Immunogenic composition for preventing or treating group a streptococcus infection

Abstract

The present disclosure relates to the field of vaccines to protect against disease caused by group A streptococcus (GAS) bacteria, and methods of using the same. In particular, the present disclosure relates to immunogenic compositions comprising a J8 peptide and a p*17 peptide.

Description

[0001] “Immunogenic composition”

[0002] Related Application Data

[0003] The present application claims priority from Australian Patent Application No. 2024903636 filed on 6 November 2024 entitled “Immunogenic composition”. The entire contents of which is hereby incorporated by reference.

[0004] Sequence Listing

[0005] The present application is filed together with a Sequence Listing in electronic form. The entire contents of the Sequence Listing are hereby incorporated by reference.

[0006] Technical field

[0007] The present disclosure relates to the field of vaccines to protect against disease caused by group A streptococcus (GAS) bacteria, and methods of using the same.

[0008] Background

[0009] Group A streptococcus (GAS) bacteria are ubiquitous pathogens that cause a wide spectrum of human diseases. GAS bacteria colonise the upper respiratory tract (URT) and skin, and can progress to invasive and immune-mediated diseases. GAS diseases include mild conditions such as tonsillitis and impetigo, to scarlet fever and more severe invasive GAS diseases such as sepsis, toxic shock, and necrotising fasciitis. GAS bacteria are responsible for an estimated 18 million cases of serious disease with more than 700 million new primary cases and 500,000 deaths per year.

[0010] GAS bacteria utilise antigenic variation as one of the main defence mechanisms to circumvent host immune responses and thus a successful vaccine needs to provide straintranscending and multicompartmental (e.g., mucosal and skin) immunity. Despite the burden of disease, there is currently no vaccine available for this organism. Thus, there remains a need for safe, effective and affordable GAS vaccine that provides cross-serotype protection.

[0011] Summary

[0012] The present disclosure is based on the unexpected finding that J8 and p* 17 peptides are recognised by the human immune system as distinct epitopes. This was a particularly surprising finding given that p*17 peptides comprise a double amino acid variant of the minimal B-cell epitope from within the pl45 portion or fragment of the M protein of GAS (i.e., the same epitope comprised within J8). This finding led the inventors to the development of a composition comprising a J8 peptide and a p*17 peptide, which demonstrated enhanced effectiveness as a result of increased cross-serotype protection and the ability to elicit both mucosal and systemic antibody responses.

[0013] The findings by the inventors support an immunogenic composition comprising:

[0014] (a) a first immunogenic protein comprising an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof, and / or a first nucleic acid molecule comprising a nucleotide sequence encoding the first immunogenic protein or a nucleotide sequence complementary thereto; and

[0015] (b) a second immunogenic protein comprising an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, and / or a second nucleic acid molecule comprising a nucleotide sequence encoding the second immunogenic protein or a nucleotide sequence complementary thereto.

[0016] In one example, the immunogenic composition comprises a third immunogenic protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14 or a fragment, variant or derivative thereof, and / or a third nucleic acid molecule comprising a nucleotide sequence encoding the third immunogenic protein or a nucleotide sequence complementary thereto.

[0017] The findings by the inventors also support an immunogenic composition comprising:

[0018] (i)(a) a first immunogenic protein comprising an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof; and

[0019] (b) a second immunogenic protein comprising an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, and / or

[0020] (ii) one or more nucleic acid molecules comprising a nucleotide sequence encoding the first and the second immunogenic protein or a nucleotide sequence complementary thereto.

[0021] In one example, the immunogenic composition comprises:

[0022] (i) a third immunogenic protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14 or a fragment, variant or derivative thereof, and / or

[0023] (ii) one or more nucleic acid molecules comprising a nucleotide sequence encoding the first, the second and the third immunogenic protein or a nucleotide sequence complementary thereto.

[0024] In one example, the first immunogenic protein is conjugated, coupled or otherwise linked to a carrier protein or a fragment, variant or derivative thereof. In one example, the second immunogenic protein is conjugated, coupled or otherwise linked to a carrier protein or a fragment, variant or derivative thereof.

[0025] In one example, the first immunogenic protein and the second immunogenic protein are conjugated, coupled or otherwise linked to a carrier protein or a fragment, variant or derivative thereof.

