can induce clinically relevant immune responses to self-antigens or non-self-antigens in mammals. Summary of the invention

[0006] The present inventors have studied the potential use of immunostimulatory peptide sequences to trigger and / or enhance immune responses to non-mammalian peptide sequences or mammalian peptide sequences. When the immune system encounters an antigen that is recognized as non-self (i.e., foreign), an immune response is usually initiated against the antigen. Some foreign antigens trigger a strong immune response, while others only trigger a weak immune response or even no immune response at all. In some cases, it may be necessary to further enhance the immune response to non-self antigens.

[0007] The inventors unexpectedly observed that when a mammalian subject is immunized with a fusion polypeptide comprising a non-mammalian antigen of interest and an immunostimulatory peptide, a faster, stronger and more balanced immune response to the antigen of interest can be obtained compared to immunization with the antigen of interest alone. Surprisingly, even when the non-self antigen of interest is an antigen (e.g., a viral antigen) that is expected to cause an immune response when used alone, an enhanced immune response is observed. During the development of protein-based treatment methods for highly contagious pathogens, this advantage is considered to be crucial. Therefore, the immunostimulatory peptides described herein can be advantageously used as part of a fusion polypeptide comprising a non-self antigen of interest to enhance the immune response to the non-self antigen.

[0008] The immunostimulatory peptides provided herein are also found to be surprisingly useful when linked to an autoantigen of interest. The inventors have unexpectedly discovered that by administering an "autoantigen" linked to a specific immunostimulatory peptide sequence as described herein, a clinically relevant immune response to the autoantigen can be triggered. This is particularly useful, for example, when the autoantigen is a disease-associated antigen (e.g., an autoantigen associated with cancer).

[0009] A variety of different immunostimulatory peptide sequences are described herein, such as SEQ ID NO:5 (also referred to herein as "CDP" or "chimeric designer peptide"); SEQ ID NO:6 (also referred to herein as "IDP1" or "iBoost designer partner 1"); SEQ ID NO:7 (also referred to herein as "IDP2" or "iBoost designer partner 2"); SEQ ID NO:8 (also referred to herein as "IDP3" or "iBoost designer partner 3"); SEQ ID NO:10 (also referred to herein as "TRXtrunc"); SEQ ID NO:11 (also referred to herein as "type 1 fimbriae protein, A"); and SEQ ID NO:12 (also referred to herein as "variant type 1 fimbriae protein, A").

[0010] The inventors considered a variety of different criteria when designing the immunostimulatory peptide sequences described herein, and they found that the ratio of bulky hydrophilic or charged amino acids to total amino acids in the immunostimulatory peptide sequence can reliably measure the efficacy of the immunostimulatory peptide sequence in stimulating an immune response to the attached antigen of interest. Other criteria they considered to further optimize the immunostimulatory peptide sequence itself include optimizing its length, isoelectric point (pI), immunogenicity (e.g., the presence of predicted T cell epitopes) and / or solubility. It is well known that some criteria are interrelated and by considering one, another may also change. For example, by increasing solubility, immunogenicity can also be increased.

[0011] The inventors have also considered these factors from the perspective of the entire fusion polypeptide (i.e., comprising the immunostimulatory peptide sequence described herein connected to the antigen sequence of interest). For example, low pI, high immunogenicity, optimal size of the fusion polypeptide, and / or high solubility may be desirable properties. Therefore, the selection of the immunostimulatory peptide sequence can be based on its expected impact on these characteristics of the entire fusion polypeptide. Based on these considerations, the best fusion polypeptide for treatment can be generated.

[0012] The inventors have found that when considering these criteria when designing an immunostimulatory peptide sequence (wherein the designed immunostimulatory peptide sequence meets at least some or all of these criteria), an immune response to almost any peptide sequence of interest (whether it is self, non-self, immunogenic or non-immunogenic) can be triggered and / or enhanced. While for some antigens of interest, it may be sufficient to consider only one of these criteria, but other antigens may need to consider two or more criteria simultaneously. Therefore, for some antigens, it is sufficient if the ratio of bulky hydrophilic or charged amino acids to total amino acids in the immunostimulatory peptide is appropriately set. On the contrary, for other antigens, it may be necessary to also reduce the overall pI of the fusion polypeptide, increase the immunogenicity of the antigen of interest, and / or increase the overall solubility of the fusion polypeptide.

[0013] In one aspect, the present invention provides a fusion polypeptide for use in vaccination or treatment of a mammalian subject, the fusion polypeptide comprising:

[0014] (i) a non-mammalian peptide sequence, wherein the peptide sequence is a sequence associated with the disease or condition for which the mammalian subject is being vaccinated or treated; and

[0015] (ii) immunostimulatory peptide sequences having a ratio of bulky hydrophilic or charged amino acids to total amino acids less than or equal to 0.55.

[0016] Suitably, the fusion polypeptide may have an isoelectric point (pi) of less than or equal to 6.

[0017] Suitably, the non-mammalian peptide sequence may be a viral peptide and the disease or condition may be a viral infection.

[0018] Suitably, the non-mammalian peptide sequence may be a mycobacterial or bacterial peptide and the disease or condition may be a mycobacterial or bacterial infection.

[0019] Suitably, the non-mammalian peptide sequence may be a yeast peptide and the disease or disorder may be a yeast infection.

[0020] Suitably, the non-mammalian peptide sequence may be a parasitic peptide and the disease or condition may be a parasitic infection.

[0021] Suitably, the non-mammalian peptide sequence may be a SARS-CoV-2 peptide and the disease or condition may be a SARS-CoV-2 infection.

[0022] Suitably, the SARS-CoV-2 peptide may be a SARS-CoV-2 receptor binding domain (RBD) peptide.

[0023] Suitably, the RBD peptide may comprise the amino acid sequence:

[0024] NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATV (SEQ ID NO: 1), or a conservative amino acid variant thereof.

[0025] Suitably, the SARS-CoV-2 peptide is an S protein peptide, optionally wherein:

[0026] (a) the S protein peptide is an S1 subdomain peptide, further optionally wherein the S1 subdomain peptide is an RBD peptide (such as a receptor binding motif (RBM) peptide), an SD1 peptide or an SD2 peptide, or a combination thereof;

[0027] (b) The S protein peptide is an S2 subdomain peptide, and further optionally, the S2 subdomain peptide is an S1 / S2 peptide, a FP+S2' peptide, or a HR1 peptide, or a combination thereof.

[0028] Suitably, the SARS-CoV-2 peptide comprises:

[0029] (a) the amino acid sequence of SEQ ID NO: 39, or a conservative amino acid variant thereof;

[0030] (b) the amino acid sequence of SEQ ID NQ:40, or a conservative amino acid variant thereof; or

[0031] (c) the amino acid sequence of SEQ ID NO: 41, or a conservative amino acid variant thereof,

[0032] Preferably, the immunostimulatory peptide comprises the amino acid sequence of SEQ ID NO:5.

[0033] Suitably, the non-mammalian peptide sequence may be an influenza virus peptide and the disease or condition may be influenza.

[0034] Suitably, the influenza virus peptide sequence is an H1 influenza virus peptide, an N1 influenza virus peptide, or an H1 / N1 influenza virus peptide.

[0035] Suitably, the influenza virus peptide comprises:

[0036] (a) the amino acid sequence of SEQ ID NO: 42, or a conservative amino acid variant thereof;

[0037] (b) the amino acid sequence of SEQ ID NO: 43, or a conservative amino acid variant thereof; or

[0038] (c) the amino acid sequence of SEQ ID NO: 44, or a conservative amino acid variant thereof,

[0039] Preferably, the immunostimulatory peptide comprises the amino acid sequence of SEQ ID NO:5.

[0040] Suitably, the immunostimulatory peptide sequence may be at least 40 amino acids and not more than 150 amino acids in length.

[0041] Suitably, the immunostimulatory peptide sequence may have an isoelectric point (pi) of less than or equal to 4.

[0042] Suitably, the immunostimulatory peptide sequence may comprise the amino acid sequence DDEDFVDEDDD (SEQ ID NO: 4) of the RRAB protein or a conservative amino acid variant thereof.

[0043] Suitably, the immunostimulatory peptide sequence may comprise at least one predicted T-cell epitope.

[0044] Suitably, the predicted T cell epitope may be selected from: a MSYB protein T cell epitope and / or a RRAB protein T cell epitope.

[0045] Suitably, the MSYB protein T cell epitope may be comprised within the amino acid sequence: NPGIDAEDANVQQFNAQKYVLQDGDIMWQV (SEQ ID NO: 2), and / or EGEFQLEPPLDTEEGRAAADE (SEQ ID NO: 3), or conservative amino acid variants thereof.

[0046] Suitably, the immunostimulatory peptide sequence may comprise the amino acid sequence:

[0047] a) DNNSLSQEVQNGSNHLENNQSQSNGGGSDSALSLSSKTAALAAATTVNDGSDGATSSAVG (SEQ ID NO: 5), or a conservative amino acid variant thereof; or

[0048] b) HHAHTMYATLEEAIDAAREEFLADNPGIDAEDANVQQFNAQKYVLQDGDIMWQVEFFADEGEEGEDDEDFVDEDDD (SEQ ID NO: 6), or a conservative amino acid variant thereof; or

[0049] c) HHAHNPGIDAEDANVQQFNAQKYVLQDGDIMWQVEGEFQLEPPLDTEEGRAAADEDDEDFVDEDDD (SEQ ID NO: 7), or a conservative amino acid variant thereof; or

[0050] d) HHAHNPGIDAEDANVQQFNAQKYVLQDGDIMWQVDDEDFVDEDDD (SEQ ID NO: 8), or a conservative amino acid variant thereof.

[0051] In one aspect, the present invention provides a fusion polypeptide for use in vaccination or treatment of a mammalian subject, the fusion polypeptide comprising:

[0052] (i) a non-mammalian peptide sequence, wherein the peptide sequence is a sequence associated with the disease or condition for which the mammalian subject is being vaccinated or treated; and

[0053] (ii) an immunostimulatory peptide sequence comprising the amino acid sequence:

[0054] a) DNNSLSQEVQNGSNHLENNQSQSNGGGSDSALSLSSKTAALAAATTVNDGSDGATSSAVG (SEQ ID NO: 5), or a conservative amino acid variant thereof; or

[0055] b) HHAHTMYATLEEAIDAAREEFLADNPGIDAEDANVQQFNAQKYVLQDGDIMWQVEFFADEGEEGEDDEDFVDEDDD (SEQ ID NO: 6), or a conservative amino acid variant thereof; or

[0056] c) HHAHNPGIDAEDANVQQFNAQKYVLQDGDIMWQVEGEFQLEPPLDTEEGRAAADEDDEDFVDEDDD (SEQ ID NO: 7), or a conservative amino acid variant thereof; or

[0057] d) HHAHNPGIDAEDANVQQFNAQKYVLQDGDIMWQVDDEDFVDEDDD (SEQ ID NO: 8), or a conservative amino acid variant thereof.

[0058] In another aspect, the present invention provides a fusion polypeptide comprising:

[0059] (i) a non-mammalian peptide sequence, wherein the peptide sequence is a sequence associated with the disease or condition for which the mammalian subject is being vaccinated or treated; and

[0060] (ii) immunostimulatory peptide sequences having a ratio of bulky hydrophilic or charged amino acids to total amino acids less than or equal to 0.55;

[0061] wherein the non-mammalian peptide sequence is linked to an immunostimulatory peptide sequence, and wherein the immunostimulatory peptide sequence comprises the amino acid sequence:

[0062] a) HHAHTMYATLEEAIDAAREEFLADNPGIDAEDANVQQFNAQKYVLQDGDIMWQVEFFADEGEEGEDDEDFVDEDDD (SEQ ID NO: 6), or a conservative amino acid variant thereof; or

[0063] b) HHAHNPGIDAEDANVQQFNAQKYVLQDGDIMWQVEGEFQLEPPLDTEEGRAAADEDDEDFVDEDDD (SEQ ID NO: 7), or a conservative amino acid variant thereof; or

[0064] c) HHAHNPGIDAEDANVQQFNAQKYVLQDGDIMWQVDDEDFVDEDDD (SEQ ID NO: 8), or a conservative amino acid variant thereof; or

[0065] d) DNNSLSQEVQNGSNHLENNQSQSNGGGSDSALSLSSKTAALAAATTVNDGSDGATSSAVG (SEQ ID NO: 5), or a conservative amino acid variant thereof.

[0066] In another aspect, the present invention provides a fusion polypeptide comprising:

[0067] (i) a non-mammalian peptide sequence, wherein the peptide sequence is a sequence associated with the disease or condition for which the mammalian subject is being vaccinated or treated; and

[0068] (ii) immunostimulatory peptide sequences;

[0069] wherein the non-mammalian peptide sequence is linked to the immunostimulatory peptide sequence, and wherein the immunostimulatory peptide sequence comprises the amino acid sequence:

[0070] a) HHAHTMYATLEEAIDAAREEFLADNPGIDAEDANVQQFNAQKYVLQDGDIMWQVEFFADEGEEGEDDEDFVDEDDD (SEQ ID NO: 6), or a conservative amino acid variant thereof; or

[0071] b) HHAHNPGIDAEDANVQQFNAQKYVLQDGDIMWQVEGEFQLEPPLDTEEGRAAADEDDEDFVDEDDD (SEQ ID NO: 7), or a conservative amino acid variant thereof; or

[0072] c) HHAHNPGIDAEDANVQQFNAQKYVLQDGDIMWQVDDEDFVDEDDD (SEQ ID NO: 8), or a conservative amino acid variant thereof; or

[0073] d) DNNSLSQEVQNGSNHLENNQSQSNGGGSDSALSLSSKTAALAAATTVNDGSDGATSSAVG (SEQ ID NO: 5), or a conservative amino acid variant thereof.

[0074] In another aspect, the present invention provides a fusion polypeptide comprising:

[0075] (i) a mammalian peptide sequence, wherein the peptide sequence is a sequence associated with a disease or condition for which the mammalian subject is being vaccinated or treated; and

[0076] (ii) immunostimulatory peptide sequences having a ratio of bulky hydrophilic or charged amino acids to total amino acids less than or equal to 0.55;

[0077] wherein the mammalian peptide sequence is linked to an immunostimulatory peptide sequence, and wherein the immunostimulatory peptide sequence comprises the amino acid sequence:

[0078] a) HHAHTMYATLEEAIDAAREEFLADNPGIDAEDANVQQFNAQKYVLQDGDIMWQVEFFADEGEEGEDDEDFVDEDDD (SEQ ID NO: 6), or a conservative amino acid variant thereof; or

[0079] b) HHAHNPGIDAEDANVQQFNAQKYVLQDGDIMWQVEGEFQLEPPLDTEEGRAAADEDDEDFVDEDDD (SEQ ID NO: 7), or a conservative amino acid variant thereof; or

[0080] c) HHAHNPGIDAEDANVQQFNAQKYVLQDGDIMWQVDDEDFVDEDDD (SEQ ID NO: 8), or a conservative amino acid variant thereof.

[0081] In another aspect, the present invention provides a fusion polypeptide comprising:

[0082] (i) a mammalian peptide sequence, wherein the peptide sequence is a sequence associated with a disease or condition for which the mammalian subject is being vaccinated or treated; and

[0083] (ii) immunostimulatory peptide sequences;

[0084] wherein the mammalian peptide sequence is linked to an immunostimulatory peptide sequence, and wherein the immunostimulatory peptide sequence comprises the amino acid sequence:

[0085] a) HHAHTMYATLEEAIDAAREEFLADNPGIDAEDANVQQFNAQKYVLQDGDIMWQVEFFADEGEEGEDDEDFVDEDDD (SEQ ID NO: 6), or a conservative amino acid variant thereof; or

[0086] b) HHAHNPGIDAEDANVQQFNAQKYVLQDGDIMWQVEGEFQLEPPLDTEEGRAAADEDDEDFVDEDDD (SEQ ID NO: 7), or a conservative amino acid variant thereof; or

[0087] c) HHAHNPGIDAEDANVQQFNAQKYVLQDGDIMWQVDDEDFVDEDDD (SEQ ID NO: 8), or a conservative amino acid variant thereof.

[0088] Suitably, the fusion polypeptide may have an isoelectric point (pi) of less than or equal to 6.

[0089] Suitably, the non-mammalian peptide sequence may be a viral peptide and the disease or condition may be a viral infection.

[0090] Suitably, the non-mammalian peptide sequence may be a mycobacterial or bacterial peptide and the disease or condition may be a mycobacterial or bacterial infection.

[0091] Suitably, the non-mammalian peptide sequence may be a yeast peptide and the disease or disorder may be a yeast infection.

[0092] Suitably, the non-mammalian peptide sequence may be a parasitic peptide and the disease or condition may be a parasitic infection.

[0093] Suitably, the mammalian peptide sequence may be associated with tumor angiogenesis.

[0094] Suitably, the mammalian peptide sequence may be vimentin (Vim), apelin, notum (dorsal plate) or timp1.

[0095] Suitably, the non-mammalian peptide sequence may be a SARS-CoV-2 peptide.

[0096] Suitably, the SARS-CoV-2 peptide may be a SARS-CoV-2 receptor binding domain (RBD) peptide.

[0097] Suitably, the RBD peptide may comprise the amino acid sequence:

[0098] NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATV (SEQ ID NO: 1), or a conservative amino acid variant thereof.

[0099] Suitably, the SARS-CoV-2 peptide is an S protein peptide, optionally wherein:

[0100] (a) the S protein peptide is an S1 subdomain peptide, further optionally, wherein the S1 subdomain peptide is an RBD peptide (such as a receptor binding motif (RBM) peptide), an SD1 peptide or an SD2 peptide, or a combination thereof;

[0101] (b) The S protein peptide is an S2 subdomain peptide, and further optionally, the S2 subdomain peptide is an S1 / S2 peptide, a FP+S2' peptide, or a HR1 peptide, or a combination thereof.

[0102] Suitably, the SARS-CoV-2 peptide comprises:

[0103] (a) the amino acid sequence of SEQ ID NO: 39, or a conservative amino acid variant thereof;

[0104] (b) the amino acid sequence of SEQ ID NQ:40, or a conservative amino acid variant thereof; or

[0105] (c) the amino acid sequence of SEQ ID NO: 41, or a conservative amino acid variant thereof.

[0106] Suitably, the non-mammalian peptide sequence is an influenza virus peptide sequence and the disease or condition is influenza.

[0107] Suitably, the influenza virus peptide sequence is an H1 influenza virus peptide, an N1 influenza virus peptide or an H1 / N1 influenza virus peptide.

[0108] Suitably, the influenza virus peptide comprises:

[0109] (a) the amino acid sequence of SEQ ID NO: 42, or a conservative amino acid variant thereof;

[0110] (b) the amino acid sequence of SEQ ID NO: 43, or a conservative amino acid variant thereof; or

[0111] (c) the amino acid sequence of SEQ ID NO: 44, or a conservative amino acid variant thereof.

[0112] Suitably, the immunostimulatory peptide sequence may be at least 40 amino acids and no more than 150 amino acids in length. Suitably, the immunostimulatory peptide sequence may be at least 40 amino acids and no more than 100 amino acids in length.

[0113] Suitably, the immunostimulatory peptide sequence may have an isoelectric point (pi) of less than or equal to 4.

[0114] In another aspect, the present invention provides a nucleic acid encoding a fusion polypeptide according to the present invention, optionally wherein the nucleic acid is an expression vector.

[0115] In another aspect, the present invention provides a pharmaceutical composition comprising a fusion polypeptide according to the present invention or a nucleic acid according to the present invention, wherein the composition further comprises a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

[0116] In another aspect, the present invention provides a fusion polypeptide according to the invention or a composition according to the invention for use as a medicament.

[0117] In another aspect, the present invention provides the use of a fusion polypeptide according to the present invention or a composition according to the present invention for vaccination or treatment of a subject.

[0118] In another aspect, the present invention provides a method for vaccinating or treating a subject, the method comprising administering to the subject a fusion polypeptide according to the present invention or a composition according to the present invention.

[0119] Various aspects of the invention are described in further detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0120] The embodiments of the present invention are further described below with reference to the accompanying drawings, wherein "CDP" corresponds to SEQ ID NO:5:

[0121] Figure 1 Conjugate vaccines targeting the RBD domain of SARS-CoV-2 are shown. (AC) Schematic representation of the RBD domain (A), chimeric designed peptide (CDP) (B), and the resulting CDP-RBD (28.9 kDa) and RBD (22.8 kDa) vaccine proteins (C). (B) The CDP consists of multiple highly immunogenic clusters derived from three different bacterial proteins (cell division protein ZapB, type 1 fimbriae protein TFP, and small heat shock protein IbpA). (C) Theoretical isoelectric point (pI) and predicted solubility fraction (Sol) of each vaccine protein. (D) Coomassie blue staining after SDS-PAGE (left) and anti-RBD Western blot (right) analysis showing RBD and CDP-RBD. (E) Size exclusion chromatogram (10 / 300GL Superdex 75, Cytivia) showing purified CDP-RBD (black arrow).

[0122] Figure 2 Protein purification and CDP-RBD sequence are shown. (A, B) Coomassie blue staining after SDS-PAGE analysis shows the eluted fractions of RBD (A) or CDP-RBD (B) during purification using Ni-NTA beads. The protein was eluted with four portions of 200mM imidazole and then with four portions of elution buffer (pH 4.5, 8M urea). (C) Amino acid sequence of CDP-RBD protein. Predicted secondary structure and B cell and T cell epitopes are shown.

[0123] Figure 3It is shown that the combined CDP-RBD vaccine induces a faster antibody response compared to the non-combined vaccine. (A) Timeline of mouse vaccination. Mice were immunized with 100 μg RBD or CDP-RBD on days 0 and 14. Serum was collected from all mice before vaccination (day 0) and on days 13, 21, 28 and 35 of the experiment (n=10 on days 13 and 21, n=5 on days 28 and 35). (B) Vaccine protein was mixed with Montanide ISA 720 and CpG 1826 oligonucleotide (MnC). (C) Anti-RBD total immunoglobulin (Ig) endpoint titers of two vaccine groups at days 13, 21, 28 and 35 of the experiment were evaluated by ELISA. The number of responding mice at each time point is provided. (D) Anti-RBD total immunoglobulin (Ig) titers on days 21 (left) and 35 (right). (E) Endpoint titer analysis of RBD-specific IgG subclasses (IgG1, IgG2a, IgG2b, IgG3) on day 21. The number of responding mice for each IgG subclass is provided. (F) Alternative virus neutralization assay. Circulating neutralizing antibodies in the serum of vaccinated mice inhibited the interaction between RBD and ACE-2 receptors. (G, H) Surface plasmon resonance (SPR) biosensor assay. Binding of mouse serum anti-RBD antibodies to commercially available RBD. Serum was diluted 1:100. Data are shown as geometric mean ± geometric SD (C, E) or mean ± SD (D, F, H). Statistical significance was determined by unpaired Mann-Whitney test or by Sidka multiple comparison test (F) after two-way ANOVA for each time point (C, H) or IgG subclass (E). (*p<0.05, **p<0.01).

[0124] Figure 4Antibody responses after vaccination with CDP-RBD are shown. (A) Anti-RBD total immunoglobulin (Ig) levels in both vaccine groups at days 0, 13, 21, and 35 of the experiment were assessed by ELISA. Sera were diluted 1:100. (B) Anti-RBD IgG subclass analysis at day 21. Mouse sera were diluted 1:100. (C) Anti-CDP total Ig levels at day 21. Mouse sera were diluted 1:100. (D) Anti-RBD total Ig endpoint titers at day 21 after co-immunization with RBD or CDP-RBD and Sepivac adjuvant. (E) Effects of different vaccine doses of CDP-RBD combined with MnC or Sep on RBD response. (F, G) Surface plasmon resonance (SPR) biosensor assay. Binding of anti-RBD antibodies to commercially available RBD in sera from mice immunized with RBD / Sep or CDP-RBD / Sep. Sera were diluted 1:100. Data are shown as mean ± SD (AD, G) or geometric mean ± geometric SD (E). Statistical significance was determined by two-way ANOVA followed by Sidka multiple comparison test (A) or unpaired Mann-Whitney test for each subclass (B), vaccine group (C), CDP-RBD protein dose (E) or time point (G). (ns; not significant, *p<0.05, ***p<0.001).

[0125] Figure 5It was shown that CDP-RBD vaccination triggered systemic T cell immunity and further improved CTL responses in mice compared with RBD. (A) Timeline and experimental procedures followed to detect splenic RBD-specific T cells in immunized mice. (B, D) Representative dot plots depicting IFNγ and TNFα expression in CD4+ T cells (B) and CD8+ T cells (D) in the presence of brefeldin A without stimulation (-) or after 5 hours of ex vivo stimulation with a mixture of SARS-CoV-2 peptides (+). (C, E) Quantification of the percentage (%) of RBD-reactive IFNγ+ (monofunctional) and IFNγ+TNFα+ (multifunctional) CD4+ (C) and CD8+ (E) T cells after vaccination with RBD / MnC or CDP-RBD / MnC regimens (n ​​= 5 mice per group). Each point represents the mean of two technical replicates. Error bars represent mean frequency ± SD. Statistical significance was determined by unpaired Student's t-test (ns; not significant, *p<0.05). (F) Heatmaps show the correlation between the different components of humoral and cellular immune responses induced in mice 21 days after immunization with RBD / MnC or CDP-RBD / MnC. The Y-axis corresponds to the percentage of antibody endpoint titers (total Ig, IgG1, IgG2a, IgG2b, IgG3) or RBD: ACE-2 binding inhibition (nAbs), while the X-axis corresponds to the percentage of RBD-specific T cells. Pearson correlation coefficient (r) values ​​were used to plot and estimate the correlation of the included features. (G, H) Representative scatter plots illustrate the correlation between the percentage of RBD-reactive CD4+ (G) or CD8+ (H) T cells and the corresponding total Ig endpoint titers. The regression line represents the linear regression of each vaccine group. Pearson correlation coefficient (r) and determination coefficient (r2) values ​​are provided for each scatter plot.

[0126] Figure 6 RBD-specific T cell responses induced after vaccination with RBD or CDP-RBD are shown. (A) Gating strategy applied during flow cytometric analysis. Gated CD4+ and CD8+ T cells were selected for further analysis. (B) Percentage (%) of IFNγ+, TNFα+, and IFNγ+TNFα+CD4+ or CD8+ T cells detected in the absence or presence of SARS-CoV-2 peptide mixture. Each point represents the mean of two technical replicates (n=5 mice per group).

[0127] Figure 7CDP-RBD was shown to elicit neutralizing antibodies and protect hamsters from SARS-CoV-2-mediated lung lesions. (A) Vaccination strategy for hamsters and schedule of SARS-CoV-2 challenge. Animals were immunized with 100 μg of CDP-RBD protein in combination with MnC as an adjuvant or with Tris-sucrose (control) on days 0 and 21. Serum was collected from all hamsters on days 0, 21, 42, 46, and 49 of the experiment. On day 42, all hamsters were challenged with SARS-CoV-2. Four days after infection (dpi), half of the animals in each group (4 of 8) were sacrificed, and on day 49 (7dpi), the remaining four animals were sacrificed. (B) Anti-RBD IgG levels on days 0, 21, 42, 46, and 49 of the experiment were assessed by ELISA. Sera were diluted 1:100. (C) Throat swabs were analyzed to measure the replication of competent SARS-CoV-2 in the first four dpi. (D) Weight progression after infection with SARS-CoV-2. On day 49, only the remaining four hamsters were weighed. (E) Alternative virus neutralization assay using hamster serum on days 0, 21, 42, 46, and 49 of the experiment. (F) Hematoxylin and eosin (HE) stained images of infected hamster lungs at seven dpi (left). The lungs of control hamsters showed strong infiltration of immune cells (asterisks). Quantification of lung lesions at four and seven dpi (right). Scale bar, 100 μm. Data are shown as mean ± SD. Statistical significance was determined at each time point by unpaired Mann-Whitney test (A) or two-way ANOVA followed by Sidak multiple comparison test (CF). (*p<0.05, **p<0.01, ****p<0.0001).

[0128] Figure 8 Antibody responses and viral loads after hamster vaccination are shown. (A) Anti-RBD IgG endpoint titers on days 21, 42, 46, and 49 of the experiment were assessed by ELISA. (B) Competent virus analysis in lung and nasal concha samples on day 46 of the experiment. (C) HE-stained lungs of control animals after SARS-CoV-2 infection. Hyperplasia of bronchial epithelial cells (BEC, double-headed arrow, left) and strong infiltration of immune cells (right) are visible. Scale bar: 100 μm (10x) or 25 μm (40x). Data are shown as geometric mean ± geometric SD (A) or mean ± SD (B). Statistical significance was determined by unpaired Mann-Whitney test for each time point (A) or organ (B).

[0129] Fig. 9IDP1 (SEQ ID NO: 6) fused to mouse vimentin (63 kDa) is shown. Most of IDP1-mVim eluted in the imidazole fractions (I1-I4). Buffer E fractions (E1-E4), supernatant after sonication of bacterial pellet (sup).

[0130] Fig.10 IDP2 (SEQ ID NO: 7) fused to mouse vimentin (62 kDa) is shown. Most of IDP2-mVim eluted in the imidazole fractions (I1-I4). Buffer E fractions (E1-E4), supernatant after sonication of bacterial pellet (sup).

[0131] Fig.11 IDP3 (SEQ ID NO: 8) fused to mouse vimentin (60 kDa) is shown. Most of IDP3-mVim eluted in the imidazole fractions (I1-I4). Buffer E fractions (E1-E4), supernatant after sonication of bacterial pellet (sup).

[0132] Fig.12 CDP (SEQ ID NO: 5) fused to mouse vimentin (61 kDa) is shown. CDP-mVim eluted in buffer E fractions (E1-E4). Pooled imidazole fractions (I), column wash (W), supernatant after sonication of bacterial pellet (sup).

[0133] Fig.13 TRXtr (SEQ ID NO: 10) fused to mouse vimentin (61 kDa) is shown. TRXtr-mVim eluted in buffer E fractions (E1-E4). Pooled imidazole fractions (I), supernatant after sonication of bacterial pellet (sup).

[0134] Fig.14 SDS-PAGE gel of eluted fractions during protein purification of each of TRXtr (SEQ ID NO: 10) and CDP (SEQ ID NO: 5) fused to mouse apelin is shown. TRXtr-apelin (16.2 kDa) eluted only in the buffer containing 100 mM EDTA (F1-F4), whereas when mouse apelin was bound to CDP (CDP-apelin (16.0 kDa)), the protein eluted in the buffer containing 200 mM imidazole.

[0135] Fig.15SDS-page gels of eluted fractions obtained during protein purification of CDP2.2 (also known as IDP2) (SEQ ID NO: 7) fused to mouse apelin (16.70 kDa) are shown. A) SDS-PAGE gel CDP2.2-apelin 16.7 kDa eluted mainly in I2 and I3. B) I2 of SDS-PAGE gel on protein blot. S (supernatant). Binding of mouse apelin to CDP2.2 (IDP2) significantly reduced the isoelectric point (pI) of the fusion protein construct and made it possible to elute the protein with 200 mM imidazole (I1-I4) ( Fig.15 A). The protein eluted mainly in the imidazole fraction, which means that imidazole competes with histidine binding and the elution is not dependent on low pH (buffer E) and therefore does not change the charge of the protein.

[0136] Fig.16 Purification of different mouse Timp1 constructs is shown. (A) Schematic diagram of the steps of protein production and purification. (B) SDS-PAGE of the pellet and supernatant of mouse Timp1 after sonication, and mouse Timp1 fused to each of TRXtr (SEQ ID NO: 10), CDP (SEQ ID NO: 5), CDP2.1 (also known as IDP1) (SEQ ID NO: 6), CDP 2.2 (also known as IDP2) (SEQ ID NO: 7), and CDP2.3 (also known as IDP3) (SEQ ID NO: 8), a thick band of the size of the fusion protein was found in the supernatant (white arrow). (C) Results of purification of 5 mL of supernatant using 200 μL Ni-agarose beads. TRXtr-Timp1 can only be eluted in buffer E by changing the charge of the protein, while the CDP-bound protein can be eluted in imidazole.

[0137] Fig.17 Shown is an SDS-page gel of eluted fractions during protein purification of CDP (SEQ ID NO: 5) fused to mouse Notum (62.0 kDa) eluted with 200 mM imidazole (I2 and I3).

[0138] Fig.18 An SDS-page gel of elution fractions during protein purification of TRXtr (SEQ ID NO: 10) fused to mouse Notum (62.3 kDa) is shown. TRXtr-Notum eluted in the imidazole and buffer E fractions.

[0139] Fig.19An SDS-page gel of elution fractions during protein purification of CDP 2.2 (SEQ ID NO: 7) fused to mouse Notum (62.9 kDa) is shown. CDP2.2-Notum was eluted mainly in the imidazole fraction.

[0140] Fig. 20 An SDS-page gel of elution fractions during protein purification of CDP 2.3 (SEQ ID NO: 8) fused to mouse Notum (60.5 kDa) is shown. CDP2.3-Notum was eluted mainly in the imidazole fraction.

[0141] Fig.21 An SDS-page gel of eluted fractions during protein purification of a CDP (SEQ ID NO: 5) fused to an influenza protein sequence is shown. Addition of CDP to the H1 / N1 sequence lowers the isoelectric point of the protein, and the protein can be eluted with imidazole (I1-I4).

[0142] Fig. 22 An SDS-page gel of elution fractions during protein purification of a control influenza construct is shown. Influenza constructs not bound to CDP could only be eluted with buffer E (E1-E4).

[0143] Fig.23 A schematic diagram of the immunization studies that have been performed in mice is shown. Left: SDS-PAGE gel showing different influenza constructs. Balb / c mice were vaccinated with 30ug of protein mixed with Sepivac adjuvant. A prime vaccination was performed on day 0 and a booster vaccination was performed on day 14. Blood samples were collected on day 0, day 7, day 21, day 28 and at the end of the experiment on day 35.

[0144] Fig.24 The levels of anti-H1 and anti-N1 antibodies in the sera of vaccinated mice are shown. CDP binding to H1, N1, or H1N1 can enhance the anti-H1 ( Fig.24 A, circle), N1( Fig.24 B, diamonds) and for H1N1 ( Fig.24 The total amount of antibodies induced after vaccination with the CDP-H1N1 protein was equal to the amount of antibodies induced with the individual constructs (CDP-H1, CDP-N1) (bottom left). Fig.25 ).

[0145] Fig.25 The levels of total anti-H1N1 antibodies in the sera of vaccinated mice are shown.

[0146] Fig.26Antibody titers induced with different influenza constructs are shown. Binding to CDP enhanced anti-H1, N1 and H1N1 antibody responses in mice.

[0147] Fig. 27 Shown are (A) a schematic diagram of the SARS-CoV2 virus, (B) a schematic diagram of the various functional domains of SARS-CoV2, namely, RBM, SD1, SD2, S1 / S2 cleavage site, FP and S2′ protease cleavage site (FP+S2′), and HR1, (C) an overview of the experiment involving BALB / c mice subcutaneously immunized with 100 μg of CDP-complete vaccine protein mixed with Sepivac 1:1 (v / v), oil-in-water adjuvant injections on days 1 and 14, and blood samples collected at time points (0, 7, 13, 21, 28, and 35 days after immunization), and (D) the corresponding antibody titers at each time point obtained using ELISA.

[0148] Fig.28 Shown are (A) colloidal Coomassie blue-stained SDS-PAGE gels of CDP-complete vaccine, CDP-conjugate vaccine, and CDP-fusion vaccine expressed in E. coli BL21 cells, and the OD levels (655 nm) of (B) anti-S1 / S2 antibodies and (C) anti-RMB, anti-SD1, anti-SD2, anti-S1 / S2, anti-FP, and anti-HR1 total Ig antibodies for each vaccine.

[0149] Fig.29 Shown (A) Spleens from mice fully immunized with CDP were isolated on day 35 and splenocytes were restimulated with RBM, SD1, SD2, S1 / S2, FP+S2', and HR1 synthetic peptides for 24 hours ex vivo. Unstimulated splenocytes treated with Brefeldin A served as negative controls. After stimulation in the presence of Brefeldin A for 5 hours, antigen-specific CD4 + and CD8 + (B) After stimulation with each peptide alone, a clear monofunctional (IFNγ + TNFα + ) and multifunctional (IFNγ + TNFα + )CD4 + T cell population. This indicates that vaccination with CDP-complete vaccine induces systemic T cell immunity and CD8 + T cell response.

[0150] Fig.30(A) Hamster weight monitored during the experiment is shown. Group 1: PBS control (circles), Group 2: CDP-complete vaccine (squares) (B) Hamster weight after SARS-CoV2 challenge (days 42 to 49). (C) Viral load in lung and nasal concha samples at D+3 after infection. Data are shown as mean ± SEM of bacterial load. Circles: PBS control, squares: CDP-complete vaccine. (D) Virus neutralization of hamster sera from D0 to D7 after the first vaccination, circles: PBS control (n=8), squares: CDP-complete vaccine (n=8). After SARS-CoV2 infection, animals immunized with CDP-complete vaccine produced neutralizing antibody responses earlier than the control group. DETAILED DESCRIPTION

[0151] The present invention provides a novel fusion polypeptide and a composition comprising the fusion polypeptide. The fusion polypeptide can trigger and / or enhance an immune response to a peptide sequence of interest contained therein, and can therefore be used for vaccination or treatment of mammalian subjects. A kit comprising the fusion polypeptide is also provided.

[0152] Vaccination and treatment of subjects

[0153] Provided herein is a fusion polypeptide for use in vaccination or treatment of a mammalian subject, the fusion polypeptide comprising:

[0154] (i) a non-mammalian peptide sequence, wherein the peptide sequence is a sequence associated with the disease or condition for which the mammalian subject is being vaccinated or treated; and

[0155] (ii) immunostimulatory peptide sequences having a ratio of bulky hydrophilic or charged amino acids to total amino acids less than or equal to 0.55.

[0156] The term "fusion polypeptide" as used herein refers to a polypeptide sequence comprising at least two parts / polypeptide parts, one of which is a sequence (antigen / epitope of interest) to which an immune response should be elicited / enhanced, and the other part (i.e., the immunostimulatory peptide sequence) is a sequence that triggers and / or enhances an immune response to the antigen / epitope of interest. The two parts are generally in their native folded conformation and are generally connected ("connected") by their respective carboxyl and amino termini.

[0157] The terms "polypeptide", "protein" and "peptide" encompass (poly)peptide analogs, which may have modifications that, for example, make them more stable (e.g. in vivo) or more permeable to cells (e.g. in vivo). Such modifications include, but are not limited to, N-terminal modifications, C-terminal modifications, peptide bond modifications, including but not limited to CH2-NH, CH2-S, CH2-S=O, O=C-NH, CH2-O, CH2-CH2, S=C-NH, CH=CH or CF=CH and / or backbone modifications. Methods for preparing peptide mimetic compounds are well known in the art and are described in detail, for example, in Quantitative Drug Design, CA Ramsden Gd., Chapter 17.2, F. Choplin, Pergamon Press (1992).

[0158] A peptide linker may be present between the two parts of the fusion polypeptide (ie, between the antigen / epitope of interest and the immunostimulatory peptide sequence).

[0159] As used herein, the term "linker peptide", "linker" or "peptide linker" refers to an amino acid sequence that connects or links at least two polypeptide parts. The linker can be a tandem repeat linker. The tandem repeat linker can be a tandem repeat of the amino acids glycine-serine. For example, the linker can have the amino acid sequence GS or GSGS, or GSGSGS (SEQ ID NO: 27).

[0160] In addition to the linker, the fusion polypeptide can further comprise an N-terminal or C-terminal tag, such as an N-terminal or C-terminal purification tag. For example, the purification tag can be a tag comprising the amino acid sequence HHAH or HHHHHH (His tag; SEQ ID NO: 26).

[0161] The fusion polypeptides described herein (comprising an immunostimulatory peptide sequence and an antigen / epitope of interest, optionally together with a linker peptide and / or an N-terminal or C-terminal tag) may have a predicted isoelectric point (pi) of less than or equal to 6.

[0162] The isoelectric point (pi) of a polypeptide is directly related to its solubility and directly affects the ability of a fusion polypeptide to elicit or enhance an immune response against the antigen of interest.

[0163] As used herein, "isoelectric point" (pI) is the pH of a solution at which the net charge of a protein becomes zero. When the solution pH is above the pI, the surface of the protein is primarily negatively charged. A protein is expected to be 50% soluble near its isoelectric point. The fusion polypeptides described herein have a pI below physiological pH (about 7.4), and since the overall charge of the protein is negative, its solubility in the body is increased, which is desirable when eliciting an immune response because precipitated / insoluble protein will not elicit an immune response. One explanation is that only particles of a certain size can be taken up by antigen presenting cells. Proteins with a pI above physiological pH do not dissolve because they are positively charged.

[0164] Methods for determining the predicted pi of a protein are well known in the art (eg, https: / / web.expasy.org / protparam / ).

[0165] In one example, the predicted pi of the fusion polypeptide described herein is less than or equal to 5.5 or less than or equal to 5.

[0166] In another example, the predicted pi of the fusion polypeptide described herein is less than or equal to 4.5, or less than or equal to 4.

[0167] By providing a fusion polypeptide as described herein, it is possible to induce an immune response to an antigen of interest, regardless of whether the antigen itself has high, medium, low or even no immunogenicity. Antigens with low or no immunogenicity will not activate the subject's immune system, or will only activate to a very small extent, which may not be sufficient to provide protection for the subject against the pathogen from which the antigen is derived. Therefore, using the fusion polypeptides described herein, the shortcomings in vaccine development and efficacy caused by the weak immune response of certain epitopes can be overcome. Therefore, using the fusion polypeptides described herein, the immunogenicity of an antigen of interest (e.g., a non-self antigen or a self antigen) can be enhanced.

[0168] The fusion polypeptide provided herein is used for administration to mammalian subjects. The fusion polypeptide can be used to induce or enhance an immune response to an antigen of interest in a mammalian subject, wherein the antigen of interest can be a "self antigen" or a "non-self antigen". Those skilled in the art can easily identify self antigens and non-self antigens according to the subject to which the fusion polypeptide is administered. For example, when the mammalian subject is human, "self antigen" corresponds to an antigen derived from / present in human protein. The term "self antigen" encompasses "neoantigens", which are self antigens but are recognized as foreign antigens by the immune system. Similarly, when the mammalian subject is human, "non-self antigen" corresponds to an antigen that is not derived from or not present in human protein. In another example, for any mammalian subject, a peptide sequence (non-mammalian peptide sequence) that is not derived from / not present in a mammal can be referred to as a "non-self antigen".

[0169] In some examples, the fusion polypeptide described herein comprises a non-mammalian peptide sequence, wherein the non-mammalian peptide sequence is a sequence associated with a disease or condition for which a mammalian subject is being vaccinated or treated. In this context, a non-mammalian peptide sequence may also be described as a peptide sequence comprising a non-mammalian antigen / epitope of interest. In other words, it may be described as a peptide sequence comprising a non-self antigen / epitope of interest. Alternatively, it may be described as a peptide sequence comprising an exogenous antigen / epitope of interest. The terms "non-self" and "exogenous" are used interchangeably herein.

[0170] The non-mammalian peptide sequence may be derived from a pathogen associated with a disease or condition. In this article, the term "associated with" refers to a pathogen, particularly a certain specific part of a pathogen, which is responsible for the onset, outbreak, exacerbation or recurrence of a disease that occurs when infected with the pathogen. This also includes secondary diseases based on infection with such pathogens.

[0171] In one example, the non-mammalian peptide sequence is a mycobacterium or bacterial peptide, and the disease or condition for which the mammalian subject is receiving vaccination or treatment is a mycobacterium or bacterial infection. In this example, the peptide can be any suitable peptide (antigen / epitope) present in the mycobacterium or bacteria for which the mammalian subject is receiving vaccination or treatment. In this context, the peptide can be a peptide from the mycobacterium or bacteria selected from the following: Mycobacterium tuberculosis (the pathogen of tuberculosis), Borrelia burgdorferi (the pathogen of Lyme disease), Bacillus anthracis (the pathogen of anthrax) and Clostridium tetani (the pathogen of tetanus). Suitable peptides are well known to those skilled in the art.

[0172] In another example, the non-mammalian peptide sequence is a yeast peptide, and the disease or condition for which the mammalian subject is being vaccinated or treated is a yeast infection. In this example, the peptide can be any suitable peptide (antigen / epitope) present in the yeast for which the mammalian subject is being vaccinated or treated. In this context, the peptide can be a peptide from Candida albicans. Suitable peptides are well known to those skilled in the art.

[0173] In a further example, the non-mammalian peptide sequence is a parasite peptide, and the disease or condition for which the mammalian subject is receiving vaccination or treatment is a parasite infection. In this example, the peptide can be any suitable peptide (antigen / epitope) present in the parasite for which the mammalian subject is receiving vaccination or treatment. In this context, the peptide can be a peptide from a parasite selected from the group consisting of Plasmodium (the causative agent of malaria), Leishmania (the causative agent of leishmaniasis), Schistosoma (the causative agent of schistosomiasis), hookworms and ticks. Suitable peptides are well known to those skilled in the art.

[0174] In one example, the non-mammalian peptide sequence is a viral peptide, and the disease or condition for which the mammalian subject is receiving vaccination or treatment is a viral infection. In this example, the viral peptide can be any suitable peptide (antigen / epitope) present in the virus for which the mammalian subject is receiving vaccination or treatment. In this context, the viral peptide can be a peptide from a virus selected from, but not limited to, coronavirus, influenza virus, human respiratory syncytial deficiency virus, dengue virus, human papillomavirus, herpes simplex virus, varicella-zoster virus, mumps virus, measles virus, rotavirus, norovirus, Zika virus, Ebola virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, and Epstein-Barr virus. Suitable peptides are well known to those skilled in the art, and include, for example, coronavirus spike protein (e.g., peptides from S1 or S2 subunits of the spike protein, such as peptides from receptor binding domain (RBD) or fusion domain), or influenza hemagglutinin or neuraminidase. In this example, when the viral infection is influenza virus, the influenza virus peptide can be an H1 influenza virus peptide, an N1 influenza virus peptide, or an H1 / N1 influenza virus peptide. For example, the influenza virus peptide can include the sequence of SEQ ID NO: 42 (H1), 43 (N1) or 44 (H1N1). The immunostimulatory peptide can include any immunostimulatory peptide disclosed herein, preferably CDP (SEQ ID NO: 5). Therefore, the fusion polypeptide can include the sequence of SEQ ID NO: 45 (CDP-H1), 46 (CDP-N1) or 47 (CDP-H1N1).

[0175] In one example, the non-mammalian peptide sequence is a SARS-CoV-2 peptide (antigen / epitope), and the disease or condition is a SARS-CoV-2 infection. For example, the SARS-CoV-2 peptide (antigen / epitope) can be a SARS-CoV-2 spike protein peptide (antigen / epitope). For example, the spike protein peptide can be an S1 subunit peptide or an S2 subunit peptide. In another example, the peptide can be a fusion domain peptide. In a further example, the SARS-CoV-2 peptide (antigen / epitope) can be a SARS-CoV-2 receptor binding domain (RBD) peptide (antigen / epitope), such as the RBD of the spike protein described in Wrapp et al., 2020. The RBD is responsible for cell entry and subsequent infection by binding to angiotensin converting enzyme 2 (ACE-2) expressed by host cells. The vast majority of neutralizing antibodies detected in Covid-19 patient sera are directed against the RBD. In this example, the SARS-CoV-2 peptide may include any one or more of the following: an RBM domain (amino acids 438-506 of the S1 subunit), an SD1 domain (amino acids 527-562 of the S1 subunit), an SD2 domain (amino acids 618-646 of the S1 subunit), an S1 / S2 domain (amino acids 674-696 of the S2 subunit), an FP+S2' domain (amino acids 786-848 of the S2 subunit), and an HR1 domain (amino acids 912-943 of the S2 subunit). The RBM domain may include the sequence of SEQ ID NO:69. The SD1 domain may include the sequence of SEQ ID NO:70. The SD2 domain may include the sequence of SEQ ID NO:71. The S1 / S2 domain may include the sequence of SEQ ID NO:72. The FP domain may include the sequence of SEQ ID NO:73. The HR1 domain may include the sequence of SEQ ID NO:74. In one example, the SARS-CoV2 peptide may comprise the sequence of SEQ ID NO:39, 40 or 41. The immunostimulatory peptide may be any immunostimulatory peptide disclosed herein, preferably CDP (SEQ ID NO:5). Thus, the fusion polypeptide may comprise the sequence of SEQ ID NO:63 (CDP-complete), SEQ ID NO:64 (CDP-binding) or SEQ ID NO:65 (CDP-fusion). The DNA sequence encoding the fusion polypeptide may, for example, comprise the sequence of SEQ ID NO:66 (CDP-complete), 67 (CDP-binding) or 68 (CDP-fusion).

[0176] It will be clear to those skilled in the art that a non-mammalian peptide sequence may include one or more antigens / epitopes. For example, a non-mammalian peptide sequence may contain two or more SARS-CoV-2 antigens / epitopes. Thus, when, for example, a non-mammalian peptide sequence is referred to as a SARS-CoV-2 peptide (antigen / epitope), this encompasses a non-mammalian peptide sequence comprising one or more (e.g., two or more) SARS-CoV-2 antigens / epitopes. The SARS-CoV-2 antigens / epitopes within a non-mammalian peptide sequence may be directly adjacent, overlapping, or may be spatially separated within the non-mammalian peptide sequence.

[0177] In one example, the RBD peptide (antigen / epitope) comprises the amino acid sequence shown in SEQ ID NO: 1, or a functional amino acid variant (or functional fragment) thereof. Such variants can be naturally occurring (e.g., allelic), synthetic, or synthetically improved functional variants of SEQ ID NO: 1. The term "variant" also encompasses homologs.

[0178] Functional variants may generally contain only conservative amino acid substitutions of one or more amino acids of SEQ ID NO: 1, or substitutions, deletions or insertions of non-critical amino acids in non-critical regions of the protein. Therefore, a functional variant of SEQ ID NO: 1 may be a conservative amino acid sequence variant of SEQ ID NO: 1, wherein the variant still comprises sequence elements necessary for obtaining protective antibodies.

[0179] Methods for identifying functional and non-functional variants (eg, functional and non-functional allelic variants) are well known to those of ordinary skill in the art.

[0180] The RBD peptides described herein may comprise an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 1, or a portion or fragment thereof. Suitably, the percent identity may be calculated as a percentage of identity to the entire length of a reference sequence (e.g., SEQ ID NO: 1), or a portion or fragment thereof.

[0181] In another example, the non-mammalian peptide sequence is an influenza virus peptide and the disease or condition is influenza. As used herein, the term "influenza" includes all types of influenza infections that may be caused by any of the four types of influenza viruses, a, b, c, and d.

[0182] The non-mammalian peptide sequence itself may be highly immunogenic or moderately immunogenic. Alternatively, it may be low immunogenic, or it may not be immunogenic at all. Due to the second part of the fusion polypeptide (immunostimulatory peptide), the immune response to the non-mammalian peptide sequence will be enhanced relative to the immune response caused by the non-mammalian peptide sequence alone.

[0183] "Immunostimulatory peptide sequences" that are responsible for triggering, initiating, enhancing, and strengthening the immune response to non-mammalian peptide sequences can also be called "immune activation peptides."

[0184] The immunostimulatory peptide sequence can be a synthetic or artificial sequence. Alternatively, the immunostimulatory peptide sequence can be a sequence existing in nature. In one example, as demonstrated in the following example, the immunostimulatory peptide sequence can be a sequence derived from one or more bacterial sequences. The immunostimulatory peptide sequence comprising one or more bacterial sequences is advantageous because it will be recognized as "foreign" by the immune system of mammals. This makes the immunostimulatory peptide sequence immunogenic.

[0185] The immunogenicity of the immunostimulatory peptide sequence can be optimized by the ratio of the bulk hydrophilic or charged amino acids to the total amino acids present in the immunostimulatory peptide sequence. The inventors have found that the ratio of the bulk hydrophilic or charged amino acids to the total amino acids in the immunostimulatory peptide sequence can reliably measure the efficacy of the immunostimulatory peptide sequence in stimulating the immune response to the connected antigen of interest. The immunostimulatory peptide sequence whose ratio of the bulk hydrophilic or charged amino acids to the total amino acids is less than or equal to 0.55 increases the solubility of the whole fusion polypeptide, which in turn causes the immune system to better recognize and increase the immunogenicity in vivo. Therefore, in the context of the present invention, it is important that the ratio of the bulk hydrophilic or charged amino acids to the total amino acids of the immunostimulatory peptide should be less than or equal to 0.55.

[0186] The term "bulky hydrophilic or charged amino acids" used herein refers to a group of amino acid residues consisting of histidine, glutamic acid, arginine, glutamine, aspartic acid and lysine.

[0187] The ratio of bulky hydrophilic or charged amino acids to total amino acids is calculated by dividing the total number of bulky hydrophilic or charged amino acids by the total number of amino acids in the immunostimulatory peptide sequence. For example, if the immunostimulatory peptide sequence has a total of 100 amino acids, 20 of which meet the above-mentioned definition of "bulky hydrophilic or charged amino acids", the ratio is 20:100=0.20. This ratio is calculated based only on the amino acids in the core immunostimulatory peptide sequence. To avoid doubt, the auxiliary tags (e.g., N-terminal or C-terminal tags, such as His tags) present in the fusion polypeptide do not constitute a part of the core immunostimulatory peptide sequence (because they are usually removed from the final fusion protein before being applied to the patient), so they should be ignored when calculating the ratio (even if they are adjacent to the core immunostimulatory peptide sequence). Such auxiliary tags are well known to those skilled in the art and are easy to identify. In addition, any auxiliary linkers (e.g., GS linkers, such as GSGS or GSGSGS (SEQ ID NO: 27) linkers) do not form part of the core immunostimulatory peptide sequence and are therefore ignored when calculating the ratio (even if they are immediately adjacent to the core immunostimulatory peptide sequence, e.g., linking the core immunostimulatory peptide sequence to an antigen of interest). Such auxiliary linkers are well known to those skilled in the art and are readily identifiable. Finally, the first methionine (encoded by the AUG start codon) is also deducted from the ratio calculation.

[0188] As shown herein, the sequence HHAH can be used as a purification tag (as an alternative to the His tag (HHHHHH; SEQ ID NO: 26)). The sequence HHAH is a naturally occurring sequence derived from the P30131|HYPF_ECOLI carbamoyltransferase HypF OS=Escherichia coli (strain K12) protein. Therefore, it is not necessary to remove this sequence before obtaining the final fusion polypeptide (as is required when using a His tag). Therefore, when calculating the ratio of bulky hydrophilic or charged amino acids to total amino acids in an immunostimulatory peptide sequence, the HHAH sequence is considered as part of the sequence, while the His tag is not considered as part of the sequence.