[0026] In one example, the third immunogenic protein is conjugated, coupled or otherwise linked to a carrier protein or a fragment, variant or derivative thereof.

[0027] In one example, the first immunogenic protein, the second immunogenic protein and the third immunogenic protein are conjugated, coupled or otherwise linked to a carrier protein or a fragment, variant or derivative thereof.

[0028] In one example, the carrier protein is selected from the group consisting of: a thyroglobulin or a fragment, variant or derivative thereof; an albumin or a fragment, variant or derivative thereof; a toxin or a fragment, variant or derivative thereof; a toxoid or a fragment, variant or derivative thereof; and a mutant cross-reactive material (CRM) or a fragment, variant or derivative thereof.

[0029] In one example, the toxin or fragment, variant or derivative thereof is selected from the group consisting of: a tetanus toxin or toxoid or a fragment, variant or derivative thereof; a diphtheria toxin or toxoid or a fragment, variant or derivative thereof; a pertussis toxin or toxoid or a fragment, variant or derivative thereof; a Pseudomonas toxin or toxoid or a fragment, variant or derivative thereof; an E. coli toxin or toxoid or a fragment, variant or derivative thereof; a Staphylococcus toxin or toxoid or a fragment, variant or derivative thereof; and a Streptococcus toxin or toxoid or a fragment, variant or derivative thereof.

[0030] In one example, the carrier protein is or comprises a diphtheria toxin (DT) or a fragment, variant or derivative thereof.

[0031] In one example, the carrier protein is or comprises a CRM197 protein or a fragment, variant or derivative thereof.

[0032] In one example, the immunogenic composition comprises an adjuvant. For example, the adjuvant is or comprises aluminium hydroxide (Alum). In another example, the adjuvant is or comprises a liposomal adjuvant.

[0033] In one example, the first immunogenic protein comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 6 or a fragment, variant or derivative thereof. In one example, the second immunogenic protein comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 4 or a fragment, variant or derivative thereof.

[0034] In one example, wherein the third immunogenic protein comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 13 or a fragment, variant or derivative thereof.

[0035] In one example, the immunogenic composition comprises a pharmaceutically acceptable diluent, carrier or excipient.

[0036] In one example, the immunogenic composition is capable of providing cross-serotype protection to a subject.

[0037] The present disclosure also provides a method of eliciting an immune response to group A streptococcus (GAS) bacteria in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of an immunogenic composition described herein, to thereby elicit the immune response.

[0038] The present disclosure also provides a method of preventing, ameliorating or treating a GAS infection, or a disease, disorder or condition associated therewith in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of an immunogenic composition described herein, to thereby prevent, ameliorate or treat the infection, disease, disorder or condition associated with the GAS bacteria in the subject.

[0039] The present disclosure also provides the use of an immunogenic composition described herein in the manufacture of a medicament for eliciting an immune response to group A streptococcus (GAS) bacteria in a subject.

[0040] The present disclosure also provides the use of an immunogenic composition described herein in the manufacture of a medicament for preventing, ameliorating or treating an infection, disease or condition associated with group A streptococcus (GAS) bacteria in a subject.

[0041] The present disclosure also provides an immunogenic composition described herein for use in a method of eliciting an immune response to group A streptococcus (GAS) bacteria in a subject.

[0042] The present disclosure also provides an immunogenic composition described herein for use in a method of preventing, ameliorating or treating an infection, disease or condition associated with group A streptococcus (GAS) bacteria in a subject.

[0043] In one example, the method further comprises the step of administering the immunogenic composition to the subject parenterally, subcutaneously, intramuscularly or intranasally. In one example, the medicament is formulated to be parenterally, subcutaneously, intramuscularly or intranasally administered to the subject.

[0044] In one example, the GAS infection, or the disease, disorder or condition associated therewith is selected from the group consisting of: a GAS bacterial skin infection, a GAS bacterial intranasal infection, an upper respiratory tract (URT) GAS bacterial infection, a mucosal GAS bacterial infection, a GAS autoimmune infection and an invasive GAS bacterial infection.