[0189] When selecting suitable immunostimulatory peptide sequences to be included in fusion polypeptides described herein, the total length of the immunostimulatory peptide sequences may also be considered. For example, short immunostimulatory peptides may be used to reduce / minimize the immune response to the immunostimulatory peptides themselves (but to shift the focus of the immune response to the antigen of interest).

[0190] In one example, the immunostimulatory peptide sequence length is at least 40 amino acids and no more than 150 amino acids. For example, the immunostimulatory peptide sequence length can be at least 40 amino acids, at least 45 amino acids, at least 50 amino acids, or at least 55 amino acids (but no more than 150 amino acids). In a further example, the immunostimulatory peptide sequence length can be at least 60 amino acids, at least 65 amino acids, or at least 70 amino acids (but no more than 150 amino acids).

[0191] In one example, the immunostimulatory peptide sequence is no longer than 150 amino acids, no longer than 145 amino acids, or no longer than 140 amino acids in length (with a minimum length of 40 amino acids).

[0192] In further examples, the immunostimulatory peptide sequence is no longer than 135 amino acids, no longer than 130 amino acids, no longer than 125 amino acids, or no longer than 120 amino acids (with a minimum length of 40 amino acids).

[0193] In a further example, the immunostimulatory peptide sequence does not exceed 100 amino acids (with a minimum length of 40 amino acids).

[0194] The length of the immunostimulatory peptide sequence may also depend on the length of the peptide sequence of interest. When the non-mammalian or mammalian peptide sequence is shorter, the attached immunostimulatory peptide should be less than 50% of the total protein. As a guideline, the suitable length of the immunostimulatory peptide sequence can be about 1 / 3 (one third) of the length of the non-mammalian or mammalian peptide sequence (i.e., the antigen of interest). However, when selecting the length, the overall solubility of the fusion protein should also be considered.

[0195] The isoelectric point of the immunostimulatory peptide sequence can also be optimized for inclusion in the fusion polypeptide described herein. As described elsewhere herein, the pI of a protein affects its solubility at physiological pH. In one example, the immunostimulatory peptide sequence has a predicted isoelectric point (pI) of less than or equal to 4.

[0196] For example, the immunostimulatory peptide sequence may have a predicted pi of less than or equal to 3.96, less than or equal to 3.8, such as less than or equal to 3.78, 3.7 or 3.63.

[0197] In one example, the immunostimulatory peptide sequence comprises the amino acid sequence of SEQ ID NO:4 or its conservative amino acid variant. The sequence increases the solubility of the fusion polypeptide because the amino acids D (aspartic acid) and E (glutamic acid) have very low pI and are negatively charged at physiological pH (pH 7.4). The immunostimulatory peptide sequence may comprise an amino acid sequence with at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to the amino acid or its part or fragment of SEQ ID NO:4. Suitably, the percentage identity may be calculated as the percentage of the identity of the entire length or its part or fragment with a reference sequence (e.g., SEQ ID NO:4).

[0198] When amino acids are replaced this may result in a change in pi and thus one skilled in the art will appreciate that modifications to this sequence alone may still provide the desired overall pi of the fusion polypeptide.

[0199] As described elsewhere herein, the pI of the fusion polypeptide described herein can be an important feature, because low pI can increase solubility and immunogenicity. An immunostimulatory peptide sequence can be selected to provide an overall pI for the fusion polypeptide, thereby resulting in high solubility and immunogenicity under physiological conditions. Therefore, for an antigen of interest (e.g., a non-mammalian peptide sequence or a mammalian peptide sequence) with a relatively high predicted pI (e.g., predicted pI of about pH 7.4, i.e., physiological conditions or higher), its solubility / immunogenicity in vivo is expected to be low, and an immunostimulatory peptide sequence with a low pI can be selected to offset this situation and reduce the overall pI of the fusion polypeptide to a more ideal level (thereby causing the antigen of interest (e.g., a non-mammalian peptide sequence or a mammalian peptide sequence) to have higher solubility and immunogenicity). An appropriate combination of an immunostimulatory peptide sequence and an antigen of interest (e.g., a non-mammalian peptide sequence or a mammalian peptide sequence) is provided herein.

[0200] A variety of immunostimulatory peptide sequences are described in detail herein, having a ratio of bulky hydrophilic or charged amino acids to total amino acids of less than or equal to 0.55 (e.g., wherein the immunostimulatory peptide sequence length may be at least 40 amino acids and no more than 150 amino acids and / or have a predicted pI of less than or equal to 4). One skilled in the art can readily identify alternative suitable immunostimulatory peptide sequences using the features described herein.

[0201] For example, an immunogenic bacterial sequence (or a complex of such bacterial sequences) can provide a suitable immunostimulatory peptide sequence having a ratio of bulky hydrophilic or charged amino acids to total amino acids less than or equal to 0.55 (e.g., wherein the immunostimulatory peptide sequence can be at least 40 amino acids and no more than 150 amino acids in length and / or have a predicted pI less than or equal to 4).

[0202] Examples of such immunostimulatory peptide sequences are provided elsewhere herein and include the bacterial protein thioredoxin (TRX UNIPROT #P0AA25), a truncated version thereof (TRXtr), type 1 fimbriae protein chain A (TFP, UNIPROT #P04128), a variant thereof (TFPv), and immunostimulatory peptides 1 to 4. These are described in detail below.

[0203] Thus, in one example, a fusion polypeptide for use in vaccination or treatment of a mammalian subject comprises:

[0204] (6) a non-mammalian peptide sequence, wherein the peptide sequence is a sequence associated with a disease or condition for which the mammalian subject is being vaccinated or treated; and

[0205] (ii) an immunostimulatory peptide sequence selected from the group consisting of:

[0206] (a) a sequence comprising SEQ ID NO: 10 or a conservative amino acid variant thereof, optionally wherein the immunostimulatory peptide sequence is no longer than 80 amino acids;

[0207] (b) a sequence comprising SEQ ID NO: 12 or a conservative amino acid variant thereof;

[0208] (c) a sequence comprising SEQ ID NO: 5 or a conservative amino acid variant thereof;

[0209] (d) a sequence comprising SEQ ID NO: 6 or a conservative amino acid variant thereof;

[0210] (e) a sequence comprising SEQ ID NO: 7 or a conservative amino acid variant thereof; or

[0211] (f) a sequence comprising SEQ ID NO: 8 or a conservative amino acid variant thereof.

[0212] In one example, the fusion polypeptide comprises:

[0213] (i) a non-mammalian peptide sequence comprising SEQ ID NO: 1, and

[0214] (ii) immunostimulatory peptide sequences having a ratio of bulky hydrophilic or charged amino acids to the remaining amino acids of less than or equal to 0.55.

[0215] In one example, the fusion polypeptide comprises:

[0216] (i) a non-mammalian peptide sequence comprising SEQ ID NO: 1, and

[0217] (ii) an immunostimulatory peptide sequence selected from the group consisting of:

[0218] (a) a sequence comprising SEQ ID NO: 10 or a conservative amino acid variant thereof, optionally wherein the immunostimulatory peptide sequence is no longer than 80 amino acids;

[0219] (b) a sequence comprising SEQ ID NO: 12 or a conservative amino acid variant thereof;

[0220] (c) a sequence comprising SEQ ID NO: 5 or a conservative amino acid variant thereof;

[0221] (d) a sequence comprising SEQ ID NO: 6 or a conservative amino acid variant thereof;

[0222] (e) a sequence comprising SEQ ID NO: 7 or a conservative amino acid variant thereof; or

[0223] (f) a sequence comprising SEQ ID NO: 8 or a conservative amino acid variant thereof.

[0224] In some advantageous examples, the immunostimulatory peptide sequence may comprise at least one (predicted) T-cell epitope.

[0225] The term "T-cell epitope" as used herein refers to -T cell epitopes, particularly CD4+T-cell epitopes. In other words, they are epitopes recognized and bound by T cell receptors (TCR) on the surface of CD4+T cells. T cell epitopes are mainly presented to TCR by histocompatibility complex (MHC) molecules. In humans, professional antigen presenting cells specifically present T-cell epitopes in the context of MHC II, while most nucleated somatic cells present T-cell epitopes in the context of MHC II. MHC class II molecules present peptides of 13-17 amino acids in length, and non-classical MHC molecules also present non-digestible epitopes, such as glycolipids.

[0226] To predict whether a peptide sequence is or contains a T-cell epitope, online tools may be used, such as those provided at the website: http: / / www.cbs.dtu.dk / services / NetMHCpan / .

[0227] The immunostimulatory peptide sequence may comprise one, two, three, four or more (predicted) T-cell epitopes. For example, the immunostimulatory peptide sequence comprises one or two (predicted) T-cell epitopes.

[0228] In one example, the immunostimulatory peptide sequence includes one or two (predicted) CD4+ T-cell epitopes.

[0229] In the immunostimulatory peptide sequence, the (predicted) T-cell epitopes may be directly adjacent / fused to each other or they may be separated by a linker or any other stuffer sequence.

[0230] In one example, the immunostimulatory peptide sequence comprises a (predicted) T-cell epitope selected from the MsyB protein (P25738|MSYB_ECOLI acidic protein MsyB OS=Escherichia coli (strain K12)) and / or the RRAB protein (P0AF90|RRAB_ECOLI ribonuclease activity regulator B OS=Escherichia coli (strain K12)). MsyB and RRAB proteins are derived from Escherichia coli and are both considered acidic proteins.

[0231] For example, the following (predicted) CD4+ T-cell epitopes within the RRAB protein (based on http: / / www.cbs.dtu.dk / services / NetMHCpan / ) may be present within the immunostimulatory peptide sequence: KLGYEVYEVTDPEELEVE (SEQ ID NO: 13), FKLGYEVYEVTDPEELEV (SEQ ID NO: 14) and / or YEVTDPEEL (SEQ ID NO: 15).

[0232] Alternatively or in addition, one or more of the following (predicted) CD4+ T-cell epitopes (based on http: / / www.cbs.dtu.dk / services / NetMHCpan / ) within the MsyB protein may be present in the immunostimulatory peptide sequence: EGEFQLEPPLDTEEG (SEQ ID NO: 16), AQKYVLQDGDIMWQV (SEQ ID NO: 17), DEGEFQLEPPLDTEE (SEQ ID NO: 18), NAQKYVLQDGDIMWQ (SEQ ID NO: 19), EPPLDTEEGRAADE (SEQ ID NO: 20), NPGIDAEDANVQQFN (SEQ ID NO: 21), LEPPLDTEEGRAAD (SEQ ID NO: 22), FQLEPPLDTYVLQDGDIM (SEQ ID NO: 23), LDTEEGRAA (SEQ ID NO: 24) and / or IDAEDANVQ (SEQ ID NO: 25).

[0233] It is clear to those skilled in the art that the immunostimulatory peptide sequence can comprise two or more of these T-cell epitopes. For example, two or more T-cell epitopes can be located in a position directly adjacent to each other, or can partially overlap in the immunostimulatory peptide sequence (where its sequence permits); for example, as shown in NPGIDAEDANVQQFNAQKYVLQDGDIMWQV (SEQ ID NO: 2) and / or EGEFQLEPPLDTEEGRAAADE (SEQ ID NO: 3).

[0234] The immunostimulatory peptide sequence described herein may comprise one (predicted) T-cell epitope from MsyB protein or one (predicted) T-cell epitope from RRAB protein, or it may comprise multiple (predicted) T-cell epitopes of MsyB protein or multiple (predicted) T-cell epitopes of RRAB protein, or it may comprise a combination of one or more (predicted) T-cell epitopes of MsyB or RRAB protein.

[0235] For example, the immunostimulatory peptide sequence can comprise one MsyB protein (predicted) T-cell epitope. In another example, the immunostimulatory peptide sequence can comprise two MsyB protein (predicted) T-cell epitopes.

[0236] In one example, the immunostimulatory peptide sequence may comprise the amino acid sequence of SEQ ID NO: 2 and / or SEQ ID NO: 3 or conservative amino acid variants thereof.

[0237] The immunostimulatory peptide sequence may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:2, or a portion or fragment thereof, and appropriately the percentage identity may be calculated as a percentage of identity to the entire length of a reference sequence (e.g., SEQ ID NO:2), or a portion or fragment thereof.

[0238] The immunostimulatory peptide sequence may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:3, or a portion or fragment thereof, and appropriately the percentage identity may be calculated as a percentage of identity to the entire length of a reference sequence (e.g., SEQ ID NO:3), or a portion or fragment thereof.

[0239] When amino acids are replaced this may result in a change in pi and thus one skilled in the art will appreciate that modifications to this sequence alone may still provide the desired overall pi of the fusion polypeptide.

[0240] In one example, the immunostimulatory peptide sequence comprises the amino acid sequences SEQ ID NO: 2 and SEQ ID NO: 3. Optionally, when the immunostimulatory peptide sequence comprises the amino acid sequences SEQ ID NO: 2 and SEQ ID NO: 3, these sequences are directly adjacent / fused to each other within the peptide sequence.

[0241] In one example, the immunostimulatory peptide sequence comprises or consists of the following amino acid sequence:

[0242] DNNSLSQEVQNGSNHLENNQSQSNGGGSDSALSLSSKTAALAAAT TVNDGSDGATSSAVG (CDP; SEQ ID NO: 5), or a conservative amino acid variant thereof.

[0243] The CDP contains the following peptides: NNSLSQEVQN (SEQ ID NO: 28) (10aa) derived from ZapB (amino acids 36-45), NHLENNQSQSNGG (SEQ ID NO: 29) (13aa) derived from IbpA (amino acids 21-33), SALSLSSTAALAAATTVN (SEQ ID NO: 30) (18aa) and GATSSAVG (SEQ ID NO: 31) (8aa) derived from TFP (amino acids 12-39 and 69-76, respectively). This is shown below:

[0244] CDP (SEQ ID NO: 5):

[0245]

[0246] A total of five amino acids within the CDP have bulky hydrophilic / charged side chains (2xE, 3xQ, and 1xH). To increase the immunogenicity of the CDP, some alanine or glycine residues were mutated to lysine or aspartic acid residues (underlined) to obtain a ratio of 0.18 bulky hydrophilic / charged amino acids to the remaining amino acids (11 bulky hydrophilic, charged aa / total 60 aa). The peptides were connected by a GS-linker (1xGS).

[0247] Thus, the immunostimulatory peptide sequence may comprise an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:5 or a portion or fragment thereof, and suitably, the percentage identity may be calculated as a percentage of identity over the entire length of the reference sequence (e.g., SEQ ID NO:5) or a portion or fragment thereof. In another example, the immunostimulatory peptide sequence comprises or consists of the following amino acid sequence:

[0248] HHAHTMYATLEEAIDAAREEFLADNPGIDAEDANVQQFNAQKYV LQDGDIMWQVEFFADEGEEGEDDEDFVDEDDD (IDP1; SEQ ID NO: 6), or a conservative amino acid variant thereof.

[0249] IDP1 comprises the native histidine-rich HHAH sequence of the E. coli protein P30131|HYPF_ECOLI carbamoyltransferase HypF OS=E. coli (strain K12) for purification purposes, the N-terminal part (amino acids 2 to 62) of the MsyB protein including the (predicted) T-cell epitope construct according to SEQ ID NO:2. In addition, it contains the amino acid sequence of the RRAB protein according to SEQ ID NO:4. IDP1 has a predicted pI of 3.63 and a molecular weight of 8813.23 Da with a solubility of 0.815. The ratio of bulky hydrophilic or charged amino acids to total amino acids is 0.47 (36 bulky hydrophilic, charged a a / Total 76 a a ).

[0250] The immunostimulatory peptide sequence may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:6, or a portion or fragment thereof, and appropriately the percentage identity may be calculated as a percentage of identity to the entire length of a reference sequence (e.g., SEQ ID NO:6), or a portion or fragment thereof.

[0251] In a further example, the immunostimulatory peptide sequence comprises or consists of the following amino acid sequence:

[0252] HHAHNPGIDAEDANVQQFNAQKYVLQDGDIMWQVEGEFQLEPPL DTEEGRAAADEDDEDFVDEDDD (IDP2; SEQ ID NO: 7), or a conservative amino acid variant thereof.

[0253] IDP2 comprises the natural histidine-rich HHAH sequence of E. coli protein P30131|HYPF_ECOLI carbamoyltransferase HypF OS=E. coli (strain K12) (used for purification purposes), two (predicted) T-cell epitope constructs of MsyB proteins according to SEQ ID NO: 2 and SEQ ID NO: 3 fused to each other. In addition, the sequence comprises the amino acid sequence according to SEQ ID NO: 4, which is fused to the (predicted) T-cell epitope construct according to SEQ ID NO: 3.

[0254] IDP2 has a predicted pI of 3.70, a molecular weight of 7616.93 Da, and a solubility of 0.815. The ratio of bulky hydrophilic or charged amino acids to total amino acids is 0.45 (30 bulky hydrophilic, charged aa / 66 aa total).

[0255] The immunostimulatory peptide sequence may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:7, or a portion or fragment thereof, and suitably the percentage identity may be calculated as a percentage of identity to the entire length of a reference sequence (e.g., SEQ ID NO:7), or a portion or fragment thereof.

[0256] In a further example, the immunostimulatory peptide sequence comprises or consists of the following amino acid sequence:

[0257] HHAHNPGIDAEDANVQQFNAQKYVLQDGDIMWQVDDEDFVDEDDD (IDP3; SEQ ID NO: 8), or a conservative amino acid variant thereof.

[0258] IDP3 comprises the E. coli protein P30131|HYPF_ECOLI carbamoyltransferase HypF OS=the native histidine-rich HHAH sequence of E. coli (strain K12) (for purification purposes), the first (predicted) T-cell epitope construct of the MysB protein according to SEQ ID NO:2 fused to the amino acid sequence according to SEQ ID NO:4.

[0259] IDP3 has a predicted pI of 3.78, a molecular weight of 5331.57 Da, and a solubility of 0.855. The ratio of bulky hydrophilic or charged amino acids to total amino acids is 0.44 (20 bulky hydrophilic, charged aa / 45 aa total).

[0260] The immunostimulatory peptide sequence may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:8, or a portion or fragment thereof, and appropriately the percentage identity may be calculated as a percentage of identity to the entire length of a reference sequence (e.g., SEQ ID NO:8), or a portion or fragment thereof.

[0261] All four sequences according to SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8 (and conservative amino acid variants thereof) represent core immunostimulatory peptide sequences as described herein. Auxiliary tags and linkers can be added to these sequences, as described elsewhere herein. For example, natural histidine-rich sequences such as HHAH (from E. coli protein P30131, HYPF_E. coli) (if not yet present) or HHHHHH can be added to the N-terminus of the sequence. In addition, those skilled in the art will appreciate that, in order to obtain synthetic proteins, methionine is required at the beginning of each sequence. In these examples, as described elsewhere herein, when calculating the ratio of bulky hydrophilic or charged amino acids to total amino acids in the immunostimulatory peptide sequence, methionine is not considered nor is the His tag considered. However, the histidine-rich sequence HHAH will be taken into account.

[0262] Alternative immunostimulatory peptide sequences that can be included in the fusion polypeptides described herein include: thioredoxin (TRX), a truncated version of thioredoxin (TRXtr), type 1 fimbriae protein (A chain) (TFP), or variant type 1 fimbriae protein (A chain) (TFPv). The use of these immunostimulatory peptide sequences as fusion polypeptides with autoantigens has been previously described. Some details about these sequences are provided below.

[0263] Thioredoxin (TRX; 11.8 kDa)

[0264] SDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIA DEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALS KGQLKEFLDANLA(108aa)(SEQ ID NO:9)

[0265] For this immunostimulatory peptide sequence, the amino acids with bulky hydrophilic / charged side chains in the TRX protein sequence are: 1xH, 5xE, 1xR, 3xQ, 11xD, 10xK. There are a total of 31 amino acids with bulky hydrophilic / charged side chains; therefore, the ratio of bulky hydrophilic / charged amino acids to total amino acids in the TRX protein is 31aa / 108aa=0.29.

[0266] Thioredoxin trunc (TRXtr; 6.2 kDa)

[0267] GKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSK GQLKEFLDANLA(58aa) (SEQ ID NQ:10).

[0268] In TRXtr, only the C-terminal portion (aa 51-109) of the full-length thioredoxin sequence is used (i.e., the last 58 C-terminal amino acids of the protein). An additional methionine is added to the N-terminus of the TRXtr protein sequence, and a GS-linker (4x glycine-serine) and a His-tag (6x histidine) are added to the C-terminus. The GS-linker is included to provide flexibility to the fusion protein construct and a His-tag for purification purposes. Compared to the full-length thioredoxin, the TRXtr sequence contains only 14 amino acids with bulky hydrophilic / charged side chains (2xE, 2xQ, 1xR, 2xD, and 7xK), resulting in a ratio of bulky hydrophilic / charged amino acids to total amino acids of 14aa / 58aa=0.24.

[0269] Type 1 fimbriae protein A chain (TFP; 13 kDa)

[0270] AATTVNGGTVHFKGEVVNAACAVDAGSVDQTVQLGQVRTASLAQEGATSSAVGFNIQLNDCDTNVASKAAFLGTAIDAGHTNVLALQSSAAGSATNVGVQILDRTGAALTLDGATFSSETTLNNGTNTI (131aa) (SEQ ID NO: 11).

[0271] The full-length TFP protein uses only the protein sequence without the signal peptide (aa 24-182; 13 kDa) and adds an N-terminal methionine. The number of amino acids with bulky hydrophilic / charged side chains in the TFP protein sequence is: 2xH, 3xE, 2xR, 7xQ, 7xD, 2xK. There are a total of 23 amino acids with bulky hydrophilic / charged side chains; therefore, the ratio of bulky hydrophilic / charged amino acids to total amino acids in the TFP protein is 23aa / 131aa=0.18.

[0272] Variant type 1 fimbriae protein A chain (TFPv; 13.3 kDa)

[0273] K ATTVNGGTVHFKGEVVNAACAVDAGSVDQTVQLGQVRTASLAQEGATSS K VGFNIQLNDCDTNVASKAAVAFLGT K IDAGHTNVLALQSSAAGS D TNVGVQILDRTGAALTLDGATFSSETTLNN D TNTI (131aa) (SEQ ID NO: 12).

[0274] By mutating a certain number of alanine (Ala, A) or glycine residues (Gly, G) to lysine or aspartic acid (underlined), a TFP variant (TFPv) protein was obtained; thereby obtaining a TFP protein with higher immunogenicity. The amino acids with bulky hydrophilic / charged side chains in the TFP variant protein sequence are: 2xH, 3xE, 2xR, 7xQ, 8xD, 5xK. There are a total of 27 amino acids with bulky hydrophilic / charged side chains; therefore, the ratio of bulky hydrophilic / charged amino acids to total amino acids in the TFP protein is 27aa / 131aa=0.21.

[0275] Fusion peptide

[0276] According to a further aspect of the present invention, there is provided a fusion polypeptide comprising:

[0277] (6) a non-mammalian peptide sequence, wherein the peptide sequence is a sequence associated with a disease or condition for which the mammalian subject is being vaccinated or treated; and

[0278] (ii) immunostimulatory peptide sequences having a ratio of bulky hydrophilic or charged amino acids to total amino acids less than or equal to 0.55;

[0279] wherein the non-mammalian peptide sequence is linked to the immunostimulatory peptide sequence, and wherein the immunostimulatory peptide sequence comprises the amino acid sequence:

[0280] a) HHAHTMYATLEEAIDAAREEFLADNPGIDAEDANVQQFNAQKYVLQDGDIMWQVEFFADEGEEGEDDEDFVDEDDD (IDP1; SEQ ID NO: 6), or a conservative amino acid variant thereof; or

[0281] b) HHAHNPGIDAEDANVQQFNAQKYVLQDGDIMWQVEGEFQLEPPLDTEEGRAAADEDDEDFVDEDDD (IDP2; SEQ ID NO: 7), or a conservative amino acid variant thereof; or

[0282] c) HHAHNPGIDAEDANVQQFNAQKYVLQDGDIMWQVDDEDFVDEDDD (IDP3; SEQ ID NO: 8), or a conservative amino acid variant thereof; or

[0283] d) DNNSLSQEVQNGSNHLENNQSQSNGGGSDSALSLSSKTAALAAATTVNDGSDGATSSAVG (CDP; SEQ ID NO: 5), or a conservative amino acid variant thereof.

[0284] In one example,

[0285] a) the non-mammalian peptide sequence is a viral peptide and the disease or condition is a viral infection; or

[0286] b) the non-mammalian peptide sequence is a mycobacterial or bacterial peptide and the disease or condition is a mycobacterial or bacterial infection; or

[0287] c) the non-mammalian peptide sequence is a yeast peptide and the disease or disorder is a yeast infection; or

[0288] d) the non-mammalian peptide sequence is a parasitic peptide and the disease or disorder is a parasitic infection.

[0289] Further details regarding viral, bacterial, mycobacterial, yeast and parasitic peptides and corresponding infections are described elsewhere herein and are equally applicable here.

[0290] In one example, the non-mammalian peptide sequence is a SARS-CoV-2 peptide and the disease or condition is a SARS-CoV-2 infection.

[0291] In one example, the non-mammalian peptide sequence is a SARS-CoV-2 peptide (antigen / epitope), and the disease or condition is a SARS-CoV-2 infection. For example, the SARS-CoV-2 peptide (antigen / epitope) can be a SARS-CoV-2 spike protein peptide (antigen / epitope). For example, the SARS-CoV-2 peptide (antigen / epitope) can be a SARS-CoV-2 receptor binding domain (RBD) peptide (antigen / epitope), such as the RBD of the spike protein described in Wrapp et al., 2020.

[0292] In one example, the RBD peptide (antigen / epitope) comprises the amino acid sequence shown in SEQ ID NO: 1, or a functional amino acid variant (or functional fragment) thereof. Such variants can be naturally occurring (e.g., allelic), synthetic, or synthetically improved functional variants of SEQ ID NO: 1. The term "variant" also encompasses homologs.

[0293] Suitable RBD peptides and functional variants are described elsewhere herein and are equally applicable here.

[0294] In another example, the non-mammalian peptide sequence is an influenza virus peptide, and the disease or condition is influenza.The term "influenza" is defined elsewhere herein and applies equally to this aspect.

[0295] Preferably, the fusion polypeptide elicits a clinically relevant immune response in a subject in the sense that the serum level of immunoglobulins against the non-mammalian peptide sequence reaches at least an ELISA-detectable level of immunoglobulins against the non-mammalian peptide sequence. An ELISA-detectable level refers to a statistically significant level of detection above background or above a control value or threshold in an ELISA protocol. Preferably, the relevant immune response involves a serum level of immunoglobulins against the non-mammalian peptide sequence that is at least 5 times the signal measured in the control serum of an unvaccinated individual, preferably the same as at least 0.1% of the total serum immunoglobulin G (IgG) level, as detected by ELISA.