[0045] In one example, the immunogenic composition comprises a first immunogenic protein described herein at a dose of at least about 30 pg to 40 pg, a second immunogenic protein described herein at a dose of at least about 30 pg to 40 pg and optionally a third immunogenic protein described herein at a dose of at least about 30 pg to 40 pg. In one example, the immunogenic composition comprises a first immunogenic protein described herein at a dose of at least about 33 pg, a second immunogenic protein described herein at a dose of at least about 33 pg and optionally a third immunogenic protein described herein at a dose of at least about 33 pg.

[0046] In one example, the immunogenic composition comprises a first immunogenic protein described herein at a dose of at least about 2 pg to 10 pg, a second immunogenic protein described herein at a dose of at least about 2 pg to 10 pg and optionally a third immunogenic protein described herein at a dose of at least about 2 pg to 10 pg. In one example, the immunogenic composition comprises a first immunogenic protein described herein at a dose of at least about 8 pg, a second immunogenic protein described herein at a dose of at least about 8 pg and optionally a third immunogenic protein described herein at a dose of at least about 8 Fg-

[0047] The immunogenic composition may further comprise about 250 pg of an adjuvant described herein. For example, the immunogenic composition may further comprise about 250 pg of Alum.

[0048] In one example, the immunogenic composition is administered to the subject at a dose of about 20 pg to about 100 pg. For example, the immunogenic composition is administered to the subject at a dose of about 20 pg to about 40 pg. In one example, the immunogenic composition is administered to the subject at a dose of about 24 pg. For example, the immunogenic composition is administered to the subject at a dose of about 90 pg to about 100 pg. In one example, the immunogenic composition is administered to the subject at a dose of about 99 pg. In one example, the immunogenic composition is administered to the subject at a first dose of about 20 pg to about 100 pg, a second dose of about 20 pg to about 100 pg and optionally a third dose of about 20 pg to about 100 pg. For example, the immunogenic composition is administered to the subject at a first dose of about 20 pg to about 40 pg, a second dose of about 20 pg to about 40 pg and optionally a third dose of about 20 pg to about 40 pg. In one example, the immunogenic composition is administered to the subject at a first dose of about 24 pg, a second dose of about 24 pg and optionally a third dose of about 24 pg.

[0049] In one example, the immunogenic composition is administered to the subject at a first dose of about 90 pg to about 100 pg, a second dose of about 90 pg to about 100 pg and optionally a third dose of about 90 pg to about 100 pg. In one example, the immunogenic composition is administered to the subject at a first dose of about 99 pg, a second dose of about 99 pg and optionally a third dose of about 99 pg.

[0050] Each dose of the immunogenic composition may further comprise about 250 pg of an adjuvant described herein. For example, each dose of the immunogenic composition may further comprise about 250 pg of Alum.

[0051] In one example, the medicament is formulated to be administered to the subject at a dose of about 20 pg to about 100 pg. For example, the medicament is formulated to be administered to the subject at a dose of about 20 pg to about 40 pg. In one example, the immunogenic composition is administered to the subject at a dose of about 24 pg. For example, the medicament is formulated to be administered to the subject at a dose of about 90 pg to about 100 pg. In one example, the immunogenic composition is administered to the subject at a dose of about 99 pg.

[0052] Particularly preferred embodiments are described herein, including in the independent claims.

[0053] Brief description of the drawings

[0054] The following figures form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these figures in combination with the detailed description of specific embodiments presented herein. It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. Figure 1. Schematic of study design of human clinical trial (n=10 per vaccination group and n=10 for placebo).

[0055] Figure 2. Assessment of vaccine J8-specific IgG in sera samples from J8 vaccinated participants. Five participants (A: GAS001; B: GAS002; C:GAS003; D: GAS004; and E:GAS005) were vaccinated intramuscularly with J8-CRM+K4S2-CRM / Alum on days 0, 21 and 42. Sera was collected before vaccination (TO) and at two weeks ± 7 days post last vaccination (T3) and tested in ELISA to assess J8-specific IgG antibody response. ELISA plates (Nunc Maxisorp) were coated with vaccine peptide J8 and blocked with 5% skim milk in PBS with 0.05% Tween-20.