[0296] According to another aspect of the present invention, there is provided a fusion polypeptide comprising:

[0297] (6) a mammalian peptide sequence, wherein the peptide sequence is a sequence associated with a disease or condition for which the mammalian subject is being vaccinated or treated; and

[0298] (ii) immunostimulatory peptide sequences having a ratio of bulky hydrophilic or charged amino acids to total amino acids less than or equal to 0.55;

[0299] wherein the mammalian peptide sequence is linked to an immunostimulatory peptide sequence, and wherein the immunostimulatory peptide sequence comprises the amino acid sequence:

[0300] a) HHAHTMYATLEEAIDAAREEFLADNPGIDAEDANVQQFNAQKYVLQDGDIMWQVEFFADEGEEGEDDEDFVDEDDD (IDP1; SEQ ID NO: 6), or a conservative amino acid variant thereof; or

[0301] b) HHAHNPGIDAEDANVQQFNAQKYVLQDGDIMWQVEGEFQLEPPLDTEEGRAAADEDDEDFVDEDDD (IDP2; SEQ ID NO: 7), or a conservative amino acid variant thereof; or

[0302] c) HHAHNPGIDAEDANVQQFNAQKYVLQDGDIMWQVDDEDFVDEDDD (IDP3; SEQ ID NO: 8), or a conservative amino acid variant thereof.

[0303] In one embodiment, the mammalian peptide sequence is associated with tumor angiogenesis. Examples of various suitable peptides associated with tumor angiogenesis are described in detail elsewhere herein.

[0304] In one embodiment, the mammalian peptide sequence is vimentin (Vim).

[0305] In one embodiment, the mammalian peptide sequence is an apelin, such as mouse apelin.

[0306] In one embodiment, the mammalian peptide sequence is notum, such as mouse notum.

[0307] In one embodiment, the mammalian peptide sequence is timp1, such as mouse timp1.

[0308] By using a fusion polypeptide comprising a mammalian peptide sequence against which an immune response should be elicited, the immune system can be triggered to produce anti-self antibodies when the fusion polypeptide is administered to a subject.

[0309] Preferably, the fusion polypeptide elicits a clinically relevant immune response in a subject in the sense that the serum level of immunoglobulins against the mammalian peptide sequence reaches at least an ELISA-detectable level against the mammalian peptide sequence. An ELISA-detectable level refers to a statistically significant level of detection above background or above a control value or threshold in an ELISA protocol. Preferably, the relevant immune response involves a serum level of immunoglobulins against the mammalian peptide sequence that is at least 5 times the signal measured in control serum of unvaccinated individuals, preferably the same as at least 0.1% of the total serum immunoglobulin G (IgG) level, as detected by ELISA.

[0310] The fusion polypeptides described herein can have a predicted isoelectric point (pi) of less than or equal to 6. As mentioned elsewhere herein, the isoelectric point (pi) of a polypeptide is directly related to its solubility and directly affects the ability of the fusion polypeptide to trigger or enhance an immune response to an antigen of interest. Isoelectric point is defined elsewhere herein and is equally applicable to this aspect.

[0311] Fusion polypeptides described herein having a pi below physiological pH (about 7.4) will have increased solubility in vivo, which is desirable when eliciting an immune response.

[0312] In one example, the predicted pi of the fusion polypeptide described herein is less than or equal to 5.5 or less than or equal to 5.

[0313] In another example, the predicted pi of the fusion polypeptide described herein is less than or equal to 4.5, or less than or equal to 4.

[0314] The immunostimulatory peptide sequences of SEQ ID NO: 5 to 8 (and variants thereof) have been discussed in detail elsewhere herein. These details apply equally to this aspect.

[0315] Suitably, the immunostimulatory peptide sequence is at least 40 amino acids and no more than 150 amino acids in length.

[0316] In one example, the length of the immunostimulatory peptide sequence is at least 40 amino acids and no more than 150 amino acids. For example, the length of the immunostimulatory peptide sequence can be at least 40 amino acids, at least 45 amino acids, at least 50 amino acids or at least 55 amino acids (but no more than 150 amino acids). In a further example, the length of the immunostimulatory peptide sequence can be at least 60 amino acids, at least 65 amino acids or at least 70 amino acids (but no more than 150 amino acids).

[0317] In one example, the immunostimulatory peptide sequence has a length of no more than 150 amino acids, no more than 145 amino acids, or no more than 140 amino acids (with a minimum length of 40 amino acids).

[0318] In further examples, the length of the immunostimulatory peptide sequence is no more than 135 amino acids, no more than 130 amino acids, no more than 125 amino acids, or no more than 120 amino acids (with a minimum length of 40 amino acids).

[0319] In a further example, the immunostimulatory peptide sequence does not exceed 100 amino acids (with a minimum length of 40 amino acids).

[0320] The length of the immunostimulatory peptide sequence may also depend on the length of the peptide sequence of interest. Detailed information on this aspect is provided elsewhere herein and is equally applicable to this aspect.

[0321] The isoelectric point of the immunostimulatory peptide sequence can also be optimized for inclusion in the fusion polypeptide described herein. As described elsewhere herein, the pI of a protein affects its solubility at physiological pH. In one example, the immunostimulatory peptide sequence has a predicted isoelectric point (pI) of less than or equal to 4.

[0322] For example, the immunostimulatory peptide sequence may have a predicted pi less than or equal to 3.8, such as less than or equal to 3.78, 3.7 or 3.63.

[0323] Any aspects discussed herein with respect to immunostimulatory peptide sequences and fusion polypeptides apply equally to these aspects.

[0324] Nucleic acids, compositions and kits

[0325] According to another aspect, provided herein is a nucleic acid encoding a fusion polypeptide according to the present invention, optionally wherein the nucleic acid is an expression vector.

[0326] The present invention also provides a pharmaceutical composition, wherein the composition comprises the fusion polypeptide of the present invention, or the nucleic acid sequence or vector according to the present invention and a pharmaceutically acceptable carrier, adjuvant, excipient or diluent. The composition may conventionally contain pharmaceutically acceptable concentrations of salt, buffer, preservative, compatible carrier, supplementary immunopotentiator (e.g., adjuvant and cytokine) and optionally other therapeutic agents or compounds.

[0327] The pharmaceutical composition described herein can be used for vaccination or treatment of a subject who needs an immune response to a certain peptide sequence. This can be an immune response triggered by a non-mammal or mammalian peptide sequence. The details of fusion polypeptides, non-mammal peptide sequences, mammalian peptide sequences, immunostimulatory peptide sequences, diseases, illnesses and treatments are described herein and are also applicable to this. The definitions and explanations provided elsewhere herein are also applicable to this aspect.

[0328] Also provided is a kit comprising the fusion polypeptide described herein, or a pharmaceutical composition comprising the fusion polypeptide or nucleic acid described herein.

[0329] Medical Uses

[0330] Also provided herein is the use of the fusion polypeptide or pharmaceutical composition described herein as a medicament. Also provided herein is the use of the fusion polypeptide or pharmaceutical composition described herein for vaccination or treatment of a subject.

[0331] Details regarding vaccination, treatment, subjects, diseases or conditions are described elsewhere herein and apply equally here.

[0332] Also provided herein is a method of vaccinating or treating a subject, the method comprising administering to the subject the fusion polypeptide described herein or the pharmaceutical composition described herein.

[0333] Also provided herein is a method for vaccinating a subject against SARS-CoV-2 using the fusion polypeptide or pharmaceutical composition described herein. In particular, provided herein is a method for vaccinating or treating a subject by administering to the subject a fusion polypeptide or pharmaceutical composition as described herein, wherein the fusion polypeptide comprises a non-mammalian peptide sequence, an RBD of SARS-CoV-2, and an immunostimulatory peptide sequence selected from the group consisting of: immunostimulatory peptide sequences 1-4 (as described herein).

[0334] General Definition

[0335] In the description and claims of this specification, the words “comprise” and “comprising” and variations thereof mean “including but not limited to”, and they are not intended to (and do not) exclude other parts, additives, components, integers or steps.

[0336] Throughout the description and claims of this specification, the singular encompasses the plural, unless the context requires otherwise. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0337] In addition, as used herein, the singular terms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. Unless otherwise indicated, nucleic acid sequences are written from left to right in 5' to 3' orientation; amino acid sequences are written from left to right in amino to carboxyl orientation. It should be understood that the present invention is not limited to the particular methods, protocols and reagents described, as these may vary depending on the context in which they are used by those skilled in the art.

[0338] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0339] As used herein, the terms "vaccinate", "vaccinating", "vaccination", "immunization" include interventions intended to reduce the risk of disease and / or reduce the likelihood of severe disease by establishing protection (i.e., immunizing the body) in collaboration with the body's natural defenses. Immunization is the process of preventing disease. But it can also mean the same thing as vaccination, i.e., vaccinating to prevent disease. This is achieved by injecting a vaccine into a subject. The vaccine can be any vaccine known to those skilled in the art, such as live attenuated vaccines, inactivated vaccines, subunit vaccines, recombinant vaccines, polysaccharide vaccines and conjugate vaccines, toxoid vaccines, mRNA vaccines or viral vector vaccines. They can be administered in any form, such as an injection (shot), liquid, pill or nasal spray. Vaccination is a preventive treatment to gain protection before and / or after encountering a pathogen.

[0340] As used herein, the terms "treat," "treating," and "treatment" include interventions performed to prevent the development of, or alter the pathology of, a condition, disorder, or symptom. Thus, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the target condition, disorder, or symptom.

[0341] As used herein, "administration" or "administering" of a pharmaceutical composition as described herein to a subject includes any route of introduction or delivery to a subject so that the composition performs its intended function. Administration can be performed by any suitable route, including oral, intranasal, intraocular, ocular, parenteral (intravenous, intramuscular, intraperitoneal, intradermal or subcutaneous), or topical. Administration includes self-administration and administration by others. The composition can be administered in a therapeutically effective amount. The phrase "therapeutically effective amount" used herein refers to a dose or plasma concentration in a subject that provides a specific pharmacological effect of administering the composition, such as treating a target disease in a target subject. A therapeutically effective amount may vary based on the route of administration and dosage form, the age and weight of the subject, and / or the disease or condition being treated.

[0342] The term "subject" as used herein refers to an individual, such as a human, dog, cat, pig, horse, mouse, cow, rat, etc., who suffers from a particular condition, disorder or symptom or is at risk of suffering from a particular condition, disorder or symptom. The subject can be a patient, i.e., a subject who needs treatment / vaccination according to the present invention. The subject may have already received vaccination / treatment for the condition, disorder or symptom. Alternatively, the subject has not received treatment / vaccination before treatment / vaccination according to the present invention. The subject preferably needs to be administered the fusion polypeptide or composition of the present invention.

[0343] The term "non-mammalian peptide sequence" as used herein refers to a peptide sequence that is not derived from a mammal or has no relation to a mammal. In particular, it refers to a peptide sequence that is considered foreign to a subject being vaccinated or treated with the methods or fusion polypeptides described herein, which means that the subject's immune system will recognize it as non-self and therefore, if possible, will produce antibodies against it. If the fusion polypeptide described herein contains a non-mammalian peptide sequence, the immunostimulatory peptide sequence should trigger / enhance an immune response against the non-mammalian peptide sequence, then the non-mammalian peptide sequence may also be referred to as a "peptide (sequence) of interest".

[0344] The term "mammalian peptide sequence" as used herein refers to any sequence associated with or derived from a mammal. Such peptide sequences will be considered as self-antigens by the subject's immune system. If the fusion polypeptide described herein comprises a mammalian peptide sequence, the immunostimulatory peptide sequence should trigger / enhance the immune response against the mammalian peptide sequence, then the mammalian peptide sequence may also be referred to as a "peptide of interest".

[0345] The term "antigen as an autoantigen" or "autoantigen" as used in the context of the present invention refers to an antigenic polypeptide portion. Preferably, such polypeptide portions are specific for tumor angiogenesis and are not specific for non-tumor-related angiogenesis (e.g., wound healing). As described herein, these portions may be conjugated to an immunogen by their full length or a portion thereof, thereby presenting or displaying at least one epitope or antigenic determinant. The autoantigen may be a structural component of blood vessels formed during tumor angiogenesis, preferably a structural component that is specific for blood vessels formed in tumor angiogenesis and is not specific for blood vessels formed in non-tumor-related angiogenesis, such as a polypeptide portion or a portion thereof, which is selected from the group formed by or selected from the group consisting of: tissue inhibitor of metalloproteinase 1 (Timp1), apelin peptide (Apln), serum amyloid A3 (Saa3), CD93 antigen (CD93), cardiac development protein with EGF-like domain 1 (Heg1), Notch 4. Aplnr, Nes, Tnc, C-domain of Tnc, Ptx3, Vimentin, PSMA, HER2, HER3, Tnfaip6, Cpz, Snail, Pmel, Arsi, Wisp1, Gpx7, Adisintegrin-like metallopeptidase with thrombospondin 1 motif 4, Pseudomonas aeruginosa ... type), endothelial cell-specific molecule 1 (Esm1), integrin alpha 5 (fibronectin receptor alpha) (Itga5), a disintegrin-like metallopeptidase (reprolysin type) with thrombospondin type 1 motif 2 (Adamts2), thrombospondin 2 (Thbs2), matrix metallopeptidase 14 (membrane inserted) (Mmp14), insulin-like growth factor binding protein 3 (Igfbp3), thrombospondin 1 (Thbsl1), fibrillin 1 (Fbn1), periostin, osteoblast-specific factor (Postn), leucine-rich repeat 17 (Lrrc17), fibrillin 2 (Fbn2), brain endothelial cell adhesion molecule (Cercam), secreted frizzled-related protein 4 (Sfrp4), C lq, and tumor necrosis factor-related protein 6 (Clqtnf6), lysyl oxidase-like 3 (Loxl3), immunoglobulin superfamily member 10 (Igsf10), secreted frizzled-related protein 2 (Sfrp2), FK506 binding protein 10 (Fkbp10), glutamine fructose-6-phosphate aminotransferase 2 (Gfpt2), carboxypeptidase X1 (Cpxm l), microfibril-associated protein 5 (Mfap5), epidermal growth factor-containing fibrillin-like extracellular matrix protein 2 (Efemp2), nephroblastoma overexpressed gene (Nov), versican (42yn), elastin (Eln), cysteine-rich protein 61 (Cyr61), sulfatase 1 (Sulf 1), nestin 2 (Nid2), CD248 antigen, endosialin (Cd248), lysyl oxidase-like 2 (Loxl2), follistatin-like 1 (Fstl1), von Willebrand factor type A, EGF and pentraxin domain-containing 1 (Svep1), laminin alpha 4 (Lama4), slit homolog 3 (Slit3), mannose receptor C type 2 (Mrc2), cytoskeleton-associated protein 4 (Ckap4), G protein-coupled receptor 133 (Gpr133), myoblastoid Bundle protein homolog 1, actin bundle protein (Fscn1), elastin microfibril interface 2 (Emilin2), scavenger receptor class A member 3 (Scara3), serine (or cysteine) peptidase inhibitor evolutionary branch B member 2 (Serpinb2), chemokine (CC motif) ligand 2 (Ccl2), insulin receptor (Insr), folate hydrolase 1 (Folh1), CD99 antigen (CD99), casein kappa (Csn3), calcitonin receptor-like (Calcrl), activin A receptor The proteins involved in the study are leukocyte antigen 2 (LEA), leukocyte antigen 2 (LEA), leukocyte antigen 2 (LEA), leukocyte antigen 3 (LEA), leukocyte antigen 4 (LEA), leukocyte antigen 5 (LEA), leukocyte antigen 6 (LEA), leukocyte antigen 7 (LEA), leukocyte antigen 8 (LEA), leukocyte antigen 2 (LEA), leukocyte antigen 10 (LEA), leukocyte antigen 20 (LEA), leukocyte antigen 21 (LEA), leukocyte antigen 3 (LEA), leukocyte antigen 50 (LEA), leukocyte antigen 11 (LEA), leukocyte antigen 12 (LEA), leukocyte antigen 13 (LEA), leukocyte antigen 25 (LEA), p3), integrin alpha 8 (Itga8), sema domain, immunoglobulin domain (Ig), short basic domain secretory (43 synthetic) 3G (Sema3g), transmembrane protein 2 (Tmem2), tumor necrosis factor (ligand) superfamily member 10 (Tnfsf10), HOP homeobox (Hopx), lactalbumin alpha (Lalba), phosphatidylinositol-3,4,5-triphosphate-dependent Rac exchange factor 2 (Prex2), mucin 15 (Muc15), rhotekin2 (Rtkn2), SRY (sex determining region Y)-box 4 (Sox4), tetraspanin 18 (Tspan18), G protein-coupled receptor 126 (Gpr126), C-type lectin domain family 1 member a (Clecla), extra domain of fibronectin-B (ED-B), extra domain of fibronectin-A (ED-A), and hairy / split enhancer associated with YRPW motif 1 (Hey1), as shown in Tables 1 and 2 below.

[0346] Table 1. List of tumor angiogenesis-specific markers

[0347]

[0348]

[0349]

[0350]

[0351] Table 2: Tumor angiogenesis specific markers coupled to mRNA / protein Genbank Acc. No.

[0352]

[0353]

[0354]

[0355]

[0356] More preferably, the polypeptide portion is specific for (i) embryonic tissue and (ii) blood vessels formed in tumor angiogenesis, or (iii) tumor cells. Even more preferably, the autoantigen is selected from the group formed or consisting of vimentin (Vim), CD93, CD99, HER2, HER3, prostate specific membrane antigen (PSMA). Based on the screening methods described herein, the skilled person will be able to determine further autoantigens that meet the above criteria.

[0357] In an alternative preferred embodiment, the autoantigen is a disease-specific antigen selected from Vim and CD99. These autoantigens have anti-tumor effects.

[0358] In another alternative preferred embodiment, the autoantigen is the CD93 antigen. This autoantigen is high in MET and is particularly beneficial in certain aspects of the invention.

[0359] In yet another alternative preferred embodiment, the autoantigen is a specific disease antigen selected from the group consisting of Mmp 14, Cercam, Sulf1, Gpr133, Scara3, Insr, Folh1, CD99, Acvrl1, ItgaS, Muc15. These autoantigens are membrane antigens.

[0360] In yet another alternative preferred embodiment of aspects of the present invention, the autoantigen is a specific disease antigen selected from the group consisting of Timpl, Apln, Saa3, Ptx3, Tnfaip6, Cpz, Pmel, Arsi, Adamts4, Thbs2, Mmp14, Igfbp3, Thbs1, Fbn1, Postn, Loxl3, Mfap5, Efemp2, Nov, 55yn, Eln, Cyr61, Sulf1, Nid2, Lama4, Slit3, Mrc2, Serpinb2, Ccl2, CD99, Tnfsf10, Lalba, Vim. These autoantigens are secretory antigens.

[0361] In yet another alternative preferred embodiment, the autoantigen is a specific disease antigen selected from the group consisting of Apln, Saa3, Vim, Pmel, Arsi, Wisp1, Fkbp10. These autoantigens are cytoplasmic targets.

[0362] In yet another alternative preferred embodiment of aspects of the present invention, the autoantigen is a specific disease antigen selected from the group consisting of Timpl, Apln, Saa3, Ptx3, Vim, Tnfaip6, Cpz, Arsi, Esml, Itga5, Aclamts2, Thbs2, Mmp14, Thbs1, Fbn1, Postn, Fbn2, Cercam, Loxl3, Mfap5, Efemp2, Nov, 55yn, Eln, Cyr61, Sulf1, Nid2, Lama4, Slit3, Mrc2, Gpr133, Scara3, Insr, Folh1, CD99, Acvrl1, Eln, Itga8, Tnfsf10, Lalba, Muc15. These autoantigens are extracellular targets.

[0363] In yet another alternative preferred embodiment of aspects of the present invention, the autoantigen is a specific disease antigen selected from the group consisting of Vim, Cd93, Heg1, Notch4, Aplnr, Nes, Tnc, Snail, Gpx7, Lrrc17, Sfrp4, C1qtnf6, Igsf10, Sfrp2, Gfpt2, Cpxm1, Cd248, Loxl2, Fstl1, Svep1, Ckap4, Emilin2, Csn3, Calcrl, Csf3r, Clca2, Clec14a, Tmem100, Cyyr1, Acer2, Trps1, Arap3, Sema3g, Tmem2, Hopx, Prex2, Rtkn2, Sox4, Tspan18, Gpr126, Clecla, Hey1. These autoantigens are involved in various disease-related processes.

[0364] In yet another alternative preferred embodiment of the present invention, the autoantigen is selected from Vim, Timpl, Apln, Saa3, Cd93, Heg1, Notch4, Aplnr, Nes, Tnc, Ptx3, Vim, Tnfaip6, Cpz, Snail, Pmel, Arsi, Wisp 1. Specific disease antigens in the group consisting of Gpx7, Adamts4, Esm1, Itga5, Adamts2, Thbs2, Mmp14, Igfbp3, Thbs1, Fbn1, Postn, Lrrc17, FFbn2, Cercam, Sfrp4, C1qtnf6, Loxl3, Igsf10, Sfrp2, Fkbp10, Gfpt2, Cpxm1, Mfap5, Efemp2, Nov, 56yn, Eln, Cyr61, Sulf1, Nid2, Cd248, Loxl2, Fstl1, Svep1, Lama4, Slit3, Mrc2, Ckap4, Gpr133, Fscn1, Emilin2, Scara3, Serpinb2, Ccl2, Insr, Folh1, CD99. These self-antigens are involved in tumor development.

[0365] In yet another alternative preferred embodiment of aspects of the present invention, the autoantigen is a specific disease antigen selected from the group consisting of vim, Csn3, Calcrl, Acvrl1, Csf3r, Clca2, Clec14a, Tmem100, Cyyrl, Eln, Acer2, Trps1, Arap3, Itga8, Sema3g, Tmem2, Tnfsf10, Hopx, Lalba, Prex2, Muc15, Rtkn2, Sox4, Tspan18, Gpr126, Clec1a, Hey1. These autoantigens are involved in metastasis.

[0366] Preferably, the autoantigen is the extra domain-B of fibronectin or vimentin (also referred to herein as Vim). More preferably, the autoantigen is the extra domain-B of human fibronectin or human vimentin.

[0367] The term "blood vessel" used herein may refer to a part of the vascular system composed of endothelial cells, extracellular matrix, and basement membrane.

[0368] The term "tumor vasculature" as used herein refers to the vascular system recruited during tumor angiogenesis to ensure blood supply to the tumor. Those skilled in the art can identify tumor vasculature based on vascular structural parameters (e.g., extracellular matrix composition and / or vascular permeability) using (electron) microscopy.

[0369] As used herein, the term "immunostimulatory peptide sequence" refers to a peptide sequence that can be considered antigenic, immunogenic, immunoactivating, etc., wherein all of these terms are used interchangeably.

[0370] As used herein, these terms refer to the ability of a (poly)peptide, molecule, compound (e.g. formulated in a vaccine) to elicit a humoral or cell-mediated immune response (or both). If an antigen / peptide sequence elicits such an immune response, it may also be referred to as an immunogen. Preferably, the immune response is a humoral immune response mediated primarily by B cells and helper T cells. Standard tests for measuring immunogenicity are well known to those skilled in the art. It has been well established that ELISPOT or ELISA can be used to determine T-cell responses, and ELISA can be used to determine humoral immune responses.

[0371] As used herein, the terms "trigger an immune response," "initiate an immune response," "stimulate the immune system," and "activate the immune system" are used interchangeably and refer to activating the immune system to respond to antigens / peptides through humoral and / or cell-mediated immune responses. The terms "enhance," "increase," or "potentiate," as used herein in this context, refer to strengthening an already existing immune response.

[0372] The terms "disease," "disorder," and "condition" are used interchangeably herein and refer to a disorder of structure or function in a human or animal, especially one that produces specific symptoms or affects a specific location and not merely the direct result of a physical injury.

[0373] Diseases that can be treated and / or prevented with the compositions described herein can be sensory diseases and diseases involving angiogenesis. Examples of diseases that can be treated and / or prevented with the compositions described herein are arthritis (e.g., rheumatoid arthritis), atherosclerosis, vascular restenosis, transplant arterial disease, warts, keloids, synovitis, osteomyelitis, asthma, nasal polyps, polypoidal choroidal vasculopathy, age-related macular degeneration, retinopathy of prematurity, diabetic retinopathy, AIDS, IBD, Crohn's disease, endometriosis, uterine bleeding, psoriasis, fibroids, cancer, or a combination thereof.

[0374] "Infectious disease" refers to a disease caused by a pathogen or microorganism (such as bacteria, viruses, protozoa, parasites, yeast, mycobacteria or fungi) entering the human body and growing and multiplying in the body. Examples of infectious diseases that can be treated and / or prevented with the compositions described herein are influenza and coronavirus disease (2019).

[0375] Cancers that can be treated using the fusion polypeptides and compositions of the invention include carcinomas, sarcomas, leukemias, lymphomas, and other types of cancer.

[0376] The term "tumor" as used herein refers to a mass of cells in a tissue that exhibits abnormal growth, which occurs when the cells proliferate faster than normal tissue and continue to grow after the stimulus that normally triggers growth has ceased. The term "tumor" as used herein includes cancer cells, necrosis, and stroma. Tumors typically exhibit a partial or complete lack of structural organization and functional coordination with normal healthy tissues, and typically form a unique mass of tissue that may be benign or malignant. Preferably, the tumor targeted by treatment or vaccination is a malignant tumor that requires tumor angiogenesis to continue to grow and eventually metastasize. More preferably, the tumor is a leukemia or a solid tumor or solid cancer, preferably selected from sarcomas, carcinomas, and lymphomas. Exemplary solid tumors include, but are not limited to, sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, osteosarcoma, angiosarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, breast cancer, Capitulum adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, testicular tumors, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, melanoma, neuroblastoma and retinoblastoma.

[0377] The term "leukemia" refers broadly to progressive malignant diseases of the blood-forming organs and is generally characterized by abnormal proliferation and development of white blood cells and their precursors in the blood and bone marrow. Leukemias are usually clinically classified based on the following factors: (1) the duration and nature of the disease - acute or chronic; (2) the type of cells involved: myeloid (myeloid), lymphoid (lymphoid), or monocytic; and (3) whether the number of abnormal cells in the blood is increased or not - leukemic or aleukemic (subleukemic). Thus, the present invention includes a method for treating leukemia, and preferably, a method for treating the following leukemias: acute non-lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, leukocytic leukemia, 58 synthetic leukemia, blast cell leukemia, bovine leukemia, chronic myeloid leukemia, cutaneous leukemia, embryonic leukemia, eosinophilic leukemia, Gross leukemia, hairy cell leukemia, hemoblastic leukemia, hemoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, Nuclear cell leukemia, leukopenic leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenic leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocyte leukemia, micromyeloid leukemia, monocytic leukemia, myeloblastic leukemia, myeloid leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeh leukemia, plasma cell leukemia, plasma cell leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling leukemia, stem cell leukemia, subleukemic leukemia, undifferentiated cell leukemia.

[0378] The terms "antigen" and "epitope" used herein are well known to those skilled in the art, and although an epitope corresponds to the (small molecule) portion of an antigen to which the immune system will produce antibodies, the two terms can be used interchangeably herein (unless the context does not allow) since they both represent the portion to which the immune system will react. Both are derived from the pathogen to which protection is desired.

[0379] Conservative variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 1, or substitutions, deletions or insertions of non-critical amino acids in non-critical regions of the protein. Therefore, a conservative variant of SEQ ID NO: 1 can be a conservative amino acid sequence of SEQ ID NO: 1, wherein the variant still contains those parts of the amino acid sequence necessary to obtain antibodies against them, which will ultimately provide protection against SARS-CoV-2 infection.