[0056] Figure 3. Assessment of antibody cross-reactivity to p*17 in sera samples from J8 vaccinated participants. Five participants (A: GAS001; B: GAS002; C:GAS003; D: GAS004; and E:GAS005) were vaccinated intramuscularly with J8-CRM+K4S2-CRM / Alum on days 0, 21 and 42. Sera was collected before vaccination (TO) and at two weeks ± 7 days post last vaccination (T3) and tested in ELISA to assess p*17-specific IgG antibody response. ELISA plates (Nunc Maxisorp) were coated with vaccine peptide p* 17 and blocked with 5% skim milk in PBS with 0.05% Tween-20.

[0057] Figure 4. Assessment of vaccine p*17-specific IgG in sera samples from p*17 vaccinated participants. Five participants (A: GAS006; B: GAS007; C:GAS009; D: GAS010; and E: GAS011) were vaccinated intramuscularly with p* 17-CRM+K4S2-CRM / Alum on days 0, 21 and 42. Sera was collected before vaccination (TO) and at two weeks ± 7 days post last vaccinated (T3) and tested in ELISA to assess p*17-specific IgG antibody response. ELISA plates (Nunc Maxisorp) were coated with vaccine peptide p* 17 and blocked with 5% skim milk in PBS with 0.05% Tween-20.

[0058] Figure 5. Assessment of antibody cross-reactivity to J8 in sera samples from p*17 vaccinated participants. Five participants (A: GAS006; B: GAS007; C:GAS009; D: GAS010; and E: GAS011) were vaccinated intramuscularly with p* 17-CRM+K4S2-CRM / Alum on days 0, 21 and 42. Sera was collected before vaccination (TO) and at two weeks ± 7 days post last vaccinated (T3) and tested in ELISA to assess J8-specific IgG antibody response. ELISA plates (Nunc Maxisorp) were coated with vaccine J8 and blocked with 5% skim milk in PBS with 0.05% Tween-20.

[0059] Figure 6. Comparative assessment of antigen-specific salivary IgG responses in mice following vaccination with Bivalent or Trivalent formulations. BALB / c mice (n=5 / group) were immunised three times intramuscularly with Bivalent or Trivalent formulations on days 0, 21, and 42. (A) J8-specific IgG levels in saliva; and (B) p*17-specific IgG levels in saliva; and (C) K4S2-specific IgG levels in saliva. NS: P > 0.05; *: P < 0.05. Statistical comparisons were performed using a Mann-Whitney t-test (compare two groups) or one-way ANOVA (to compare three groups) in GraphPad Prism 10: ns p > 0.05.

[0060] Figure 7. Comparative assessment of antigen-specific serum IgG responses in mice following vaccination with Bivalent or Trivalent formulations. BALB / c mice (n=5 / group) were immunised three times intramuscularly with Bivalent or Trivalent formulations on days 0, 21, 42. (A) J8-specific IgG levels in serum; and (B) p*17-specific IgG levels in serum; and (C) K4S2-specific IgG levels in serum. NS: P > 0.05; *: P < 0.05. Statistical comparisons were performed using a Mann-Whitney t-test (compare two groups) or one-way ANOVA (to compare three groups) in GraphPad Prism 10: ns p > 0.05.

[0061] Figure 8. Assessment of the protective efficacy of Bivalent or Trivalent vaccine formulations. BALB / c mice (n=10 / group) were immunised three times intramuscularly on days 0, 21, and 42. Two weeks post-last- boost, the mice were infected via the URT with S. pyogenes 5448AP, covR / S mutant at 5 * 10A6 cfu / mouse. On day 2 post-infection all surviving mice were euthanized and (A) nasal shedding; (B) throat swabs; (C) NALT; and (D) lungs were collected to determine S. pyogenes burden, data are represented as the geomean ± geometric SD (cfu / tissue) on a Log 10 scale. Statistical analysis was performed using One-way ANOVA on GraphPad Prism 10: *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

[0062] Figure 9. Flow cytometric assessment of IgG binding to whole GAS. GAS2031 (clinical isolate, covR / S wt, Ml) was incubated overnight with sera from naive mice or mice vaccinated with PBS / Alum, J8-DT+K4S2-DT / Alum (J8Bivalent), pl7-DT+K4S2-DT / Alum (pl7Bivalent), or J8-DT+pl7-DT+K4S2-DT / Alum (Trivalent). BALB / c mice (n=3 / group) were immunized on days 0, 21, and 42, with sera collected on day 65 for binding assessment to GAS2031. After incubation, bacteria were washed with PBS and incubated for 1 hour with anti-mouse IgGl (PE Dazzle) secondary antibody. Samples were analysed using LSR Fortessa and FlowJo software. (A) Histogram showing sera binding of vaccinated groups vs. PBS / Alum group; (B) Histogram showing vaccinated groups vs. naive group; (C) Geometric mean fluorescence intensity (MFI) for anti -IgGl (PE Dazzle) secondary antibody; and (D) Frequency of anti-mouse IgGl positive cells relative to the parent population.

[0063] Figure 10. Flow cytometric assessment of IgG binding to whole GAS. GAS5448AP (animal passaged, covR / S mt, Ml) was incubated overnight with sera from naive mice or mice vaccinated with PBS / Alum, J8-DT+K4S2-DT / Alum (J8Biv), pl7-DT+K4S2-DT / Alum (pl7Biv), or J8-DT+pl7-DT+K4S2-DT / Alum (Triv). BALB / c mice (n=3 / group) were immunized on days 0, 21, and 42, with sera collected on day 65 for binding assessment to GAS5448AP. After incubation, bacteria were washed with PBS and incubated for 1 hour with anti-mouse IgGl (PE Dazzle) secondary antibody. Samples were analysed using LSR Fortessa and Flow Jo software. (A) Histogram showing sera binding of vaccinated groups vs. PBS / Alum group; (B) Histogram showing vaccinated groups vs. naive group; (C) Geometric mean fluorescence intensity (MFI) for anti -IgGl (PE Dazzle) secondary antibody; and (D) Frequency of anti-mouse IgGl positive cells relative to the parent population.

[0064] Figure 11. Flow cytometric assessment of IgG binding to whole GAS. GASM59 (clinical isolate, covR / S wt, M59) was incubated overnight with sera from naive mice or mice vaccinated with PBS / Alum, J8-DT+K4S2-DT / Alum (J8Biv), pl7-DT+K4S2-DT / Alum (pl7Biv), or J8-DT+pl7-DT+K4S2-DT / Alum (Triv). BALB / c mice (n=3 / group) were immunized on days 0, 21, and 42, with sera collected on day 65 for binding assessment to GASM59. After incubation, bacteria were washed with PBS and incubated for 1 hour with antimouse IgGl (PE Dazzle) secondary antibody. Samples were analysed using LSR Fortessa and FlowJo software. (A) Histogram showing sera binding of vaccinated groups vs. PBS / Alum group; (B) Histogram showing vaccinated groups vs. naive group; (C) Geometric mean fluorescence intensity (MFI) for anti -IgGl (PE Dazzle) secondary antibody; and (D) Frequency of anti-mouse IgGl positive cells relative to the parent population.

[0065] Figure 12. Dose-dependent saliva IgG responses following 1-, 2-, or 3 -dose trivalent vaccine in BALB / c mice. Mice were immunised with a single (DO), two (DO and 21) or three doses (DO, 21 and 42) of Trivalent vaccine. Saliva samples were collected on day 50 and tested for antigen-specific IgG by ELISA. The endpoint titer was defined as the highest dilution with an absorbance exceeding three standard deviations above the mean of naive control wells. Data are presented as mean ± standard error of the mean (SEM). Statistical comparisons were performed using one-way ANOVA in GraphPad Prism 10: *, P < 0.05; **, P < 0.01; ***, PO.OOl; ****, P < 0.0001.