[0380] Conservative substitutions are well known to those skilled in the art and also encompass variants in which amino acids with similar properties are substituted for one another. For example, leucine and isoleucine are both aliphatic, branched-chain hydrophobes. Similarly, aspartic acid and glutamic acid are both small negatively charged residues. Based on the structure of the amino acids and the general chemical characteristics of the side chains, amino acids can be divided into the following six groups:

[0381] Aliphatic (glycine, alanine, valine, leucine, isoleucine)

[0382] Containing hydroxyl groups or sulfur / selenium (serine, cysteine, selenocysteine, threonine, methionine)

[0383] ·Cyclic (proline)

[0384] Aromatic (phenylalanine, tyrosine, tryptophan)

[0385] Alkaline (histidine, lysine, arginine)

[0386] Acidic and their amides (aspartic acid, glutamic acid, asparagine, glutamine).

[0387] Replacement of an amino acid by another amino acid from the same group can be considered conservative.

[0388] Calculations of sequence homology or identity (the terms are used interchangeably herein) between sequences are performed as follows.

[0389] In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, it is necessary to align the sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences to achieve optimal alignment, and non-homologous sequences can be ignored for comparison purposes). In a preferred embodiment, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the two molecules are identical at that position (amino acid or nucleic acid "identity" as used herein is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced to achieve optimal alignment of the two sequences.

[0390] Mathematical algorithms can be used to complete sequence comparison and percent identity determination between two sequences. In a preferred embodiment, the Needleman et al. (1970, J. Mol. Biol. 48: 444-453) algorithm is used, using the BLOSUM62 matrix or PAM250 matrix and gap weights 16, 14, 12, 10, 8, 6 or 4 and length weights 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences, which algorithm has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com). In another preferred embodiment, the GAP program in the GCG software package (available at http: / / www.gcg.com) is used, using the NWSgapdna.CMP matrix and gap weights 40, 50, 60, 70 or 80 and length weights 1, 2, 3, 4, 5 or 6 to determine the percent identity between two nucleotide sequences. A particularly preferred parameter set (and the parameter set that should be used when the practitioner is unsure what parameters should be applied to determine whether a molecule is within the sequence identity or homology constraints of the present invention) is the BLOSUM 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0391] Alternatively, the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Meyers et al. (1989, CABIOS 4: 11-17), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4, which has been incorporated into the ALIGN program (version 2.0).

[0392] The nucleic acid and protein sequences described herein can be used as a "query sequence" to search public databases, for example, to identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990, J. Mol. Biol. 215: 403-410). BLAST nucleotide searches can be performed using the NBLAST program, score = 100, word length = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed using the XBLAST program, score = 50, word length = 3 to obtain amino acid sequences homologous to the protein molecules of the invention. In order to obtain gapped alignments for comparison, Gap BLAST can be utilized as described in Altschul et al. (1997, Nucl. Acids Res. 25: 3389-3402). When using BLAST and Gap BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See<http: / / www.ncbi.nlm.nih.gov> .

[0393] The polypeptides described herein may have an amino acid sequence that is sufficiently or substantially identical to the amino acid sequence of SEQ ID NO: 1 to 22. The term "sufficiently identical" or "substantially identical" as used herein means that a first amino acid or nucleotide sequence contains a sufficient or minimum number of amino acid residues or nucleotides that are identical or equivalent (e.g., have similar side chains) to a second amino acid or nucleotide sequence, such that the first and second amino acid or nucleotide sequences have a common structural domain or a common functional activity. For example, amino acid or nucleotide sequences containing a common structural domain having at least about 60% or 65% identity, possibly 75% identity, more likely 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are defined herein as being sufficiently or substantially identical.

[0394] As used herein, "pharmaceutically acceptable" refers to a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject along with the selected neurotoxin without causing any adverse biological effect or interacting in a deleterious manner with any other ingredients of the pharmaceutical composition in which it is contained.

[0395] Excipients are natural or synthetic substances formulated with active ingredients (e.g., neurotoxins provided herein), and their purpose is to increase the volume of the formulation or enhance the therapeutic effect of the active ingredient in the final dosage form, such as promoting the absorption or dissolution of the drug. Excipients are also useful in the manufacturing process to help handle the relevant active substances, such as by promoting powder fluidity or non-stickiness, and also help in vitro stability, such as preventing denaturation during the expected shelf life. Pharmaceutically acceptable excipients are well known in the art. Therefore, a person of ordinary skill in the art can easily identify suitable excipients. For example, suitable pharmaceutically acceptable excipients include water, saline, aqueous glucose, glycerol, ethanol, etc.

[0396] Adjuvants are pharmacological and / or immunological agents that can modify the effects of other agents in a formulation. Pharmaceutically acceptable adjuvants are well known in the art and include cell penetrating peptides. Thus, one of ordinary skill in the art can readily identify suitable adjuvants.

[0397] Diluents are diluents. Pharmaceutically acceptable diluents are well known in the art and include water or saline. Thus, one of ordinary skill in the art can readily identify suitable diluents.

[0398] A carrier is nontoxic to the recipient at the dosage and concentration employed and is compatible with the other ingredients in the formulation. The term "carrier" refers to an organic or inorganic ingredient, natural or synthetic, that is combined with the active ingredient to facilitate application. Pharmaceutically acceptable carriers are well known in the art and include serum albumin. Thus, one of ordinary skill in the art can readily identify suitable carriers.

[0399] The term "nucleic acid" as used herein generally refers to an oligomer or polymer (preferably a linear polymer) of any length mainly composed of nucleotides. Nucleotide units generally include a heterocyclic base, a sugar group and at least one (e.g., one, two or three) phosphate group, including modified or substituted phosphate groups. The heterocyclic base can especially include purine and pyrimidine bases, such as adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U), which are widely present in naturally occurring nucleic acids, other naturally occurring bases (e.g., xanthine, inosine, hypoxanthine) and chemically or biochemically modified (e.g., methylated), non-natural or 62 synthetic bases. The sugar group can especially include a pentose (pentofuranosyl) group, such as ribose and / or 2-deoxyribose commonly found in naturally occurring nucleic acids, or arabinose, 2-deoxyarabinose, threose or hexose sugar groups, and modified or substituted sugar groups. Nucleic acids as described herein can include naturally occurring nucleotides, modified nucleotides or mixtures thereof. Modified nucleotides may include modified heterocyclic bases, modified sugar moieties, modified phosphate groups, or combinations thereof. Phosphate groups or sugar modifications may be introduced to improve stability, resistance to enzymatic degradation, or some other useful properties. The term nucleic acid further preferably encompasses DNA, RNA, and DNA RNA hybrids, specifically including hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g., chemically synthesized) DNA, RNA, or DNA RNA hybrids. Nucleic acids may be naturally occurring, such as present in nature or separated from nature; or may be non-naturally occurring, such as recombinant (i.e., produced by recombinant DNA technology), and / or partially or entirely chemically or biochemically synthesized. Nucleic acids may be double-stranded, partially double-stranded, or single-stranded. In adenovirus, the linear nucleic acids that form the viral genome typically have a polypeptide derived from a terminal protein (pTP) precursor that is covalently coupled to the 5' end of each polynucleotide chain. When single-stranded, nucleic acids may be sense strands or antisense strands. In addition, nucleic acids may be circular or linear. The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof.

[0400] The term "vector" is well known in the art and is used herein to refer to a nucleic acid molecule, such as double-stranded DNA. In one example, an exogenous nucleic acid sequence is inserted into the vector. The vector can be appropriately used to transport the inserted nucleic acid molecule to a suitable host cell. The vector generally contains all necessary elements that allow the inserted nucleic acid molecule to be transcribed and preferably the transcript to be translated into a polypeptide. The vector generally contains all necessary elements so that once the vector enters a suitable host cell, the vector can replicate independently of the host chromosomal DNA or simultaneously with the host chromosomal DNA; multiple copies of the vector and its inserted nucleic acid molecule can be generated. The vector of the present invention can be an additional vector (i.e., it will not be integrated into the host cell genome) or it can be a vector integrated into the host cell genome. This definition includes non-viral vectors and viral vectors. Non-viral vectors include, but are not limited to, plasmid vectors (e.g., pMA-RQ, pUC vectors, bluescript vectors (pBS) and pBR322 or derivatives thereof that do not contain bacterial sequences (minicircles)), transposon-based vectors (e.g., PiggyBac (PB) vectors or Sleeping Beauty (SB) vectors), etc. Larger vectors such as artificial chromosomes (bacteria (BAC), yeast (YAC) or humans (HAC)) can be used to accommodate larger inserts. In a specific example, the vector described herein can therefore be a plasmid vector. Such a plasmid vector can be present in a cell. In one example, a cell comprising a vector (e.g., a plasmid as described herein) can therefore be provided, the vector comprising a nucleic acid sequence as described herein. Therefore, a cell comprising a nucleic acid sequence of the present invention can be provided.

[0401] The expression vector of the present invention is preferably suitable for inserting the nucleic acid molecule of the present invention into prokaryotic cells (including bacterial cells), or more preferably, into eukaryotic host cells (such as yeast cells and mammalian cells). Particularly preferred are expression vectors pET-21a or pCMV4. Further expression vectors compatible with the present invention are TOPO, CMV or pcDNA3.1.

[0402] The vector defined herein may also be a viral vector. "Viral vector" is defined as a recombinantly produced virus or viral particle, which contains a polynucleotide that can be delivered to a host cell in vivo, in vitro or in vitro. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors (AAV), alphavirus vectors, etc. Usually, but not necessarily, viral vectors are replication-defective because they lose the ability to reproduce in a given cell because the viral genes necessary for replication have been eliminated from the viral vector. However, some viral vectors are also suitable for specific or preferential replication in a given cell (such as a cancer cell), and are generally used to trigger (cancer) cell-specific (tumor) lysis. These viral vectors are referred to herein as "oncolytic viruses". Virions are non-limiting examples of vectors comprising viral and non-viral elements, particularly they combine liposomes with inactivated HIV or influenza viruses (Yamada et al., 2003). Another example includes viral vectors mixed with cationic lipids.

[0403] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention relates. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide a general dictionary of many of the terms used in the present invention for those skilled in the art. Although any methods and materials similar or equivalent to those described herein can be used to practice the present invention, preferred methods and materials are described herein. Therefore, the terms defined immediately below will be more fully described by reference to the entire specification.

[0404] Aspects of the invention are demonstrated by the following non-limiting examples.

[0405] Example

[0406] In an embodiment, the sequence of the CDP corresponds to SEQ ID NO: 5 described above.

[0407] Example Section A

[0408] Production of RBD-targeted conjugate vaccines according to the present invention

[0409] In order to achieve strong immune recognition of the RBD domain of SARS-CoV-2 ( Figure 1 A), whose sequence was combined with an engineered chimeric design peptide (CDP) ( Figure 1 B). The CDP has selected clusters of amino acids with bulky hydrophilic or charged side chains derived from three different bacterial (E. coli) proteins, namely the cell division protein ZapB (uniprot#P0AF36), type I fimbriae protein (A chain) (TFP, uniprot#P04128), and the small heat shock protein IbpA (uniprot#P0C054). The resulting binding protein CDP-RBD and the RBD alone ( Figure 1 C) and then purified as described previously. Notably, the addition of the CDP component to the RBD increased the solubility of the final conjugated protein ( Figure 1 C) and facilitates purification ( Figure 2 The purified protein was verified by SDS-PAGE and Western blot analysis using a commercial anti-RBD antibody ( Figure 1 D). In addition, size exclusion chromatography confirmed the purity of CDP-RBD ( Figure 1 E). Finally, computer analysis of the CDP-RBD protein sequence revealed α-helical or β-strand domains, as well as predicted B cell- and CD4 + / CD8 + T cell epitopes ( Figure 2 C), indicating strong immune recognition.

[0410] CDP-RBD induced a faster and stronger antibody response than RBD alone

[0411] To evaluate the in vivo immunogenicity of CDP-RBD and compare it to its unconjugated RBD counterpart, BALB / c mice were immunized subcutaneously with 100 μg of each purified protein by priming on day 0 and boosting on day 14 ( Figure 3 A). Montanide ISA 720 (water-in-oil emulsion) supplemented with CpG 1826 oligonucleotide (Toll-like receptor 9 agonist) was selected as the vaccine adjuvant (referred to as MnC) based on its previously reported safety and efficacy ( Figure 3 B) Mouse sera were collected before immunization (day 0) and on days 13, 21, 28 and 35 ( Figure 3 A) and used for ELISA detection of anti-RBD antibodies. No antibodies against the RBD domain were present in the pre-immune sera (day 0). By day 13, seroconversion was detectable in several mice in each group ( Figure 3 C and Figure 4 A), and by day 21, all mice immunized with CDP-RBD mounted a strong immunoglobulin (Ig) response to RBD. At this time, only 50% of RBD-immunized mice showed a strong RBD-specific humoral response ( Figure 3 C、3D left and Figure 4 A), whereas no anti-RBD antibodies were detected in 2 / 10 mice that received the unconjugated RBD vaccine ( Figure 3 C). This result confirms that RBD is moderately immunogenic. Importantly, although the difference in total anti-RBD Ig levels between the two vaccine groups appeared to disappear at later time points ( Figure 4 A), but evaluation of antibody titers showed that even three weeks after the booster injection, RBD-immunized mice still lagged behind CDP-RBD mice ( Figure 2 C, 2D right). These findings suggest that vaccination against RBD using the iBoost platform resulted in a faster and stronger anti-RBD antibody response relative to RBD alone.

[0412] Next, the production of IgG subclasses was investigated. IgG1 and IgG2b represent T helper (Th) 2-skewed immune responses, while IgG2a and IgG3 are associated with the induction of Th1 responses. By day 21, mice immunized with CDP-RBD produced significantly higher levels of anti-RBD IgG1 and IgG3 relative to mice vaccinated with RBD ( Figure 3 E and Figure 4 B). In addition, in the CDP-RBD group, the IgG2a response showed a more robust and homogeneous trend ( Figure 3 E and Figure 4 B). Interestingly, a higher number of mice were able to mount full IgG1, IgG2a, IgG2b, and IgG3 responses following immunization with CDP-RBD rather than unconjugated RBD ( Figure 3 E). For example, RBD-specific IgG3 was measured in 7 / 10 CDP-RBD-immunized mice, whereas none of the RBD-injected mice were found to be IgG3 positive ( Figure 3 E and Figure 4 B). Thus, the CDP-RBD vaccine showed signs of an improved Th1-based immune response (indicated by higher IgG2a and IgG3 titers) as early as seven days after the second vaccination (day 21) and simultaneously ensured a more balanced Th1 and Th2 response (based on the presence of all IgG subtypes).

[0413] Given that strong immune recognition is achieved after RBD binds to CDP, it is speculated that CDP-RBD recipient mice will also produce anti-CDP antibodies. On day 21, CDP-specific antibodies were only detected in the CDP-RBD group ( Figure 4 C). Considering the improved RBD-specific antibody responses observed in mice immunized with the CDP-RBD conjugate vaccine, immune responses to the bacterial CDP fusion partner do not appear to have a negative impact on the immunogenicity of the RBD antigen. Furthermore, to exclude the possibility that the differences between the two vaccine groups were due to the adjuvant used rather than the presence of a specific CDP, mice were immunized in the same experiment with RBD or CDP-RBD in combination with the alternative adjuvant Sepivac (Sep). Figure 3 A). Sepivac is a water-in-oil adjuvant, unlike Montanide, an oil-in-water emulsion, which is currently being tested for multiple influenza and COVID-19 vaccines. Again, CDP-RBD / Sep outperformed the RBD / Sep vaccine in terms of anti-RBD total Ig induction ( Figure 4 D), similar to CDP-RBD / MnC, indicating that CDP is a key component in enhancing humoral immune responses. Importantly, CDP-RBD, whether combined with MnC or Sep, was able to induce a potent antibody response against RBD, even at protein levels below 100 μg ( Figure 4 E). When combined with Sepivac, CDP-RBD induced a potent immune response even at a protein concentration of 10 μg ( Figure 4 E). Alternative neutralization assay showed that sera from CDP-RBD-immunized mice exhibited higher inhibition of RBD:ACE-2 binding compared with sera from RBD-immunized mice from day 21 ( Figure 3 F). This emphasizes that excellent neutralization capacity was achieved one week after the second vaccination with the CDP-RBD conjugate vaccine. Consistent with this, surface plasmon resonance (SPR) analysis showed that sera from mice vaccinated with CDP-RBD had significantly higher binding levels to immobilized RBD than sera from RBD-vaccinated mice ( Figure 3 G, H).

[0414] Vaccination with CDP-RBD induced systemic T cell immunity and enhanced CD8 + T cell response

[0415] In addition to antibodies, antigen-specific T cells are also critical for clearing SARS-CoV-2 infection. Therefore, the next goal was to determine whether CDP-RBD induced a stronger T cell response in mice compared to unconjugated RBD. To assess this, spleens were removed from vaccinated mice on day 21, as this is the time point at which differences in antibody responses and neutralizing capacity began to emerge between the two vaccine groups. Splenocytes were restimulated in vitro using a panel of 15 amino acid-long peptides covering the S1 domain of the spike protein, including the RBD. Figure 5 A). Unstimulated splenocytes were used as negative control. After 5 h of stimulation in the presence of brefeldin A, the induction of interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) expression ( Figure 5 B, 5D), antigen-specific CD4 + and CD8 + The frequency of T cells ( Figure 6 A). More specifically, monofunctional (IFNγ + TNFα + ) and multifunctional (IFNγ + TNFα + )CD4 + Clear populations of T cells ( Figure 5 B, 5C and Figure 6 B), but no obvious discernible differences were found between the two vaccine groups. In contrast, monofunctional and polyfunctional RBD-specific CD8 + The percentage of T cells was significantly higher ( Figure 5 D, 5E and Figure 6 B), indicating that binding to CDP had a positive effect on systemic cytotoxic T cell (CTL) responses. To further clarify the link between humoral and cellular immune responses in these vaccine groups, the correlation between anti-RBD antibodies and RBD-specific T cells was tested ( Figure 5 F). Interestingly, multiple components of the antibody response were found to be positively correlated with T cell responses in CDP-RBD-immunized mice (multifunctional CD8 + T cells), while IgG1 was the major IgG subtype, which was shown to be highly associated with T cells in the case of RBD recipients ( Figure 5 F-3H). Taken together, these data suggest that the combined technology is advantageous for inducing coordinated and fine-tuned antibody and T cell responses very rapidly after the second vaccination.

[0416] CDP-RBD vaccine protects against COVID-19 in hamsters

[0417] In addition, it was investigated whether the CDP-RBD vaccine could provide protection against SARS-CoV-2 infection in addition to inducing RBD-specific antibody and T cell responses. Therefore, Syrian hamsters were injected intramuscularly with CDP-RBD plus MnC or with Tris-sucrose alone (control) on days 0 and 21. Three weeks after the booster vaccination (day 42 of the experiment), all hamsters were given an intranasal 10 4 SARS-CoV-2 virus particles were challenged, which is equivalent to the median tissue culture infectious dose (TCID50), and serum samples were collected at four and seven days post-infection (dpi) ( Figure 7 A). The results showed that the CDP-RBD vaccine induced a high antibody titer against RBD after a single vaccine dose (day 21), and booster vaccination further increased the anti-RBD antibody titer ( Figure 7 B and Figure 8 A). As expected, control hamsters did not show anti-RBD antibodies after vaccination. However, seven days after challenge with SARS-CoV-2, control hamsters also showed elevated anti-RBD antibodies, although these did not reach the antibody titers detected in CDP-RBD-vaccinated hamsters ( Figure 7 B and Figure 8 A). In agreement with the higher antibody titers, CDP-RBD vaccinated hamsters showed faster viral clearance from the throat. More precisely, at 2 and 3 dpi, significantly lower numbers of replicating viruses were found ( Figure 7 C). In addition, at 4 dpi, lower amounts of virus were found in the lungs and nasal turbinates of CDP-RBD-inoculated hamsters ( Figure 8 B). After infection with SARS-CoV-2, both groups lost approximately 5% of their body weight within the first 4 dpi. Nevertheless, CDP-RBD-vaccinated hamsters maintained their body weight from this point on (day 46), whereas control-vaccinated hamsters lost significantly more body weight ( Figure 7 D) To elucidate whether CDP-RBD-induced antibodies have neutralizing capacity, an alternative neutralization assay was performed ( Figure 7 E). Antibodies from CDP-RBD-vaccinated animals blocked the binding of RBD to ACE-2 on the day of viral challenge (day 42). Neutralization continued to be significantly improved at 4 dpi compared with controls. One week after viral challenge (day 49), both groups of hamsters showed a high degree of inhibition of binding between RBD and ACE-2 ( Figure 7E). Surprisingly, histopathological analysis of lung tissue revealed typical COVID-19 features such as congestion, emphysema, hemorrhage, bronchioloalveolar hyperplasia, and inflammation or edema in all control hamsters but not in CDP-RBD-vaccinated hamsters. Up to 40% of lung tissue showed lesions at 7 dpi in control hamsters, while CDP-RBD-vaccinated hamsters showed no signs of SARS-CoV-2 pathology ( Figure 7 F).

[0418] discuss

[0419] Here, it is proposed to enhance the immunogenicity of viral antigens by combining them with bacterial chimeric sequences. A key component of the present invention is CDP (chimeric design peptide), which is composed of bacterial protein sequences rich in highly immunogenic amino acid clusters. CDP should also be conducive to improving the immune response to key but only moderately immunogenic viral sequences (such as the RBD domain of SARS-CoV-2 spike protein). Therefore, CDP is combined with the RBD sequence, and this causes the immune recognition of RBD antigens to be significantly improved.

[0420] It has been observed that immunization of mice with the conjugated CDP-RBD vaccine produced a faster, stronger and more balanced IgG response compared to vaccination with unconjugated RBD. Inducing an early immune response is considered crucial when targeting highly infectious pathogens such as SARS-CoV-2, especially in pandemic situations. In addition, a mature response may reduce the risk of COVID-19 mortality associated with Th2 / Th1 cytokine differences. Here, an improved IgG response in the CDP-RBD group was observed. Notably, only CDP-RBD-immunized mice were able to produce IgG3, an IgG subclass associated with complement activation. In addition, vaccination with CDP-RBD enhanced the skewing towards a Th1 immune response (based on IgG2a and IgG3 levels), which is essential during viral infection and is also a prerequisite for the success of many vaccine candidates. Regardless of the technology used, the production of vaccine-induced neutralizing antibodies is largely dependent on CD4 with a Th1 phenotype. + T cells. Relatedly, it has been found that the combination of CDP and RBD can significantly improve serum neutralization capacity and binding to RBD in mice from day 21 compared to RBD alone. In addition, using SPR analysis, we found that CDP-RBD-derived serum antibodies bound more strongly to immobilized RBD, indicating higher affinity and consistent with enhanced inhibition of ACE-2:RBD binding.

[0421] In addition to humoral immunity, the induction of cellular immune responses is also crucial for protection against the virus, especially since antibodies have been shown to wane soon after infection or vaccination. In addition to their quantity, the functionality of SARS-CoV-2-specific T cells determines the timing and efficiency of viral clearance from the body and is inversely correlated with disease severity. Moreover, certain worrisome SARS-CoV-2 variants were unable to escape recognition by S-specific memory T cells, as often happens with antibodies. Therefore, there is particular interest in the generation of SARS-CoV-2-specific T cells after vaccination. Established methods for the ex vivo assessment of antigen-reactive T cells in mice or humans involve the quantification of cells expressing the Th1 cytokines IFNγ and TNFα after restimulation with an overlapping pool of peptides corresponding to the target antigen. In this study, CDP-RBD-immunized mice exhibited CD4 + and CD8 + RBD-specific T cells were detected in the two groups, indicating the successful induction of systemic cellular responses one week after the booster vaccination. + The frequencies of T cells were similar. Nevertheless, CDP-RBD increased reactive monofunctional and polyfunctional CD8 + The frequency of T cells, which are supported by Th1 cells and play a key role in killing infected cells when they encounter their cognate antigens, suggests that the use of the present invention can improve the potential cytotoxic T cell response and thereby increase protection in vaccinated individuals, as CD8 specific for certain coronavirus epitopes + T cells were associated with the development of mild COVID-19. Splenic RBD-specific CD8 + The proportion of T cells is less than that of CD4 + This is consistent with previous reports showing that memory responses in the context of SARS-CoV-2 are more skewed toward CD4 + T cell compartments, not CD8 + T cell compartment. Finally, CDP-RBD-immunized mice showed better correlation of antibody responses with cellular responses compared with RBD-immunized mice. This suggests that presenting viral antigens in the context of a conjugate vaccine with a selected foreign partner has the potential to mobilize both branches of the adaptive immune response in a more robust and coordinated manner than presenting viral antigens alone.

[0422] In addition to the improved immune responses in mice, the data also showed that CDP-RBD can prevent severe COVID-19 in a hamster model. CDP-RBD-vaccinated hamsters showed strong antibody responses, no lung lesions and reduced weight loss after SARS-CoV-2 challenge compared with controls. Protection was achieved despite a reduction of ∼0.5log10 TCID50 in the amount of competent virus in the throat (per mL) and nasal turbinates (per gram of tissue). However, CDP-RBD-vaccinated animals had a reduction of ∼3log10 TCID50 / g of competent virus in the lungs at 4 dpi, indicating that protection against lung lesions strongly depended on viral load in the lungs rather than in the upper respiratory tract. This observation is consistent with SARS-CoV-2 infection in humans, where viral replication in the lower respiratory tract is the main cause of increased mortality. Moreover, the reduction of ∼3log10 TCID50 / g in the lungs is comparable to the reduction in specific variants of concern after vaccination with the licensed ChAdOx1nCoV-19 (AZD1222) vaccine. Consistent with this observation, the two animals that showed no signs of lung lesions on day 46 of the experiment were the same animals that showed a strong reduction in competent virus in the lungs (~5log10TCID50 / g) and high levels of neutralizing antibodies (~100% inhibition of ACE-2:RBD binding). CDP-RBD-stimulated neutralizing antibody levels were comparable to those of natural infection one week after challenge. While natural infection produced higher neutralizing antibody levels after seven days, in CDP-RBD-immunized hamsters, moderate neutralization was already achieved three weeks after the second vaccination / on the day of viral challenge. In addition, a significant increase in neutralizing capacity was observed at 4dpi, emphasizing the importance of early manifestation of neutralizing antibodies for viral clearance and patient survival.

[0423] In general, protein subunit vaccines are a mature technology for many years with proven efficacy and safety, especially pneumococcal polysaccharide vaccines or MenACWY vaccines. In addition, protein vaccines are relatively stable within the range of 2-8°C suitable for refrigeration, and are therefore easy to distribute and more economical to implement, especially in countries with limited resources. The invention described herein not only induces faster and stronger B cell and T cell immune responses, but also allows specific targeting of key epitopes within key viral proteins (such as the receptor binding domain of the spike protein). In addition, the present invention provides the possibility of displaying domains of different viral variants (e.g., δ and ω SARS-CoV-2 variants) or even conserved sequences (e.g., between SARS-CoV-1, -2 and MERS-CoV), regardless of their original immunogenicity. In this way, the present invention can promote the design and further development of multi-epitope vaccines that can induce more targeted immune responses and thus provide defense against new transmission variants. In summary, the present invention represents a promising method for future vaccination strategies against different viruses and pathogenic microorganisms.