[0066] Figure 13. Dose-dependent serum IgG responses following trivalent vaccine in BALB / c mice. Mice were immunised with a single (DO), two (DO and 21) or three doses (DO, 21 and 42) of Trivalent vaccine. Sera samples were collected on day 65 and tested for antigenspecific IgG by ELISA. (A) J8-specific, (B) p*17-specific, and (C) K4S2-specific IgG levels were measured by ELISA. The endpoint titer was defined as the highest dilution with an absorbance exceeding three standard deviations above the mean of naive control wells. Data are presented as mean ± standard error of the mean (SEM). Statistical comparisons were performed using one-way ANOVA in GraphPad Prism 10: *, P < 0.05; **, P < 0.01; ***, PO.OOl; ****, P < 0.0001. Figure 14. Assessment of vaccine efficacy following URT infection with 5448AP (Ml). (A) clinical scores, (B) percent weight loss, (C) survival proportions, (D) nasal shedding scores (0=0, 1=1-10, 2=10-100, 3=100-1000 and 4 = 1000+) and (E) throat swabs and tissues (NALT, nasal turbinates, lungs, spleen and blood) were collected to determine S. pyogenes burden, data are represented as the geomean ± geometric SD (cfu / tissue) on a Log 10 scale. Statistical analysis was performed using non-parametric Mann-Whitney t-test corrected for multiple comparisons (Holm-Sidak) on GraphPad Prism 10: ns, P>0.05; *, P < 0.05; **, P < 0.01.

[0067] Figure 15. Sustained immunogenicity in human participants after vaccination. (A-B) Combined Stage 1 and 2 data (J8-CRM + K4S2-CRM cohort; n = 10). (A) J8- and (B) K4S2- specific serum IgG (OD, 450 nm dilution) data with samples collected pre-vaccination (TO), two weeks post-third vaccination (T3), and six months post-third vaccination (T4). (C-D) Combined Stage 1 and 2 (p*17-CRM + K4S2-CRM* cohort; n = 10) showing (C) p*17-and (D) K4S2-specific serum IgG responses. Data represent serum IgG measured at a 1 :80 dilution. Net OD values were calculated by subtracting secondary-antibody control OD values from raw readings. Data represent averaged duplicates. Statistics performed using Dunnett’s multiple comparisons test. Non-significant values are indicated as (ns). Significantly different values are indicated as (*) when p < 0.05, (**) when p < 0.005, (***) when p < 0.0005 and (****) when p < 0.0001.

[0068] Figure 16. Quantification of antigen-specific IgG in human participants. Quantitative ELISA was used to measure antigen-specific IgG concentrations in serum samples from trial participants. Quantification was performed using a standard curve generated with purified antigen-specific IgG (J8 or p*17) and corresponding peptide antigens. The antigen-specific IgG antibodies were purified from pooled Fijian plasma samples known to be positive for J8 and / or p*17-specific IgG. The purification process involved two steps: first, total IgG was isolated using a Protein G column; second, antigen-specific IgG was purified using an affinity column (SulfoLink column) coupled with the respective antigen peptide. Data are presented for both Stage 1 and Stage 2 participants in (A-J) the J8-CRM + K4S2-CRM cohort and (K- T) the p*17-CRM + K4S2-CRM cohort, across all time points - pre-vaccination (TO), post- first, second, and third booster doses (Tl, T2, and T3), and six months after the third booster (T4). Some data for participants GAS001, GAS002, and GAS005 are missing due to the unavailability of samples.

[0069] Figure 17. Strep A competition flow binding assay with participant sera. Strep A strain 2031 (Ml) was incubated with sera from participants GAS006, GAS007, and GAS011 collected at TO, T3, and T4. After incubation, unbound sera were washed off and the bacteria incubated overnight (O / N) with mouse p*17 monoclonal antibody (mAb), which recognises the same epitope within the C3 repeat region of the M-protein (pl45 region). Following O / N incubation, unbound p*17 mAb was washed off and detection was performed using an antimouse IgGl PE-Dazzle® 594 secondary antibody (A). Reduced p*17 binding at later time points indicates increased vaccine-specific IgG occupancy of the M-protein. (A) Schematic of the competition binding assay. (B-D) Representative flow cytometry plots and quantification (mean fluorescence intensity, MFI) for (B) GAS006, (C) GAS007, and (D) GAS011, showing progressive reduction of p*17 binding from TO to T4. Data are represented as mean + standard error of the mean (SEM). Statistical analysis was performed using a Mann-Whitney t-test on GraphPad Prism 10: *, P < 0.05.

[0070] Figure 18. Immunisation of human participants induced long-term bactericidal antibodies. (A)(i) Participant sera pre- (TO), 2-weeks (T3) and 6-months (T4) post third vaccination were incubated with Strep A strain 2031 (Ml), (ii) combined with baby rabbit complement, and (iii) differentiated neutrophil -like HL-60 cells. Following incubations to allow phagocytosis and CFU growth O / N, (iv) CFU were counted and (v) used to calculate an opsonic titre, indicating the highest dilution where test serum CFU < control serum CFU. Open circles indicate negative control; solid circles indicate positive control. (B i-iv) Opsonic titres from vaccinees GAS004 (J8-CRM+K4S2-CRM) and 006, 007, 009, and 011 (P*17- CRM+K4S2-CRM) are shown. For GAS004, T4 samples were not available at the time of analysis and thus not included.

[0071] Key to the Sequence Listing

[0072] SEQ ID NO: 1 Amino acid sequence of full length M protein SEQ ID NO: 2 Amino acid sequence of pl 45 peptide SEQ ID NO: 3 Amino acid sequence of pl 45 minimal epitope SEQ ID NO: 4 Amino acid sequence of p*17 peptide SEQ ID NO: 5 Amino acid sequence of p*17 minimal epitope SEQ ID NO: 6 Amino acid sequence of J8 peptide SEQ ID NO: 7 Amino acid sequence of J8 peptide variant #1 SEQ ID NO: 8 Amino acid sequence of J8 peptide variant #2 SEQ ID NO: 9 Amino acid sequence of J8 peptide variant #3 SEQ ID NO: 10 Amino acid sequence of J8 peptide variant #4 SEQ ID NO: 11 Amino acid sequence of J14 peptide SEQ ID NO: 12 Amino acid sequence of the S2 peptide of SpyCEP

[0073] SEQ ID NO: 13 Amino acid sequence of K4S2 peptide

[0074] SEQ ID NO: 14 Amino acid sequence of SpyCEP peptide

[0075] SEQ ID NO: 15 Amino acid sequence of CRM 197 protein

[0076] SEQ ID NO: 16 Amino acid sequence of diphtheria toxin

[0077] SEQ ID NO: 17 Nucleotide sequence of pl 45 minimal epitope

[0078] SEQ ID NO: 18 Nucleotide sequence of p*17 variant #1

[0079] SEQ ID NO: 19 Nucleotide sequence of p*17 variant #2

[0080] SEQ ID NO: 20 Nucleotide sequence of p*17 variant #3

[0081] SEQ ID NO: 21 Nucleotide sequence of p*17 variant #4

[0082] SEQ ID NO: 22 Nucleotide sequence of p*17 variant #5

[0083] SEQ ID NO: 23 Nucleotide sequence of p*17 variant #6

[0084] SEQ ID NO: 24 Nucleotide sequence of p*17 variant #7

[0085] SEQ ID NO: 25 Nucleotide sequence of p*17 variant #8

[0086] SEQ ID NO: 26 Nucleotide sequence of p*17 variant #9

[0087] SEQ ID NO: 27 Nucleotide sequence of p*17 variant #10

[0088] SEQ ID NO: 28 Nucleotide sequence of p*17 variant #11

[0089] SEQ ID NO: 29 Nucleotide sequence of p*17 variant #12

[0090] SEQ ID NO: 30 Nucleotide sequence of p*17 variant #13

[0091] SEQ ID NO: 31 Nucleotide sequence of J8 peptide

[0092] SEQ ID NO: 32 Nucleotide sequence of J14 peptide

[0093] SEQ ID NO: 33 Nucleotide sequence of S2 peptide variant #1

[0094] SEQ ID NO: 34 Nucleotide sequence of S2 peptide variant #2

[0095] SEQ ID NO: 35 Nucleotide sequence of S2 peptide variant #3

[0096] SEQ ID NO: 36 Nucleotide sequence of S2 peptide variant #4

[0097] SEQ ID NO: 37 Nucleotide sequence of S2 peptide variant #5

[0098] SEQ ID NO: 38 Nucleotide sequence of K4S2 peptide

[0099] SEQ ID NO: 39 Nucleotide sequence of CRM 197 protein

[0100] SEQ ID NO: 40 Nucleotide sequence of diphtheria toxin

[0101] SEQ ID NO: 41 Nucleotide sequence of p*17 minimal epitope

[0102] Detailed description

[0103] General Techniques and Definitions

[0104] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in genomics, immunology, molecular biology, immunohistochemistry, biochemistry, oncology, and pharmacology).