[0424] Materials and Methods

[0425] Vaccine design

[0426] The genome of Escherichia coli (E. coli) (strain K12) was examined and three proteins rich in clusters of amino acids with hydrophilic or charged side chains were identified: type 1 fimbriae protein TFP, cell division protein ZapB, and small heat shock protein IbpA. These immunogenic clusters were fused to form a single peptide, called a chimeric designed peptide (CDP), with a total length of 60 amino acids (5.8 kDa). For the RBD, a sequence encoding amino acids 330 to 524 (a total of 194 amino acids) of the reported SARS-CoV-2 genome (GenBank: MN908947.3) was used. In addition, a (GS)3-linker (GSGSGS) was added between the CDP and RBD domains and a terminal His6-tag (HHHHHH) was added to facilitate proper antigen display and vaccine purification, respectively.

[0427] Vaccine protein production

[0428] As previously described (Huijbers et al., 2018), RBD and CDP-RBD proteins were expressed and purified. In brief, sequences encoding RBD or CDP-RBD (codons optimized for expression in E. coli) were synthesized by Genscript (USA) and inserted between the Nde1 and Xho1 restriction sites of the pET21a (+) expression vector. For protein expression, 10 ng of plasmid DNA was transfected into competent BL21 (DE3) E. coli (Sigma-Aldrich, #69450) by heat shock method. Individual clones were cultured overnight at 200 rpm and 37 ° C in a non-humidified shaker (Laboshake) in LB medium supplemented with ampicillin (100 μg / ml). 1 mM isopropyl β-d-1-thiogalactoside (IPTG) (Serva, #26600.04) was added to the overnight culture diluted 1:2.6 to induce protein expression. After 4 hours, the bacteria were harvested and the bacterial pellet (equivalent to 50 ml culture volume) was dissolved in 5 ml of sonication buffer containing 0.5 M EDTA (Sigma-Aldrich, #E5134), 1% N-lauroylsarcosine (Sigma-Aldrich, #L5125), 1% phenylmethanesulfonyl fluoride solution (PMSF) (Sigma-Aldrich, #93482) and 6 M urea (Acros Organics, #10665572) in PBS (VWR, #L0500-500). Proteins were released from the bacteria by high frequency vibration (ultrasound) for 15 cycles on ice, 20 seconds "on" and 30 seconds "off" per cycle (Soniprep 150MSE, amplitude 22-24 microns). For protein purification, the supernatant of the sonicated sample (after centrifugation at 4500 rpm for 20 minutes) was mixed with 200 μl of 50% Ni-NTA agarose slurry (Qiagen, #30210) and incubated overnight at 4°C on a roller test bench. The next day, the Ni-NTA agarose beads were washed five times with a wash buffer containing 1 M NaCl (VWR, #27788.366) and 0.05% Tween-20 (Sigma-Aldrich, #P7949) in PBS. Subsequently, the beads were transferred to a column, which was a syringe with a glass filter (Sartorius, #13400-100------K).The protein was eluted in two steps: 100 μl of each of the first four fractions was eluted in 1M Tris-Cl (pH 8.0), 100 mM NaCl, 200 mM imidazole (JT Baker, #1747.0100), 1 mM PMSF; 100 μl of each of the next four fractions was eluted in 10 mM Tris-Cl (pH 4.5), 100 mM NaH2PO4 (Merck, #1.06346.1000), 8 M urea. CDP-RBD was eluted in all eight fractions, while RBD was eluted only in the last four fractions containing 8 M urea. The purified protein fractions were pooled and dialyzed stepwise against 2 M urea (4 M, 3 M, 2.5 M, 2 M urea). The final protein concentration was determined by BCA assay (Thermo Scientific, #23235). SDS-PAGE and Coomassie blue staining (. Brilliant Blue G 250, Serva, #17524.02), Western blot analysis, and size exclusion chromatography to confirm the purity and identity of the protein.

[0429] Western blotting

[0430] The identity of the protein was confirmed by Western blotting. Approximately 20 μg of purified protein was loaded onto a Mini- TGX TM Precast protein gel (Biorad, #4561094, 4561096) and perform gel electrophoresis. Subsequently, the protein was transferred to an immobilon PVDF membrane (Millipore, #IPFL00010). The membrane was blocked with PBS containing 0.05% Tween-20 (PBS-T) and 5% BSA fraction V (Roche, #10735086001) for 1 hour at room temperature. Rabbit anti-RBD antibody (R&D systems, #MAB10540, 1 / 250 dilution) was used as the primary antibody and overnight at 4°C. The next day, wash 5 times with 0.05% PBS-T, and then the secondary antibody goat anti-mouse IRDye 800CW (LI-COR Biosciences, #926-32210, 1 / 10000 dilution) was added to the membrane for 30 minutes. After washing five times with PBS-T and once with PBS, proteins were visualized using an Odyssey infrared imaging system (model 9120, LI-COR Biosciences). A prestained Spectrum Multicolor Broad Spectrum Protein Ladder (Thermo Fisher, 26634) was used as a size reference.

[0431] Analytical gel filtration assay

[0432] Analytical gel filtration was performed using a Superdex 75 10 / 300GL column (Cytiva). The Pure system was run in 2 M urea in phosphate buffered saline (PBS, pH 7.4) at a flow rate of 0.5 ml / min. Each injection contained 100 μl of sample at a concentration of 2 g l -1 The absorbance was monitored at 280 nm.

[0433] B. CD4 + and CD8 + Prediction of T cell epitopes

[0434] Linear B cell epitopes in the CDP-RBD sequence were predicted by BepiPred using an epitope threshold of 0.55. + For T cell epitopes, we utilized the NetMHCIIpan4.0 server. Specifically, all overlapping 15 amino acid length peptides contained in the CDP-RBD sequence were tested for binding affinity to murine H-2 alleles (H-2-Iau, H-2-Ied, H-2-Iek) as well as multiple common human HLA-DP, HLA-DQ, and HLA-DR alleles. Similarly, we identified a predicted 8 amino acid length CD8 in CDP-RBD using the NetMHCpan 4.1 server. + T cell epitopes. Binding to murine H-2 alleles (H-2-Db, H-2-Dd, H-2-Dq, H-2-Kb, H-2-Kd, H-2-Kk, H-2-Kq, H-2-Ld, H-2-Lq) and different human HLA class I alleles was examined.

[0435] Mouse vaccination

[0436] All mouse experiments were approved by the Dutch nationalethics board Centrale Commissie Dierproeven (CCD, registration number AVD11400202010545) and were performed in accordance with the Dutch animal experiment guidelines and laws. Two independent immunization studies (n=5 in each group) were performed using 8-week-old female BALB / c OlaHsd mice (Envigo). One study was terminated on day 21 and the other on day 35. After 2 weeks of adaptation, mice received a primary immunization on day 0 and a booster immunization on day 14 ( Figure 3A). Each mouse was injected subcutaneously in the left groin with 100 μg of purified CDP-RBD or RBD. The protein was mixed with Montanide ISA 720 (Seppic, #36059VFL2R3) and 50 μg CpG oligo 1826 (Eurogentec, #1826) in a ratio of 40:50:10, abbreviated as MnC. For concentration studies, each mouse was injected subcutaneously in the left groin with 100, 30, 10 or 3 μg of purified RBD or CDP-RBD in combination with Montanide ISA 720 and CpG (40:50:10, MnC) or Sepivac SWE (Seppic, #1849101, 50:50). Blood samples were collected from the tail vein before immunization (day 0) and on days 13, 21, 28 and 35. One mouse from the CDP-RBD MnC group was sacrificed due to an open wound at the vaccine injection site between days 28 and 35. All remaining mice were sacrificed on day 35.

[0437] Detection of anti-RBD antibodies in mouse sera by ELISA

[0438] After blood samples were coagulated overnight at 4°C, they were centrifuged twice at 7000rpm (10min, 4°C). Serum was collected and stored at -20°C until further use. Flat-bottomed 96-well plates (Thermo Fisher Scientific, #442404) were coated with recombinant RBD (GenScript, #Z03483) for 1 hour at 37°C, with a final concentration of 2 μg / mL (total Ig plates) or 0.5 μg / mL (IgG1, IgG2a, IgG2b and IgG3 plates) in PBS. The plates were washed once with PBS (PBS-T) containing 0.1% Tween-20 and blocked for 1 hour at 37°C with 1% skim milk powder (Santa Cruz, #SC-2325) in PBS-T. After washing once with PBS-T, serially diluted (1 / 100 to 1 / 72900 in blocking solution) mouse serum was added and incubated at 37°C for 45 minutes. A monoclonal antibody (R&D Systems, 1 / 500 dilution) against the RBD of SARS-CoV-2 was used as a positive control. The plate was washed four times with PBS-T. Biotinylated goat anti-mouse total Ig (Dako, #E0433), IgG1 (Southern Biotech, #1070-08), IgG2a (Southern Biotech, #1080-08), IgG2b (Southern Biotech, #1090-08) and IgG3 (Southern Biotech, #1100-08) antibodies diluted in PBS-T (1 / 2000) were incubated at 37°C for 45 minutes. After washing four times with PBS-T, the plate was incubated with streptavidin-HRP (Dako, #P0397) diluted in PBS-T (1 / 2000) at 37°C for 30 minutes. The plate was washed four times with PBS-T and developed with TMB (Sigma, #T0440) for 10 minutes. The absorbance was measured at 655 nm on a microplate reader (BioTek Synergy HTX). Actual values ​​were obtained by subtracting the blank (wells treated identically to the sample wells except that blocking solution was added instead of mouse serum) OD values ​​from the actual OD values.

[0439] Neutralization assay

[0440] To measure the neutralizing capacity of the induced anti-RBD antibodies, the SARS-CoV-2 surrogate virus neutralization assay from Genscript (USA, #L00847) was performed according to the manufacturer's instructions.

[0441] SPR biosensor assay

[0442] Surface plasmon resonance (SPR) biosensor assays have been performed using a Biacore T200 (GE Healthcare) with a CM5 sensor chip (Cytiva). According to the manufacturing protocol, RBD (R&D systems) with a concentration of ~6 μg / ml was immobilized at a density of ~2 kRU in 10 mM acetate buffer pH 4.5 using an amine coupling kit (Cytiva) at a flow rate of 5 μl / min. For binding analysis, serum samples were diluted 1:100 in PBS buffer pH 7.4 supplemented with 0.05% Tween-20 and then injected into the sensor chip surface at 25 ° C at a flow rate of 30 μl / min for 240 seconds. After the injection, the complex formed was tracked for 180 seconds to dissociate. After each cycle, the chip surface was regenerated by injecting 10 mM Gly, pH 2, and 0.5 M urea for 30 seconds.

[0443] Detection of anti-CDP antibodies in mouse sera by ELISA

[0444] CDP was fused to a truncated (first 58 C-terminal amino acids) bacterial thioredoxin (uniprot #P0AA25) (TRXtr-CDP). TRXtr-CDP was produced in BL21 (DE3) E. coli (Novagen) as described above and used to coat flat-bottom 96-well plates (Thermo Fisher Scientific) at a final concentration of 2 μg / mL in PBS for 1 hour at 37°C. The plates were washed and blocked as described above. Mouse serum was diluted in 100% BL21 extract, serial dilutions were added and incubated at 37°C for 45 minutes. To detect anti-CDP antibodies, biotinylated goat anti-mouse total Ig (Dako) antibody diluted in PBS-T (1 / 2000) was incubated at 37°C for 45 minutes. The plates were washed four times with PBS-T and then incubated with streptavidin-HRP (Dako) diluted in PBS-T (1 / 2000) for 30 minutes at 37°C. Plates were further washed, developed and analyzed as described above.

[0445] Splenocyte isolation and restimulation

[0446] Spleens from mice immunized with RBD / MnC or CDP-RBD / MnC were isolated on day 35, cut into small pieces and mechanically dissociated. Splenocytes were passed through a 70 μm cell strainer (Corning, #431751) and spun at 1500 rpm (brake 5, room temperature) for 5 minutes in a Rotina 420R (Hettich) centrifuge. Red blood cells were lysed after incubation with ammonium chloride-potassium lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 100 mM EDTA, pH = 7.4) for 3 minutes at room temperature. The spleen cells were washed once and then resuspended in RPMI-1640 (Gibco, #L0495-500) medium supplemented with 10% FCS (Biowest, #S0750), 1% Pen / Strep (Gibco, #15140-122), 1% L-glutamine (BrunschwigChemie BV, #HN08.2) and 50 μM 2-mercaptoethanol (Gibco, #31350-010). The isolated spleen cells were seeded in U-bottom 96-well plates (Greiner Bio-One, #650180) (2*10 6 Cells were cultured in 4% paraformaldehyde (400 μg / mL) and restimulated with a SARS-CoV-2 peptide cocktail (Miltenyi Biotec, #130-127-041) in the presence of Brefeldin A (BioLegend, #420604) for 5 hours ex vivo. Peptides of 15 amino acids in length with 11 amino acids overlapping, covering the S1 domain (containing the RBD) of the spike glycoprotein, were used. Unstimulated splenocytes treated with Brefeldin A were used as negative controls.

[0447] Identification of RBD-specific T cells using flow cytometry

[0448] After restimulation, cells were transferred to a V-bottom 96-well plate (Greiner Bio-One, #651180) and washed once with PBS. Cells were suspended in TruStain Fc blocking solution (BioLegend, #101320) for 10 minutes at room temperature. Subsequently, cells were incubated on ice for 20 minutes with anti-mouse CD4-AF700 (BioLegend, #100429, 1 / 200 dilution), anti-mouse CD8b-AF488 (BioLegend, #126627, 1 / 200 dilution) and Zombie Aqua fixable viability dye (BioLegend, #423101, 1 / 200 dilution) diluted in PBS. Cells were washed once with PBS and fixed on ice for 15 minutes with 4% paraformaldehyde (PFA; Electron Microscopy Sciences, #15710). After fixation, cells were washed once with PBS and permeabilized using intracellular staining permeabilization wash buffer (BioLegend, #421002) according to the manufacturer's instructions. The cell suspension was then incubated with anti-mouse IFNγ-APC (BioLegend, #505305, 1 / 200 dilution) and TNFα-PE (BioLegend, #506305, 1 / 200 dilution) diluted in intracellular staining permeabilization wash buffer at room temperature for 30 minutes. The cells were washed twice with permeabilization wash buffer, resuspended in 100 μl PBS, and transferred to a FACS tube. Fluorescence intensity and the percentage of IFNγ- and TNFα-expressing cells were measured using an LSRII (BD Biosciences) flow cytometer. Data analysis was performed using FlowJo V10 software (Tree Star).

[0449] Hamster

[0450] Male Syrian hamsters (Golden Hamsters) were purchased from Janvier (France). At the start of the experiment, the animals were 9 weeks old and weighed 98-114 g. During the acclimation and vaccination phases, hamsters were housed in type 2 cages with a maximum of two animals per cage under DM (BSL)-II conditions. On the day of virus inoculation, the hamsters were transferred to standard extended type 2 group cages with two animals per cage under BSL-III conditions (isolators), using sawdust as bedding and cage enrichment. For all invasive animal procedures, nasal and intramuscular administration, blood sampling, throat swab collection and euthanasia, the animals were sedated with isoflurane (3-4% / O2). Hamsters were vaccinated by the intramuscular route (im) as planned ( Figure 7A). Collect blood by orbital bleeding. Immediately transfer blood sample for serum into appropriate tubes containing coagulation activator. Collect serum, heat treat, aliquot and store frozen.

[0451] Hamster vaccination

[0452] On the 0th day and the 21st day, animals were vaccinated by intramuscular injection (im) using a Tris-sucrose vaccine (as a control) or 100 μg CDP-RBD (mixed with Montanide ISA720 and 67 μg CpG oligo 1826, MnC), with a total injection volume of 100 μl. A syringe equipped with a 29G (0.33x12.7 mm) needle was used to inject two hind legs of hamsters. In brief, the hind limbs were extended and the inoculum was injected into the outer thigh (biceps femoris) with a short-distance fluid motion, avoiding the tail muscle to prevent the risk of damaging the sciatic nerve. The animals were returned to the cage and monitored during recovery.

[0453] SARS-CoV-2 vaccination

[0454] On day 42, all hamsters were inoculated intranasally (in) with a total volume of 100 μl of inoculum at 10 4 The animals were challenged with a median tissue culture infectious dose (TCID50) of SARS-CoV-2 virus particles (BetaCoV / Munich / BavPat1 / 2020, European Virus Archive Global). On the fourth day after the challenge, half of the animals in each group were sacrificed by exsanguination under isoflurane anesthesia and autopsied. On the seventh day after the challenge, the remaining half of the animals in each group were sacrificed by exsanguination under isoflurane anesthesia and autopsied.

[0455] Detection of anti-RBD antibodies in hamster serum by ELISA

[0456] ELISA was performed as described above. A biotinylated goat anti-hamster IgG antibody (Southern Biotech, #6060-08, 1 / 2000 dilution) was used to detect hamster antibodies.

[0457] Sampling after inoculation

[0458] In the challenge phase of the study, samples were collected from the respiratory tract every day. In short, pharyngeal swabs (FLOQSwabs, COPAN Diagnostic Inc., Italy) were used to sample the pharynx by rubbing the swabs against the back of the animal's throat to soak the swabs with saliva. Subsequently, the swabs were placed in a test tube containing 1.5 mL of virus transport medium (Eagles minimum essential medium, containing Hepes buffer, sodium bicarbonate solution, L-glutamine, penicillin, streptomycin, BSA fraction V and amphotericin B), divided into three parts and stored.

[0459] Detection of replication-competent viruses

[0460] The virus titer in the confluent layer of Vero E6 cells was determined by four replicates of 10-fold serial dilutions. To this end, serial dilutions of samples (throat swabs and tissue homogenates) were prepared and incubated on Vero E6 monolayers at 37°C for 1 hour. The Vero E6 monolayers were washed and incubated at 37°C for 5 or 6 days. The plates were scored based on cytopathic effect (CPE) by scoring using the viability marker WST8. Therefore, a WST-8 stock solution was prepared and added to the plates. 20 μL of the solution (containing 4 μL of ready-to-use WST-8 solution from the kit and 16 μL of infection medium, diluted 1:5) was added to each well and incubated at room temperature for 3-5 hours. Subsequently, the absorbance (OD) of the plate at 450nm was measured using a microplate reader. 450 ), and the visual results of the positive control CPE were used to set the limit of WST-8 staining (OD value related to CPE). The virus titer (log10TCID50 / ml or / g) was calculated using the Spearman-Karber method.

[0461] Lung histopathology

[0462] Tissue samples (trachea, left lung, and left turbinate) were collected, aerated, and / or stored in 10% formalin. After fixation, tissues from the left lung and left turbinate, gastrointestinal tract were embedded in paraffin. Tissue sections were stained with hematoxylin / eosin for histological examination. Histopathological evaluation included aspects such as congestion, emphysema, presence of foreign bodies, hemorrhage, bronchioloalveolar hyperplasia and inflammation, and edema.

[0463] Statistical analysis

[0464] The significance of differences between experimental groups was assessed by unpaired Mann-Whitney test, unpaired Student's t test, or two-way ANOVA followed by Sidka's multiple comparisons using Prism (GraphPad) software. P values ​​< 0.05 were considered significant.

[0465] By fusing an iBoost designed partner (IDP) to vimentin, the overall pI of the fusion protein was reduced

[0466] Proteins with a His tag (6x histidine) can be eluted from Ni-agarose with an excess of imidazole because imidazole competes with histidine for binding to Ni-agarose. If the protein binds to Ni-agarose by charge, it cannot be eluted from Ni-agarose by imidazole, but only by changing to a low pH to make the protein positively charged, which can then be eluted from Ni-agarose.

[0467] By fusing IDP1 (SEQ ID NO:6), IDP2 (SEQ ID NO:7), or IDP3 (SEQ ID NO:8) to mouse vimentin, the overall isoelectric point (pi) of the protein was lowered.

[0468] The isoelectric points of the different constructs described herein are as follows: the overall pI of IDP1-mVim is 4.65; the overall pI of IDP2-mVim is 4.71; the overall pI of IDP3-mVim is 4.81; the overall pI of CDP (SEQ ID NO: 5)-Vim is 5.09; and the overall pI of TRXtr (SEQ ID NO: 10)-Vim is 5.48. For the full-length sequences of these constructs, see Seq ID NO: 34 to 38. For these constructs, mouse vimentin (UniProtKB-P20152 (VIME MOUSE)) was used.

[0469] Lowering the overall pI of the fusion protein can enhance the solubility of vimentin, which means that it can be eluted with imidazole buffer. For the fusion proteins CDP-Vim and TRXtr-Vim, the protein could not be eluted with imidazole buffer, but only with buffer E.

[0470] Example Section B

[0471] Purified mammalian peptide apelin, combined with immunostimulatory peptides of SEQ ID NO: 5 (CDP), 6 (CDP2.1), 7 (CDP2.2) and 10 (TRXtr). The mouse-derived mammalian peptide apelin was combined with the immunostimulatory peptides disclosed herein (i.e., SEQ ID NO: 5 ("CDP"), 6 ("CDP2.1"), 7 ("CDP2.2") and 10 ("TRXtr" or "TRXtrunc")).

[0472] Regarding protein purification, TRXtr-apelin was not eluted using the four 200 mM imidazole fractions (pooled on the gel), and was only eluted in the buffer containing 100 mM EDTA (F1-F4). The EDTA buffer can strip nickel ions from the agarose beads. When apelin was bound to CDP, the protein was observed to elute in the four 200 mM imidazole fractions (pooled on the gel) and the buffer E fraction ( Fig.14 ). It was observed that expression of unconjugated apelin could never be induced in E. coli and the protein could not be purified.

[0473] Binding of mouse apelin to CDP2.2 (SEQ ID NO: 7) significantly reduced the isoelectric point (pI) of the fusion protein construct and allowed elution of the protein with 200 mM imidazole (I1-I4) ( Fig.15 ). The protein eluted mainly in the imidazole fraction, which means that imidazole competes with histidine binding and that the elution is independent of low pH (buffer E) and therefore does not change the charge of the protein.

[0474] The following table summarizes various properties of mouse apelin and mouse apelin fused to the C-terminus of each of TRXtr (SEQ ID NO: 10), CDP (SEQ ID NO: 5), CDP2.1 (SEQ ID NO: 6), CDP2.2 (SEQ ID NO: 7), and CDP 2.3 (SEQ ID NO: 8):

[0475] Table 1

[0476]

[0477] The mammalian peptide Timpl was purified in combination with the immunostimulatory peptides CDP (SEQ ID NO: 5), CDP2.1 (SEQ ID NO: 6), CDP2.2 (SEQ ID NO: 7), CDP2.3 (SEQ ID NO: 8) and TRXtr (SEQ ID NO: 10).

[0478] Protein production and purification of mouse Timp1 and mouse Timp1 fused to each of the following immunostimulatory peptides were performed: CDP (SEQ ID NO: 5), CDP2.1 (SEQ ID NO: 6), CDP2.2 (SEQ ID NO: 7), CDP2.3 (SEQ ID NO: 8), and TRXtr (SEQ ID NO: 10) ( Fig.16 A). After ultrasonic treatment, the precipitate and supernatant were loaded into SDS-PAGE gel. A thick band at the size of the fusion protein was found in the supernatant ( Fig.16The white arrow in B indicates that only a low concentration of protein remained in the pellet. Using 5 mL of supernatant and 200 μL of Ni-agarose beads resulted in a low concentration of TRXtr-Timp1 eluted in the buffer E fraction, while the highest concentrations of CDP-Timp1, CDP2.1-Timp1, CDP2.2-Timp1, and CDP2.3-Timp1 ( Fig.16 C).

[0479] It was observed that binding of Timp1 to CDP or CDP2.1, CDP2.2 and CDP2.3 lowered the isoelectric point of the fusion protein, thereby facilitating purification with 200 mM imidazole. The following table summarizes various properties of mouse Timp1 and mouse Timp1 fused to the C-terminus of each of TRXtr (SEQ ID NO: 10), CDP (SEQ ID NO: 5), CDP2.1 (SEQ ID NO: 6), CDP2.2 (SEQ ID NO: 7) and CDP2.3 (SEQ ID NO: 8):

[0480] Table 2

[0481]

[0482] Purification of mammalian peptide backplate proteins that bind to the immunostimulatory peptides CDP (SEQ ID NO: 5), CDP2.1 (SEQ ID NO: 6), CDP2.2 (SEQ ID NO: 7), CDP2.3 (SEQ ID NO: 8) and TRXtr (SEQ ID NO: 10)

[0483] Fusion polypeptides of mouse Notum fused with CDP (SEQ ID NO: 5), CDP2.1 (SEQ ID NO: 6), CDP2.2 (SEQ ID NO: 7), CDP2.3 (SEQ ID NO: 8) and TRXtr (SEQ ID NO: 10) were successfully purified and eluted using 200 mM imidazole. Fig.17 ).

[0484] The following table summarizes various properties of mouse Notum and mouse Notum fused to the C-terminus of each of TRXtr (SEQ ID NO: 10), CDP (SEQ ID NO: 5), CDP2.1 (SEQ ID NO: 6), CDP2.2 (SEQ ID NO: 7), and CDP2.3 (SEQ ID NO: 8):

[0485] Table 3

[0486]

[0487] Purification of influenza peptides from hemagglutinin (H) and / or neuraminidase (N) bound to the immunostimulatory peptide CDP (SEQ ID NO: 5).

[0488] Figures 21 to 26 Data generated using these constructs are shown. Addition of CDP to the H1 / N1 sequence lowered the isoelectric point of the protein and the protein could be eluted with imidazole (I1-I4). Unbound H1 / N1 protein could only be eluted with buffer E (E1-E4).

[0489] The following table summarizes various properties of the influenza constructs when fused to CDP (SEQ ID NO: 5):

[0490] Table 4

[0491] M pI H1N1 49.2 kDa 7.62 H1 24.9 kDa 6.90 N1 24.8 kDa 8.34 CDP-H1N1 55.4 kDa 6.43 CDP-H1 31.0 kDa 6.05 CDP-N1 31.2 kDa 6.58

[0492] Example Section C

[0493] Production of a vaccine for non-mammalian peptides conjugated to an immunostimulatory peptide of SEQ ID NO: 5 (CDP)

[0494] The inventors further designed, developed and characterized the vaccines disclosed herein, utilizing the immunostimulatory peptides of the present invention to direct the immune response to SARS-CoV-2, namely, accessible epitopes of the RBM (a small portion of the RBD, called the receptor binding motif (RBM), which contains all the residues that directly interact with ACE-2), the SD1, SD2, S1 / S2, FP+S2' and HR1 functional subdomains of the S (spike) glycoprotein.

[0495] Epitope selection and vaccine production

[0496] Based on the extensive work provided by the global research community over the past few years, the functional domains of the S protein of SARS-CoV-2 were screened. A total of six functional epitopes were identified, of which three epitopes were located in the S1 subunit of the S protein and three epitopes were located in the S2 subunit of the S protein ( Fig. 27 A). The first three selected epitopes are RBM, SD1, and SD2, which play an important role in recognition and binding to the host cell receptor ACE-2 (Henderson et al., 2020; Lan et al., 2020). The three selected epitopes located in the S2 domain are the S1 / S2 cleavage site, FP and S2′ protease cleavage site (FP+S2′), and HR1, which coordinate the fusion machinery and viral entry into host cells. All six selected epitopes were further examined for antibody accessibility. Only segments / portions within the selected epitopes that showed high antigen accessibility were selected to generate new vaccines ( Fig. 27A).

[0497] The selected epitopes were linked in the order described above (RBM, SD1, SD2, S1 / S2, FP+S2', HR1) with a flexible GSGSGS linker, and the CDP sequence described elsewhere herein (SEQ ID NO: 5) was conjugated to the N-terminus ( Fig. 27 B). This vaccine is further referred to as CDP-complete ( Fig. 27 B). To characterize the immunogenicity of epitopes of the S1 and S2 subdomains, the inventors also generated vaccines in which CDPs were bound to three selected epitopes of the S1 subdomain (RBM, SD1, SD2) or three selected epitopes of the S2 subdomain (S1 / S2, FP+S2', HR1). These vaccines are referred to as CDP-bound and CDP-fused ( Fig. 27 B).

[0498] All three vaccines were expressed in Escherichia coli BL21 and subsequently purified by immobilized metal chelate affinity chromatography (IMAC) as previously described (Huijbers et al., 2018)(Blanas et al., 2022). SDS-Page with colloidal Coomassie blue staining confirmed the purity and correct size of the produced vaccines ( Fig.28 A).

[0499] Inducing specific humoral responses against selected epitopes of the S protein

[0500] First, it was determined whether the multi-epitope vaccine disclosed herein can induce immune responses against multiple functional domains of the SARS-CoV-2 S protein at the same time. BALB / c mice were subcutaneously immunized with 100 μg of CDP-complete vaccine protein mixed with the water-in-oil adjuvant Sepivac at 1:1 (v / v). Injections were performed on days 1 and 14, and blood samples were collected at time points (0 days, 7, 13, 21, 28, and 35 days after immunization) to assess antibody titers by ELISA ( Fig. 27 C).

[0501] The CDP-complete vaccine elicited higher total immunoglobulin (Ig) titers against the full-length S1 and S2 subunits of the S protein 35 days after the first immunization ( Fig. 27 D). In more detail, all mice showed high antibody titers against RBD, RBM, SD2, as well as FP+S2' and HR1 epitopes. Only the SD1 epitope showed a more dispersed antibody response, with only two of the five mice showing specific antibody titers.

[0502] Similar antibody responses against epitopes of the S1 subunit were observed in mice immunized with the CDP-conjugate vaccine ( Fig. 27D). Again, high antibody titers were detected against the S1 protein, RBD, RBM and SD2. The SD1 epitope triggered antibodies in only one of the five mice. As expected, immunization with CDP-conjugation did not induce antibodies against the S2 protein and its epitopes S1 / S2, FP+S2′ or HR1.

[0503] The CDP-fusion vaccine elicited high antibody titers against the S2 protein and FP+S2′ epitopes ( Fig. 27 D). Analysis of the HR1 epitope showed that three out of five mice showed specific antibodies. Interestingly, a specific antibody response was also detected against the S1 subunit protein. Sequence alignment of the commercial full-length S1 protein used for ELISA coating with the CDP-fusion vaccine showed that there was a 12 amino acid overlap in the S1 / S2 epitope (aa674-685), which may be responsible for the antibody response.

[0504] In addition, all mice in the three vaccine groups elicited antibodies against the bacterial CDP domain. Interestingly, none of the mice showed S1 / S2 epitope-specific antibodies. This was not due to insufficient S1 / S2 peptide coating, as the commercial anti-S1 / S2 antibody showed a strong signal, indicating that S1 / S2 peptide coating was effective and no antibodies were elicited after vaccination ( Fig.28 B).

[0505] To reveal the kinetics of the antibody response, sera from immunized mice between day 0 and day 35 were analyzed at different time points and showed high total Ig titers against RBM and FP+S2′ epitopes only after a single vaccination and 13 days after the primary immunization. Specific antibodies against SD2 and HR1 epitopes were detected after two vaccinations and 21 days after the primary immunization, and specific antibodies against SD1 epitopes were detected 28 days after the primary immunization ( Fig.28 C).

[0506] These findings demonstrate for the first time that multi-epitope vaccines based on the technology disclosed herein can induce high antibody titers against multiple selected epitopes of the SARS-CoV-2 S protein.

[0507] Induce systemic epitope-specific T cell responses

[0508] SARS-CoV-2-specific T cells are directly involved in the rapid clearance of virus-infected cells and counteract the development of mild disease symptoms. Therefore, it is extremely important to induce a potent antigen-specific T cell response after vaccination. To investigate whether vaccination with CDP-complete would elicit systemic T cell immunity against selected functional epitopes of the S protein, spleens of immunized mice were harvested on day 35. Splenocytes were restimulated ex vivo with RBM, SD1, SD2, S1 / S2, FP+S2', and HR1 peptides, and the frequency of antigen-specific T cells was quantified based on Th1- and Th2-specific cytokine production ( Fig.29 A) Unstimulated splenocytes were used as negative control. Interestingly, after vaccination, mice developed polyfunctional CD4 + and CD8 + In particular, antigen-specific T helper (Th) cells with distinct Th1 and Th2 cytokine profiles were detected. Fig.29 B). Th1-skewed cells were mainly characterized by interleukin 2 (IL-2) and tumor necrosis factor alpha (TNFα) production, while Th2-skewed cells showed strong IL-4 induction. In contrast, CD8+ T cells induced after vaccination showed a strong interferon gamma (IFNg) and TNFa profile, indicating their potent cytotoxic potential. Based on these findings, vaccination with the conjugate CDP-complete vaccine ensures a potent cellular response against functional epitopes that do not induce detectable antibody responses, such as the S1 / S2 subdomains.

[0509] CDP-complete vaccine in the SARS-CoV2 hamster challenge model

[0510] Hamsters (n=8 per group) were vaccinated twice (day 0 and day 21) with 100 μl of CDP-complete vaccine (containing 100 μg of protein mixed with Sepivac adjuvant 1:1 (v / v)) or PBS mixed with adjuvant (PBS control). The body weight of the hamsters was monitored during the experiment ( Fig.30 A). On day 42, hamsters were challenged with SARS-CoV2 and body weight was further monitored ( Fig.30 B). After hamsters were infected with SARS-CoV-2, the body weight of infected hamsters decreased from day 43 to day 49, with a decrease of about -13% in group 1 and about -6% in group 2. In addition, for group 2, the rate of weight loss was slower than that of group 1. These results demonstrate that CDP-complete appears to limit the effect of SARS-CoV-2 on body weight loss. Viral loads were measured in lung and nasal turbinates samples at D+3 after infection ( Fig.30C). Significantly lower viral loads were found in the lungs and nasal turbinates of CDP-complete vaccinated hamsters. On day 45 (day 3 after viral challenge), 5 of 8 hamsters vaccinated with CDP-complete developed neutralizing antibody responses, whereas no antibody responses were seen in the PBS control group ( Fig.30 D) Both groups had detectable neutralizing antibody responses against SARS-CoV2 at day 49. Thus, animals immunized with the CDP-complete vaccine developed a neutralizing antibody response earlier than the control group following SARS-CoV2 infection.

[0511] Materials and methods

[0512] Vaccine design

[0513] Each recombinant vaccine protein begins with a His6-tag (HHHHHH) followed by the iBoostCDP sequence at its N-terminus. The CDP consists of bacterial amino acid clusters with hydrophilic or charged side chains fused to a single peptide with a total length of 60 amino acids (5.8 kDa). The selected SARS-CoV-2 S protein epitope is linked to the CDP via a (GS)3-linker (GSGSGS) and is expressed as Fig. 27 The sequences shown in B are connected to each other. The sequence of the S protein epitope is based on the reported SARS-CoV-2 genome (GenBank: MN908947.3) and is located at: RBM: aa438-506, SD1: aa527-562, SD2: aa618-646, S1 / S2: aa674-696, FP+S2': aa786-848 and HR1: aa912-943.

[0514] Vaccine protein production

[0515] The coding sequence of the recombinant vaccine protein is synthesized by Genscript (USA) and cloned between the NdeI and XhoI restriction enzyme sites of the pET21a expression vector. 10ng of plasmid DNA was used to transform competent Escherichia coli BL21 (DE3) bacteria (Sigma-Aldrich) using the heat shock method. Single clones were selected and cultured overnight at 37°C in a non-humidified oscillator (200rpm, Laboshake) in LB-medium supplemented with ampicillin (100 μg / ml). The next day, the culture was diluted 1:2.6 and 1mM isopropyl β-d-1-thiogalactoside (IPTG) (Serva) was used to induce protein expression for four hours. Bacteria were harvested by centrifugation and the bacterial pellet (equivalent to 50 ml culture volume) was dissolved in 5 ml ultrasonic buffer containing 0.5 M EDTA (Sigma-Aldrich), 1% N-lauroylsarcosine (Sigma-Aldrich), 1% phenylmethanesulfonyl fluoride solution (PMSF) (Sigma-Aldrich) and 6 M urea (Acros Organics) in PBS (VWR). Subsequently, the protein was released on ice by ultrasonic treatment (amplitude 22-24 microns), which was carried out for 15 cycles, each cycle being 20 seconds ultrasonic treatment and then suspending for 30 seconds (Soniprep 150MSE). Subsequently, the sample was pelleted by centrifugation (4500 rpm, 20 minutes), and the supernatant containing the protein was mixed with 200 μl 50% Ni-NTA agarose slurry (Qiagen). After incubation overnight on a roller test bench at 4°C, the beads were spun and the supernatant was frozen for additional purification rounds. The Ni-NTA beads were washed five times with 25 mL of a wash buffer containing 1 M NaCl (VWR), 0.05% Tween-20 (Sigma-Aldrich), and PBS, and then stacked in a 1 mL syringe (BD Biosciences) with a glass filter (Sartorius). Four fractions (100 μl each) of 1 M Tris Cl (pH 8.0), 100 mM NaCl, 200 mM imidazole (JT Baker), 1 mM PMSF were used, followed by four fractions (100 μl each) of 10 mM Tris Cl (pH 4.5), 100 mM NaH2PO4 (Merck), 8 M urea to elute the vaccine protein. The purified protein fractions were pooled and step-dialyzed against PBS. SDS PAGE and Coomassie blue staining ( The purity and identity of the vaccine protein were confirmed by Brilliant Blue G 250, Serva). The final protein concentration was determined by BCA assay (Thermo Scientific) and the vaccine was diluted to a final concentration of 2 mg / ml.

[0516] Mouse experiments

[0517] Eight-week-old female BALB / c OlaHsd mice were obtained from Envigo (The Netherlands) and acclimatized for two weeks at a temperature of 20-24°C, a humidity of 45-65%, and a 12 / 12-hour light-dark cycle. After acclimatization, mice received a primary immunization on day 1 and a booster immunization on day 14 ( Fig. 27 C). Each injection was performed subcutaneously in the left groin with 100 μl of vaccine emulsion consisting of 100 μg of purified vaccine protein and Sepivac SWE (Seppic) adjuvant mixed in a 50:50 (v / v) ratio. Blood samples were collected from the tail vein before immunization (day 0) and on days 7, 13, 21, 28, and 35. All mice were sacrificed on day 35. All mouse experiments were approved by the Central Committee for Animal Experimentation of the Netherlands National Ethics Committee (CCD, registration number AVD11400202010545) and the VU / Vumc Animal Welfare Agency (Working Agreement No. 10545-ANG21-01) and were in accordance with the Dutch guidelines and laws for animal experiments.

[0518] ELISA for antibody detection

[0519] The total Ig titer of binding to RBD, S1 subunit, S2 subunit or selected epitopes (RBM, SD1, SD2, S1 / S2, FP+S2', HR1) in the serum of immune mice was measured by ELISA. Therefore, blood samples were allowed to coagulate overnight at 4°C and centrifuged twice the next day (7000rpm, 10 minutes, 4°C). Flat-bottomed 96-well ELISA plates (Thermo Fisher Scientific) were coated with 50 μl of purified protein in PBS at a final concentration of 2 μg / mL (for RBD, S1, S2 subunits) or 4 μg / mL (for selected S protein peptides) at 37°C for 1 hour. Order S protein peptides (Proteogenix, France) and reconstitute them to a final concentration of 10 mg / ml according to the manufacturer's instructions. Next, the plate was washed once with PBS containing 0.1% Tween-20 (PBS-T) and blocked with 1% skim milk powder (Santa Cruz) in PBS-T for 1 hour at 37°C. After washing once, a three-fold dilution of mouse serum in blocking solution (1 / 100 to 1 / 72900) was added to the plate and incubated at 37°C for 45 minutes. The plate was washed and a secondary antibody biotinylated goat anti-mouse total Ig (Dako, #E0433) diluted in PBS-T (1 / 2000) was added to the wells and incubated at 37°C for 45 minutes. After washing four times, the plate was incubated at 37°C for 30 minutes with streptavidin-HRP (Dako, #P0397) diluted in PBS-T (1 / 2000). The plate was washed and developed with TMB (Sigma, #T0440) for 10 minutes. The absorbance at 655nm was read on a microplate reader (BioTek Synergy HTX). The actual OD value was calculated by subtracting the blank value (wells treated in the same manner as the wells in the sample except that blocking solution was added instead of mouse serum) from the sample value.

[0520] Splenocyte isolation and peptide restimulation

[0521] The spleen of mice fully immunized with CDP- was isolated on day 35, cut into small pieces and mechanically dissociated. Splenocytes were passed through a 70 μm cell strainer (Corning, #431751) and spun at 1500 rpm (brake 5, room temperature) for 5 minutes in a Rotina 420R (Hettich) centrifuge. Red blood cells were incubated with ammonium chloride-potassium lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 100 mM EDTA, pH = 7.4) for 3 minutes at room temperature for lysis. Splenocytes were washed once and stored frozen in RPMI-1640 (Gibco, #L0495-500) medium containing 10% DMSO. Splenocytes were thawed and resuspended in RPMI-1640 medium supplemented with 10% FCS (Biowest, #S0750), 1% Pen / Strep (Gibco, #15140-122), 1% L-glutamine (Brunschwig Chemie BV, #HN08.2) and 50 μM 2-mercaptoethanol (Gibco, #31350-010). They were seeded in U-bottom 96-well plates (Greiner Bio-One, #650180) (2*10 6 Cells / well) and restimulated with RBM, SD1, SD2, S1 / S2, FP+S2' and HR1 synthetic peptides ex vivo for 24 hours. Unstimulated splenocytes treated with Brefeldin A were used as negative controls.

[0522] Epitope-specific T cell characterization based on flow cytometry

[0523] After stimulation again, the cells were transferred to a V-bottom 96-well plate (Greiner Bio-One, #651180) and washed once with PBS. The cells were resuspended in TruStain Fc blocking solution (BioLegend, #101320) for 10 minutes at room temperature. Subsequently, the cells were incubated on ice with anti-mouse CD4-AF700 (BioLegend, #100429, 1 / 200 dilution), anti-mouse CD8b-AF488 (BioLegend, #126627, 1 / 200 dilution) and Zombie Aqua fixable viability dye (BioLegend, #423101, 1 / 200 dilution) diluted in PBS for 20 minutes. The cells were washed once with PBS and fixed with 4% paraformaldehyde (PFA; Electron Microscopy Sciences, #15710) on ice for 15 minutes. After fixation, cells were washed once with PBS, and permeabilization was performed according to the manufacturer's instructions using intracellular dyeing permeable washing buffer (BioLegend, #421002). Then, at room temperature, cell suspension was incubated for 30 minutes with anti-mouse IFNγ-APC (BioLegend, #505305, 1 / 200 dilution), TNFα-PE (BioLegend, #506305, 1 / 200 dilution), IL-2-PE-Cy7 (BioLegend, #503832, 1 / 200 dilution), IL-4-PerCP / Cy5.5 (BioLegend, #504123, 1 / 200 dilution) and IL-5 (BioLegend, #504311, 1 / 200 dilution) diluted in intracellular dyeing permeable washing buffer. Cells were washed twice with permeable washing buffer, resuspended in 100 μl PBS and transferred to FACS tubes. Fluorescence intensity and the percentage of IFNγ-, TNFα-, IL-2-, IL-4-, and IL-5-expressing cells were measured using an LSRII (BD Biosciences) flow cytometer. The percentage of each stimulation condition was subtracted from the percentage of the non-stimulation condition for each mouse. Data analysis was performed using FlowJo V10 software (Tree Star).

[0524] Hamster breeding

[0525] During the acclimation and vaccination phases, hamsters were housed in type 2 cages with a maximum of two animals per cage under DM (BSL)-II conditions. On the day of viral inoculation, hamsters were transferred to standard extended type 2 group cages with two animals per cage under BSL-III conditions (isolators), using sawdust as bedding and cage enrichment. For all invasive animal procedures, nasal and intramuscular administration, blood sampling, throat swab collection and euthanasia, animals were sedated with isoflurane (3-4% / O2). Hamsters were vaccinated by the intramuscular route (im) on days 0 and 21 as planned. Blood was collected by orbital bleeding. Blood samples for serum were immediately transferred to appropriate test tubes containing coagulation activators. Serum was collected, heat treated, aliquoted and frozen.

[0526] Hamster vaccination

[0527] On days 0 and 21, animals were vaccinated intramuscularly (im) with PBS (as control) or with 100 μg of CDP-complete mixed 1:1 (v / v) with Sepivac (Seppic) adjuvant in a total injection volume of 100 μL.

[0528] SARS-CoV-2 vaccination

[0529] On day 42, 50 μL of SARS-CoV-2WT variant (090621_SARS-CoV-2_UVE / SARS-CoV-2 / 2020 / FR / 702) suspension (at a concentration of 2x106 PFU / mL) was roughly evenly distributed and dripped into each nostril of all hamsters using a fine pipette cone to challenge the hamsters. On day 3 after the challenge, half of the animals in each group were killed by bleeding under isoflurane anesthesia and autopsied. On day 7 after the challenge, the remaining half of the animals in each group were killed by bleeding under isoflurane anesthesia and autopsied.

[0530] References

[0531] Cohen,K.W.,Linderman,S.L.,Moodie,Z.,Czartoski,J.,Lai,L.,Mantus,G.,Norwood,C.,Nyhoff,L.E.,Edara,V.V.,Floyd,K.,et al.(2021).Longitudinal analysisshows durable and broad immune memory after SARS-CoV-2 infection withpersisting antibody responses and memory B and T cells.Cell Rep Med 2,100354.10.1016 / j.xcrm.2021.100354.

[0532] Gattinger,P.,Niespodziana,K.,Stiasny,K.,Sahanic,S.,Tulaeva,I.,Borochova,K.,Dorofeeva,Y.,Schlederer,T.,Sonnweber,T.,Hofer,G.,et al.(2022).Neutralization of SARS-CoV-2 requires antibodies againstconformationalreceptor-binding domain epitopes.Allergy 77,230-242.10.1111 / all.15066.

[0533] Henderson,R.,Edwards,R.J.,Mansouri,K.,Janowska,K.,Stalls,V.,Gobeil,S.M.C.,Kopp,M.,Li,D.,Parks,R.,Hsu,A.L.,et al.(2020).Controlling the SARS-CoV-2 spike glycoprotein conformation.Nat Struct Mol Biol 27,925-933.10.1038 / s41594-020-0479-4.

[0534] Hoffmann, M., Kleine-Weber, H., Schroeder, S., Kruger, N., Herrler, T., Erichsen, S., Schiergens, T., Herrler, G., Wu, NH, Nitsche, A., et al of ClinicallyProven Protease Inhibitor.Cell 181,271–280e278.10.1016 / j.cell.2020.02.052.

[0535] An improved conjugate vaccine technology: induction of antibody responses to the tumor vasculature.Vaccine36,3054–3060.10.1016 / j.vaccine.2018.03.064.

[0536] Koppisetti, RK, Fulcher, YG, and Van Doren, SR(2021).Fusion Peptideof SARS-CoV-2 Spike Rearranges into a Wedge Inserted in Bilayered Micelles.JAm Chem Soc 143,13205-13211.10.1021 / jacs.1c05435.

[0537] Lan,J.,Ge,J.,Yu,J.,Shan,S.,Zhou,H.,Fan,S.,Zhang,Q.,Shi,X.,Wang,Q.,Zhang,L.,and Wang,X.(2020).Structure of the SARS-CoV-2spike receptor-bindingdomain bound to the ACE2 receptor.Nature 581,215-220.10.1038 / s41586-020-2180-5.

[0538] Nelde,A.,Bilich,T.,Heitmann,J.S.,Maringer,Y.,Salih,H.R.,Roerden,M.,Lubke,M.,Bauer,J.,Rieth,J.,Wacker,M.,et al.(2021).SARS-CoV-2-derived peptidesdefine heterologous and COVID-19-induced Tcell recognition.Nat Immunol 22,74-85.10.1038 / s41590-020-00808-x.

[0539] Niu,L.,Wittrock,K.N.,Clabaugh,G.C.,Srivastava,V.,and Cho,M.W.(2021).AStructural Landscape of Neutralizing Antibodies Against SARS-CoV-2ReceptorBinding Domain.Front Immunol 12,647934.10.3389 / fimmu.2021.647934.

[0540] Perez-Potti,A.,Lange,J.,and Buggert,M.(2021).Deciphering the ins andouts of SARS-CoV-2-specific T cells.Nat Immunol 22,8-9.10.1038 / s41590-020-00838-5.

[0541] Rabi, FA, Al Zoubi, MS, Kasasbeh, GA, Salameh, DM, and Al-Nasser, AD(2020).SARS-CoV-2 and Coronavirus Disease

[0542] Rossler, A., Riepler, L., Bante, D., von Laer, D., and Kimpel, J. (2022).SARS-CoV-2Omicron Variant Neutralization in Serum from Vaccinated and ConvalescentPersons.N Engl J Med 386,698-700.10.1056 / NEJMc2119236.

[0543] Tenforde, MW, Self, WH, Adams, K, Gaglani, M, Ginde, AA, McNeal, T, Ghamande, S, Douin, DJ, Talbot, HK, Casey, JD, et al DiseaseSeverity.JAMA326,2043–2054.10.1001 / jama.2021.19499.

[0544] van Beijnum,JR,Huijbers,EJM,van Loon,K,Blanas,A,Akbari,P,Roos,A,Wong,TJ,Denisov,SS,Hackeng,TM,Jimenez,CR,et al.(2022).Extracellular vimentin mimics VEGF and is a target for anti-angiogenic immunotherapy.Nat Commun 13,2842.10.1038 / s41467-022-30063-7.

[0545] Xia, S., Zhu, Y., Liu, M., Lan, Q., Xu, W., Wu, Y., Ying, T., Liu, S., Shi, Z., Jiang, S., and Lu, L. (2020). 17.765-767.10.1038 / s41423-020-0374-2.

[0546] Zhao , P. , Praissman , JL , Grant , OC , Cai , Y. , Xiao , T. , Rosenbalm , KE , Aoki , K. , Kellman , BP , Bridger , R. , Barouch , DH , et al ACE2Receptor.bioRxiv.10.1101 / 2020.06.25.172403.

[0547] sequence

[0548] The receptor binding domain (RBD) of SARS-CoV-2 (GenBank: MN908947.3) sequence is from amino acids 330 to 524 (SEQ ID NO: 1):

[0549] NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPD DFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATV

[0550] CD4 T-cell epitope construct 1 sequence from MsyB protein of Escherichia coli (SEQ ID NO: 2):

[0551] NPGIDAEDANVQQFNAQKYVLQDGDIMWQV

[0552] CD4 T-cell epitope construct 2 sequence from MsyB protein of Escherichia coli (SEQ ID NO: 3):

[0553] EGEFQLEPPLDTEGRADE

[0554] Sequence of RRAB protein from Escherichia coli (SEQ ID NO: 4):

[0555] DDEDFVDEDDD

[0556] Immunostimulatory peptide sequence [original CDP] (SEQ ID NO: 5):

[0557] DNNSLSQEVQNGSNHLENNQSQSNGGGSDSALSLSSKTAALAAATTVNDGSDGATSSAVG

[0558] Immunostimulatory peptide sequence [IDP1 or CDP2.1] (SEQ ID NO: 6):

[0559] HHAHTMYATLEEAIDAAREEFLADNPGIDAEDANVQQFNAQKYVLQDGDIMWQVEFFADEGEEGEDDEDFVDEDDD

[0560] Immunostimulatory peptide sequence [IDP2 or CDP2.2] (SEQ ID NO: 7):

[0561] HHAHNPGIDAEDANVQQFNAQKYVLQDGDIMWQVEGEFQLEPPLDTEEGRAAADEDDEDFVDEDDD

[0562] Immunostimulatory peptide sequence [IDP3 or CDP2.3] (SEQ ID NO: 8):

[0563] HHAHNPGIDAEDANVQQFNAQKYVLQDGDIMWQVDDEDFVDEDDD

[0564] Thioredoxin (TRX) sequence (SEQ ID NO:9):

[0565] SDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLA

[0566] Thioredoxin truncated (TRXtrunc) sequence (SEQ ID NO: 10):

[0567] GKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLA

[0568] Type 1 fimbriae protein, A chain sequence (SEQ ID NO: 11):

[0569] AATTVNGGTVHFKGEVVNAACAVDAGSVDQTVQLGQVRTASLAQEGATSSAVGFNIQLNDCDTNVASCAAVAFLGTAIDAGHTNVLALQSSAAGSATNVGVQILDRTGAALTLDGATFSSETTLNGTNTI

[0570] Variant type 1 pilin, A chain sequence (SEQ ID NO: 12):

[0571] CATTVNGGTVHFKGEVVNAACAVDAGSVDQTVQLGQVRTASLAQEGATSSKVGFNIQLNDCDTNVASKAAVAFLGTKIDAGHTNVLALQSSAAGSDTNVGVQILDRTGAALLDGATFSSETTLNNDTNTI

[0572] CD4 T-cell epitope 1 sequence of RRAB protein from Escherichia coli (SEQ ID NO: 13):

[0573] KLGYEVYEVTDPEELEVE

[0574] CD4 T-cell epitope 1 sequence of RRAB protein from Escherichia coli (SEQ ID NO: 14):

[0575] FKLGYEVTDPEELEV

[0576] CD4 T-cell epitope 1 sequence of RRAB protein from Escherichia coli (SEQ ID NO: 15):

[0577] YEVTDPEEL

[0578] CD4 T-cell epitope sequence from MsyB protein of Escherichia coli (SEQ ID NO: 16):

[0579] EGEFQLEPPLDTEEG

[0580] CD4 T-cell epitope sequence of MsyB protein from Escherichia coli (SEQ ID NO: 17):

[0581] AQKYVLQDGDIMWQV

[0582] CD4 T-cell epitope sequence of MsyB protein from Escherichia coli (SEQ ID NO: 18):

[0583] DEGEFQLEPPLDTEE

[0584] CD4 T-cell epitope sequence of MsyB protein from Escherichia coli (SEQ ID NO: 19):

[0585] NAQKYVLQDGDIMWQ

[0586] CD4 T-cell epitope sequence from MsyB protein of Escherichia coli (SEQ ID NQ:20):

[0587] APPLE GRADES

[0588] CD4 T-cell epitope sequence of MsyB protein from Escherichia coli (SEQ ID NO: 21):

[0589] NPGIDAEDANVQQFN

[0590] CD4 T-cell epitope sequence of MsyB protein from Escherichia coli (SEQ ID NO: 22):

[0591] LEPPLDTEEGRAD

[0592] CD4 T-cell epitope sequence from MsyB protein of Escherichia coli (SEQ ID NO: 23):

[0593] FQLEPPLDTYVLQDGDIM

[0594] CD4 T-cell epitope sequence from MsyB protein of Escherichia coli (SEQ ID NO: 24):

[0595] LDTEEGRAA

[0596] CD4 T-cell epitope sequence from MsyB protein of Escherichia coli (SEQ ID NO: 25):

[0597] IDAEDNVQ

[0598] His tag (SEQ ID NO: 26):

[0599] HHHHHHH

[0600] Linker (SEQ ID NO: 27):

[0601] GSGSGS

[0602] ZapB (amino acids 36-45) (SEQ ID NO: 28):

[0603] NNSLSQEVQN

[0604] IbpA (amino acids 21-33) (SEQ ID NO: 29):

[0605] NHLENNQSQSNGG

[0606] TFP sequence (amino acids 12-39) (SEQ ID NO: 30):

[0607] SALSLSSTAALAAAATTVN

[0608] TFP sequence (amino acids 69-76) (SEQ ID NO: 31):

[0609] GATSSAVG

[0610] TRXtrunc-Vimentin protein sequence (540aa) (linker and his tag are underlined, Vimentin sequence is Bold) (SEQ ID NO:34):

[0611]

[0612] CDP-m Vimentin protein (542aa) (linker and his-tag are underlined, vimentin sequence is in bold) (SEQ ID NO:35):

[0613]

[0614] IDP1-m Vimentin protein (linker underlined, Vimentin sequence in bold) (SEQ ID NO: 36):

[0615]

[0616] IDP2-m Vimentin protein (linker underlined, Vimentin sequence in bold) (SEQ ID NO: 37):

[0617]

[0618]

[0619] IDP3-m Vimentin protein (linker underlined, Vimentin sequence in bold) (SEQ ID NO: 38):

[0620]

[0621] SARS-CoV-2 sequences used in "CDP-Complete" (RBM-GS-Linker-SD1-GS-Linker-SD2-GS-Linker- S1 / S2-GS-Linker-FP-GS-Linker-HR1-Linker is underlined and SARS-CoV-2 sequence is italicized) (SEQ ID NO: 39)

[0622]

[0623] SARS-CoV-2 sequences used in "CDP-binding" (RBM-GS-Linker-SD1-GS-Linker-SD2-Linker band Underlined, SARS-CoV2 sequence in italics) (SEQ ID NO: 40)

[0624]

[0625] SARS-CoV-2 sequences used in "CDP-Fusion" (S1 / S2-GS-Linker-FP-GS-Linker-HR1 protein sequence) Column-linker is underlined, SARS-CoV-2 sequence is italicized) (SEQ ID NO:41)

[0626]

[0627] HI (SEQ ID NO: 42)

[0628] CNIAGWILGNPECESLSTARSWSYIVETSNSDNGTCYPGDFINYEELREQLSSVSSFERFEIFPKTSSWPNHDSDNGVTAACPHAGAKSFYKNLIWLVKKGKSYPKINQTYINDKGKEVLVLWGIHHPPTIADQQSLYQNADAYVFVGTSRYSKKFKPEIATRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVAPRYAFTMERDAG

[0629] N1 (SEQ ID NO:43)

[0630] WLTIGISGPDSGAVAVLKYNGIITDTIKSWRNKILRTQESECACVNGSCFTIMTDGPSDGQASYKIFRIEKGKIIKSVEMKAPNYHYEECSCYPDSSEITCVCRDNWHGSNRPWVSFNQNLEYQMGYICSGVFGDNPRPNDKTGSCGPVSSNGANGVKGFSFKYGNGVWIGRTKSISSRKGFEMIWDPNGWTGTDNKFSKKQDIVGINEWSGYS

[0631] H1 / N1 (SEQ ID NO:44)

[0632] CNIAGWILGNPECESLSTARSWSYIVETSNSDNGTCYPGDFINYEELREQLSSVSSFERFEIFPKTSSWPNHDSDNGVTAACPHAGAKSFYKNLIWLVKKGKSYPKINQTYINDKGKEVLVLWGIHHPPTIADQQSLYQNADAYVFVGTSRYSKKFKPEIATRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVAPRYAFTMERDAGGSGSGSELWLTIGISGPDSGAVAVLKYNGIITDTIKSWRNKILRTQESECACVNGSCFTIMTDGPSDGQASYKIFRIEKGKIIKSVEMKAPNYHYEECSCYPDSSEITCVCRDNWHGSNRPWVSFNQNLEYQMGYICSGVFGDNPRPNDKTGSCGPVSSNGANGVKGFSFKYGNGVWIGRTKSISSRKGFEMIWDPNGWTGTDNKFSKKQDIVGINEWSGYS

[0633] CDP-H1 (CDP sequence in bold, linker underlined, H1 sequence in italics) (SEQ ID NO:45)

[0634]

[0635] Number of amino acids: 284

[0636] Molecular weight: 31030.97 (31.0 kDa)

[0637] Theoretical pI: 6.05

[0638] CDP-N1 (CDP sequence in bold, linker underlined, N1 sequence in italics) (SEQ ID NO:46)

[0639]

[0640] Number of amino acids: 290

[0641] Molecular weight: 31158.15 (31.2 kDa)

[0642] Theoretical pI: 6.58

[0643] CDP-H1N1 (CDP sequence in bold, linker underlined, H1N1 sequence in italics) (SEQ ID NO: 47)

[0644]

[0645] Number of amino acids: 506

[0646] Molecular weight: 55401.19 (55.4 kDa)

[0647] Theoretical pI: 6.42

[0648] His-TRXtr-m-apelin protein (TRXtr sequence is bold, linker is underlined, m-apelin is italic) (SEQ ID NO:48)

[0649]

[0650] Number of amino acids: 149

[0651] Molecular weight: 16104.81Da (16.1kDa)

[0652] Theoretical pI: 10.80

[0653] His-CDP-m-apelin protein (CDP sequence in bold, linker underlined, m-apelin in italics) (SEQ ID NO:49)

[0654]

[0655] Number of amino acids: 151

[0656] Molecular weight: 15834.59Da (15.8kDa)

[0657] Theoretical pI: 9.88

[0658] CDP2.1-Apelin (CDP2.1 / IDP1 sequence is in bold, linker is underlined, Apelin is in italics) (SEQ ID NO:50)

[0659]

[0660] Number of amino acids: 161

[0661] Molecular weight: 17898.88

[0662] Theoretical pI: 4.53

[0663] Solubility: 0.599

[0664] CDP2.2-Apelin (CDP2.2 / IDP2 in bold, linker underlined, Apelin in italics) (SEQ ID NO:51)

[0665]

[0666] Number of amino acids: 151

[0667] Molecular weight: 16702.59

[0668] Theoretical pI: 4.73

[0669] Solubility: 0.648

[0670] CDP2.3-Apelin (CDP2.3 / IDP3 sequence is in bold, linker is underlined, Apelin is in italics) (SEQ ID NO:52)

[0671]

[0672] Number of amino acids: 130

[0673] Molecular weight: 14417.23

[0674] Theoretical pI: 5.73

[0675] Solubility: 0.470

[0676] TRXtr-Notum (TRXtr is bold, linker is underlined, Notum is italic) (SEQ ID NO: 53)

[0677]

[0678] Number of amino acids: 558

[0679] Molecular weight: 62281.72Da (62kDa)

[0680] Theoretical pI: 7.90

[0681] CDP-mNotum (CDP is bold, linker is underlined, mNotum is italic) (SEQ ID NO: 54)

[0682]

[0683]

[0684] Number of amino acids: 560

[0685] Molecular weight: 62011.51Da (62kDa)

[0686] Theoretical pI: 6.27

[0687] CDP2.1-mNotum (CDP2.1 / IPD1 sequence is in bold, linker is underlined, mNotum is in italics) (SEQ ID NO:55)

[0688]

[0689] Number of amino acids: 570

[0690] Molecular weight: 64075.80Da (64kDa)

[0691] Theoretical pI: 5.00

[0692] CDP2.2-mNotum (CDP2.2 / IPD2 sequence in bold, linker underlined, mNotum in italics) (SEQ ID NO:56)

[0693]

[0694]

[0695] Number of amino acids: 560

[0696] Molecular weight: 62879.51Da (63kDa)

[0697] Theoretical pI: 5.14

[0698] CDP2.3-mNotum (CPD2.3 / IPD3 sequence in bold, linker underlined, mNotum in italics) (SEQ ID NO:57)

[0699]

[0700] Number of amino acids: 539

[0701] Molecular weight: 60594.15Da (60.5kDa)

[0702] Theoretical pI: 5.49

[0703] TRXtr-Timp1 (TRXtr is bold, linker is underlined, Timp1 is italic) (SEQ ID NO: 58)

[0704]

[0705] Number of amino acids: 255

[0706] Molecular weight: 27955.14Da (27kDa)

[0707] Theoretical pI: 9.30

[0708] CDP-Timp1 (CDP in bold, linker underlined, Timp1 in italics) (SEQ ID NO:59)

[0709]

[0710] Number of amino acids: 257

[0711] Molecular weight: 27684.93Da (28kDa)

[0712] Theoretical pI: 8.46

[0713] CDP2.1-Timp1 (CDP2.1 is bold, linker is underlined, Timp1 is italic) (SEQ ID NO: 60)

[0714]

[0715] Number of amino acids: 267

[0716] Molecular weight: 29749.22Da (30kDa)

[0717] Theoretical pI: 4.95

[0718] CDP2.2-Timp1 (CDP2.2 is bold, linker is underlined, Timp1 is italic) (SEQ ID NO: 61)

[0719]

[0720] Number of amino acids: 257

[0721] Molecular weight: 28552.93Da (28.5kDa)

[0722] Theoretical pI: 5.23

[0723] CDP2.3-Timp1 (CDP2.3 is bold, linker is underlined, Timp1 is italic) (SEQ ID NO: 62)

[0724]

[0725] Number of amino acids: 236 /

[0726] Molecular weight: 26267.57Da (26kDa)

[0727] Theoretical pI: 6.13

[0728] CDP-complete (His tag-CDP-GS-linker-RBM-GS-linker-SD1-GS-linker-SD2-GS-linker-S1 / S2-GS-Linker-FP-GS-Linker-HR1 protein sequence (357aa)-CDP is in bold, the linker is underlined and the SARS-CoV-2 sequence is Listed in italics) (SEQ ID NO:63)

[0729]

[0730] Theoretical pI / Mw: 8.89 / 37458.79

[0731] CDP-binding (His tag-CDP-GS-linker-RBM-GS-linker-SD1-GS-linker-SD2 protein sequence-CDP is in bold, the linker is underlined and the SARS-CoV-2 sequence is in italics) (221aa) (SEQ ID NO:64)

[0732]

[0733]

[0734] Theoretical pI / Mw: 7.91 / 23095.91

[0735] CDP-fusion (His tag-CDP-GS-linker-S1 / S2-GS-linker-FP-GS-linker-HR1 protein sequence-CDP in bold, linker underlined, SARS-CoV-2 sequence in italics) (205aa) (SEQ ID NO: 65)

[0736]

[0737] Theoretical pI / Mw: 6.71 / 21124.75

[0738] DNA sequence for "CDP-complete" (His tag-CDP-GS-linker-RBM-GS-linker-SD1-GS-linker- SD2-GS-Linker-S1 / S2-GS-Linker-FP-GS-Linker-HR1) (1089 nt) (SEQ ID NO: 66)

[0739] CATATGCACCATCACCATCACCATGATAACAACTCACTGTCGCAGGAAGTTCAAAATGGTTCTAACCACTTAGAAAACAACCAGAGCCAGAGTAATGGCGGCGGTTCTGATTCGGCTCTGTCCCTCAGTTCTAAAACAGCGGCTCTGGCCGCTGCCACGACGGTTAATGATGGTTCTGATGGAGCAACCAGTTCTGCTGTCGGT GGTTCTGGTTCTGGTAGC GGATCCAGCAACAACCTGGATAGCAAAGTGGGCGGCAACTATAACTATCTGTATCGCCTGTTTCGCAAAAGCAACCTGAAACCGTTTGAACGCGATATTAGCACCGAAATTTATCAGGCGGGCAGCACCCCGTGCAACGGCGTGGAAGGCTTTAACTGCTATTTTCCGCTGCAGAGCTATGGCTTTCAGCCGACCAACGGCGTGGGCTATCAG GGTTCTGGTTCTGGTAGC CCGAAAAAAAGCACCAACCTGGTGAAAAACAAATGCGTGAACTTTAACTTTAACGGCCTGACCGGCACCGGCGTGCTGACCGAAAGCAACAAAAAATTTCTGCCGTTT GGTTCTGGTTCTGGTAGC ACCGAAGTGCCGGTGGCGATTCATGCGGATCAGCTGACCCCGACCTGGCGCGTGTATAGCACCGGCAGCAACGTGTTTCAGACCCGC GGTTCTGGTTCTGGTAGC TATCAGACCCAGACCAACAGCCCGCGCCGCGCGCGCAGCGTGGCGAGCCAGAGCATTATTGCGTATACC GGTTCTGGT TCTGGTAGC AAACAGATTTATAAAACCCCGCCGATTAAAGATTTTGGCGGCTTTAACTTTAGCCAGATTCTGCCGGATCCGAGCAAACCGAGCAAACGCAGCTTTATTGAAGA

[0740]

[0741] Additional information - (restriction sites are italicized, his-tag is bold, linker is underlined, 3x stop is underlined and bold): pET21a(+)-Nde1-His-tag (18nt)-CDP (180nt)-GS-linker (18nt)-BamHI-RBM (207nt)-GS-linker (18nt)-SD1 (108nt)-GS-linker (18nt)-SD2 (87nt)-GS-linker (18nt)-S1 / S2 (69nt)-GS-linker (18nt)-FP (189nt)-GS-linker (18nt)-HR1 (96nt)-3x stop Xho1-pET21a

[0742] DNA sequence for "CDP binding" (His tag-CDP-GS-linker-RBM-GS-linker-SD1-GS-linker- SD2) (681 nt) (SEQ ID NO: 67)

[0743]

[0744] Additional information - (restriction sites are italicized, his-tag is bold, linker is underlined, 3x stop is underlined and bold): pET21a(+)-Nde1-His-tag (18nt)-CDP (180nt)-GS-linker (18nt)-BamHI-RBM (207nt)-GS-linker (18nt)-SD1 (108nt)-GS-linker (18nt)-SD2 (87nt)-3x stop Xho1-pET21a

[0745] DNA sequence for "CDP-fusion" (His tag-CDP-GS-linker-S1 / S2-GS-linker-FP-GS-linker- HR1) (SEQ ID NO:68)

[0746]

[0747] Additional information - (restriction sites are italicized, his-tag is bold, linker is underlined, 3x stop is underlined and bold): pET21a(+)-Nde1-His-tag (18nt)-CDP (180nt)-GS-linker (18nt)-BamHI-S1 / S2 (69nt)-GS-linker (18nt)-FP (189nt)-GS-linker (18nt)-HR1 (96nt)-3x stop Xho1-pET21a

[0748] RBM of SARS-CoV-2 (SEQ ID NO:69)

[0749] SNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQ

[0750] SD1 of SARS-CoV-2 (SEQ ID NO:70)

[0751] PKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPF

[0752] SD2 of SARS-CoV-2 (SEQ ID NO:71)

[0753] TEVPVAIHADQLTPTWRVYSTGSNVFQTR

[0754] S1 / S2 of SARS-CoV-2 (SEQ ID NO:72)

[0755] YQTQTNSPRRARSVASQSIIAYT

[0756] FP of SARS-CoV-2 (SEQ ID NO:73)

[0757] KQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGF IKQYGDCLGDIAARD

[0758] HR1 of SARS-CoV-2 (SEQ ID NO:74)

[0759] TQNVLYENQKLIANQFNSAIGKIQDSLSSTAS。

Claims

1. A fusion polypeptide for use in vaccination or treatment of a mammalian subject, the fusion polypeptide comprising: (i) a non-mammalian peptide sequence, wherein The peptide sequence is a sequence associated with the disease or condition for which the mammalian subject is being vaccinated or treated; and (ii) an immunostimulatory peptide sequence having a ratio of bulky hydrophilic or charged amino acids to total amino acids less than or equal to 0.

55.

2. The fusion polypeptide for use according to claim 1, wherein The fusion polypeptide has an isoelectric point (pI) of less than or equal to 6.

3. The fusion polypeptide for use according to claim 1 or claim 2, wherein: a) the non-mammalian peptide sequence is a viral peptide and the disease or condition is a viral infection; or b) the non-mammalian peptide sequence is a mycobacterial or bacterial peptide and the disease or condition is a mycobacterial or bacterial infection; or c) the non-mammalian peptide sequence is a yeast peptide and the disease or disorder is a yeast infection; d) the non-mammalian peptide sequence is a parasitic peptide and the disease or disorder is a parasitic infection.

4. The fusion polypeptide for use according to any one of claims 1 to 3, wherein The non-mammalian peptide sequence is a SARS-CoV-2 peptide, and the disease or condition is a SARS-CoV-2 infection.

5. The fusion polypeptide for use according to claim 4, wherein: The SARS-CoV-2 peptide is a SARS-CoV-2 receptor binding domain (RBD) peptide.

6. The fusion polypeptide for use according to claim 5, wherein: The RBD peptide contains the amino acid sequence: NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATV (SEQ ID NO: 1), or a conservative amino acid variant thereof.

7. The fusion polypeptide for use according to any one of claims 4 to 6, wherein The SARS-CoV-2 peptide is an S protein peptide, optionally wherein: (a) the S protein peptide is an S1 subdomain peptide, further optionally wherein the S1 subdomain peptide is an RBD peptide (such as a receptor binding motif (RBM) peptide), an SD1 peptide or an SD2 peptide, or a combination thereof; (b) the S protein peptide is an S2 subdomain peptide, and further optionally, the S2 subdomain peptide is an S1 / S2 peptide, a FP+S2' peptide, or a HR1 peptide, or a combination thereof.

8. The fusion polypeptide for use according to claim 7, wherein: The SARS-CoV-2 peptide comprises: (a) the amino acid sequence of SEQ ID NO: 39, or a conservative amino acid variant thereof; (b) the amino acid sequence of SEQ ID NO: 40, or a conservative amino acid variant thereof; or (c) the amino acid sequence of SEQ ID NO: 41, or a conservative amino acid variant thereof.

9. The fusion polypeptide for use according to any one of claims 1 to 3, wherein The non-mammalian peptide sequence is an influenza virus peptide sequence and the disease or disorder is influenza.

10. The fusion polypeptide for use according to claim 9, wherein: The influenza virus peptide sequence is an H1 influenza virus peptide, an N1 influenza virus peptide, or an H1 / N1 influenza virus peptide.

11. The fusion polypeptide for use according to claim 10, wherein: The influenza virus peptide comprises: (a) the amino acid sequence of SEQ ID NO: 42, or a conservative amino acid variant thereof; (b) the amino acid sequence of SEQ ID NO: 43, or a conservative amino acid variant thereof; or (c) the amino acid sequence of SEQ ID NO: 44, or a conservative amino acid variant thereof.

12. A fusion polypeptide for use according to any one of the preceding claims, wherein The immunostimulatory peptide sequence is at least 40 amino acids and no more than 150 amino acids in length.

13. A fusion polypeptide for use according to any one of the preceding claims, wherein The immunostimulatory peptide sequence has an isoelectric point (pI) of less than or equal to 4.

14. A fusion polypeptide for use according to any one of the preceding claims, wherein The immunostimulatory peptide sequence comprises the amino acid sequence DDEDFVDEDDD (SEQ ID NO: 4) of the RRAB protein or a conservative amino acid variant thereof.

15. A fusion polypeptide for use according to any one of the preceding claims, wherein The immunostimulatory peptide sequence comprises at least one predicted T cell epitope.

16. The fusion polypeptide for use according to claim 15, wherein: The predicted T cell epitope is selected from: MSYB protein T cell epitope and / or RRAB protein T cell epitope.

17. The fusion polypeptide for use according to claim 16, wherein: The MSYB protein T cell epitope is contained in the amino acid sequence: NPGIDAEDANVQQFNAQKYVLQDGDIMWQV (SEQ ID NO: 2), and / or EGEFQLEPPLDTEEGRAAADE (SEQ ID NO: 3), or conservative amino acid variants thereof.

18. A fusion polypeptide for use according to any one of the preceding claims, wherein The immunostimulatory peptide sequence comprises the amino acid sequence: a) DNNSLSQEVQNGSNHLENNQSQSNGGGSDSALSLSSKTAALAAATTVNDGSDGATSSAVG (SEQ ID NO: 5), or a conservative amino acid variant thereof; or b) HHAHTMYATLEEAIDAAREEFLADNPGIDAEDANVQQFNAQKYVLQDGDIMWQVEFFADEGEEGEDDEDFVDEDDD (SEQ ID NO: 6), or a conservative amino acid variant thereof; or c) HHAHNPGIDAEDANVQQFNAQKYVLQDGDIMWQVEGEFQLEPPLDTEEGRAAADEDDEDFVDEDDD (SEQ ID NO: 7), or a conservative amino acid variant thereof; or d) HHAHNPGIDAEDANVQQFNAQKYVLQDGDIMWQVDDEDFVDEDDD (SEQ ID NO: 8), or a conservative amino acid variant thereof.

19. A fusion polypeptide comprising: (i) a non-mammalian peptide sequence, wherein The peptide sequence is a sequence associated with the disease or condition for which the mammalian subject is being vaccinated or treated; and (ii) an immunostimulatory peptide sequence having a ratio of bulky hydrophilic or charged amino acids to total amino acids less than or equal to 0.55; wherein the non-mammalian peptide sequence is linked to the immunostimulatory peptide sequence, and wherein the immunostimulatory peptide sequence comprises the amino acid sequence: a) HHAHTMYATLEEAIDAAREEFLADNPGIDAEDANVQQFNAQKYVLQDGDIMWQVEFFADEGEEGEDDEDFVDEDDD (SEQ ID NO: 6), or a conservative amino acid variant thereof; or b) HHAHNPGIDAEDANVQQFNAQKYVLQDGDIMWQVEGEFQLEPPLDTEEGRAAADEDDEDFVDEDDD (SEQ ID NO: 7), or a conservative amino acid variant thereof; or c) HHAHNPGIDAEDANVQQFNAQKYVLQDGDIMWQVDDEDFVDEDDD (SEQ ID NO: 8), or a conservative amino acid variant thereof; or d) DNNSLSQEVQNGSNHLENNQSQSNGGGSDSALSLSSKTAALAAATTVNDGSDGATSSAVG (SEQ ID NO: 5), or a conservative amino acid variant thereof.

20. A fusion polypeptide comprising: (i) a mammalian peptide sequence, wherein The peptide sequence is a sequence associated with the disease or condition for which the mammalian subject is being vaccinated or treated; and (ii) an immunostimulatory peptide sequence having a ratio of bulky hydrophilic or charged amino acids to total amino acids less than or equal to 0.55; wherein the mammalian peptide sequence is linked to the immunostimulatory peptide sequence, and wherein the immunostimulatory peptide sequence comprises the amino acid sequence: d) HHAHTMYATLEEAIDAAREEFLADNPGIDAEDANVQQFNAQKYVLQDGDIMWQVEFFADEGEEGEDDEDFVDEDDD (SEQ ID NO: 6), or a conservative amino acid variant thereof; or e) HHAHNPGIDAEDANVQQFNAQKYVLQDGDIMWQVEGEFQLEPPLDTEEGRAAADEDDEDFVDEDDD (SEQ ID NO: 7), or a conservative amino acid variant thereof; or f) HHAHNPGIDAEDANVQQFNAQKYVLQDGDIMWQVDDEDFVDEDDD (SEQ ID NO: 8), or a conservative amino acid variant thereof.

21. The fusion polypeptide according to claim 19 or 20, wherein: The fusion polypeptide has an isoelectric point (pI) of less than or equal to 6.

22. The fusion polypeptide according to claim 19 or 21, wherein: a) the non-mammalian peptide sequence is a viral peptide and the disease or condition is a viral infection; or b) the non-mammalian peptide sequence is a mycobacterial or bacterial peptide and the disease or condition is a mycobacterial or bacterial infection; or c) the non-mammalian peptide sequence is a yeast peptide and the disease or disorder is a yeast infection; d) the non-mammalian peptide sequence is a parasitic peptide and the disease or disorder is a parasitic infection.

23. The fusion polypeptide according to claim 20, wherein The mammalian peptide sequence is associated with tumor angiogenesis.

24. The fusion polypeptide according to claim 20, wherein The mammalian peptide sequence is vimentin (Vim), apelin, notum or timp1.

25. The fusion polypeptide according to any one of claims 19 and 21 to 22, wherein The non-mammalian peptide sequence is a SARS-CoV-2 peptide.

26. The fusion polypeptide according to claim 25, wherein The SARS-CoV-2 peptide is a SARS-CoV-2 receptor binding domain (RBD) peptide.

27. The fusion polypeptide according to claim 26, wherein The RBD peptide contains the amino acid sequence: NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATV (SEQ ID NO: 1), or a conservative amino acid variant thereof.

28. The fusion polypeptide according to any one of claims 25 to 27, wherein The SARS-CoV-2 peptide is an S protein peptide, optionally wherein: (a) the S protein peptide is an S1 subdomain peptide, further optionally wherein the S1 subdomain peptide is an RBD peptide (such as a receptor binding motif (RBM) peptide), an SD1 peptide or an SD2 peptide, or a combination thereof; (b) the S protein peptide is an S2 subdomain peptide, and further optionally, the S2 subdomain peptide is an S1 / S2 peptide, a FP+S2' peptide, or a HR1 peptide, or a combination thereof.

29. The fusion polypeptide according to claim 28, wherein The SARS-CoV-2 peptide comprises: (a) the amino acid sequence of SEQ ID NO: 39, or a conservative amino acid variant thereof; (b) the amino acid sequence of SEQ ID NQ:40, or a conservative amino acid variant thereof; or (c) the amino acid sequence of SEQ ID NO: 41, or a conservative amino acid variant thereof.

30. The fusion polypeptide according to any one of claims 19 and 21 to 22, wherein The non-mammalian peptide sequence is an influenza virus peptide sequence and the disease or disorder is influenza.

31. The fusion polypeptide according to claim 30, wherein The influenza virus peptide sequence is an H1 influenza virus peptide, an N1 influenza virus peptide, or an H1 / N1 influenza virus peptide.

32. The fusion polypeptide according to claim 31, wherein The influenza virus peptide comprises: (a) the amino acid sequence of SEQ ID NO: 42, or a conservative amino acid variant thereof; (b) the amino acid sequence of SEQ ID NO: 43, or a conservative amino acid variant thereof; or (c) the amino acid sequence of SEQ ID NO: 44, or a conservative amino acid variant thereof.

33. The fusion polypeptide according to any one of claims 19 to 32, wherein The immunostimulatory peptide sequence is at least 40 amino acids and no more than 150 amino acids in length.

34. The fusion polypeptide according to any one of claims 19 to 33, wherein The immunostimulatory peptide sequence has an isoelectric point (pI) of less than or equal to 4.

35. A nucleic acid encoding a fusion polypeptide according to any one of claims 19 to 34, optionally wherein, The nucleic acid is an expression vector.

36. A pharmaceutical composition comprising the fusion polypeptide according to any one of claims 19 to 34 or the nucleic acid according to claim 35, wherein The composition further comprises a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

37. The fusion polypeptide according to any one of claims 19 to 34 or the composition according to claim 36 for use as a medicament.

38. Use of the fusion polypeptide according to any one of claims 19 to 34 or the composition according to claim 36 for vaccination or treatment of a subject.

39. A method for vaccinating or treating a subject, the method comprising: The fusion polypeptide according to any one of claims 19 to 34 or the composition according to claim 36 is administered to the subject.

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