[0105] The present disclosure is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology and immunology. Such procedures are described, for example in Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Fourth Edition (2012), whole of Vols I, II, and III; DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, Second Edition., 1995), IRL Press, Oxford, whole of text; Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed, 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, ppl-22; Atkinson et al, pp35-81; Sproat etal, pp 83-115; and Wu etal, pp 135-151; 4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text; Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, whole of text; Perbal, B., A Practical Guide to Molecular Cloning (1984) and Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), whole of series.

[0106] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0107] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the disclosure, as described herein.

[0108] Each feature of any particular aspect or embodiment or example of the present disclosure may be applied mutatis mutandis to any other aspect or embodiment or example of the present disclosure.

[0109] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter. As used herein, the singular forms of “a”, “and” and “the” include plural forms of these words, unless the context clearly dictates otherwise. For example, a reference to “a bacterium” includes a plurality of such bacteria, and a reference to “an allergen” is a reference to one or more allergens.

[0110] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0111] Throughout this specification, the word “comprise’ or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0112] By “consisting essentially of’, in the context of an amino acid sequence, is meant the recited amino acid sequence together with an additional one, two or three amino acids at the N- and / or C-terminus. Similarly, in the context of a nucleic acid sequence, by “consisting essentially of’ is meant the recited nucleotide sequence together with an additional one, two or three base pairs at the 3’ and / or 5’ end thereof.

[0113] Throughout the present specification, various aspects and components of the disclosure can be presented in a range format. The range format is included for convenience and should not be interpreted as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range, unless specifically indicated. For example, description of a range such as from 1 to 5 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 5, from 3 to 5 etc., as well as individual and partial numbers within the recited range, for example, 1, 2, 3, 4, 5, 5.5 and 6, unless where integers are required or implicit from context. This applies regardless of the breadth of the disclosed range. Where specific values are required, these will be indicated in the specification.

[0114] The term “substantially” does not exclude “completely” (e.g., a composition which is “substantially free” from Y may be completely free from Y).

[0115] The term “about” in relation to a numerical value x is optional and means, for example, any number within 1, 5 or 10% of the referenced number. In certain embodiments, the term “about” encompasses the exact number recited.

[0116] All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference.

[0117] For the present disclosure, the database accession number or unique identifier provided herein for a gene or protein, as well as the gene and / or protein sequence or sequences associated therewith, are incorporated by reference herein.

[0118] Immunogenic proteins

[0119] The inventors have surprisingly shown that an immunogenic composition or a vaccine comprising the combination of a p* 17 protein, or a fragment, variant or derivative thereof, and a J8 peptide, or a fragment, variant or derivative thereof, is capable of providing cross-serotype reactivity and protection against GAS bacteria more broadly. These data suggest that the minimal epitopes of these immunogenic proteins (i.e., the minimal epitopes provided in SEQ ID NOs: 3 and 5 respectively), despite only differing by two amino acid residues, are surprisingly capable of eliciting different humoral or B-cell mediated antibody responses that specifically recognise each of these minimal epitopes. The immunogenic composition may optionally further include a Streptococcus pyogenes cell envelope proteinase (SpyCEP) protein or a fragment, variant or derivative thereof, such as an S2 protein or a K4S2 protein described herein.

[0120] The M protein of GAS bacteria represents a key virulence factor and is a clinically relevant marker for strain identification. The M protein coats GAS bacteria and acts as the primary antigen and determinant of type-specific immunity. This protein is generally considered essential for GAS virulence, providing antiphagocytic functions critical to survival in human tissues and fluids.

[0121] As used herein, a “pl45 protein” is an immunogenic peptide derived from the conserved C-repeat region of an M protein from GAS. An exemplary amino acid sequence of a pl 45 protein is set forth in SEQ ID NO: 2. A minimal epitope of a pl45 protein is also set forth in SEQ ID NO: 3. This minimal epitope comprises a peptide with minimal B and T cell epitopes from within the pl 45 protein, which was identified as devoid of potentially deleterious T cell autoepitopes, but which contained an opsonic B cell epitope.

[0122] As used herein, a “p*17 peptide” comprises an amino acid sequence at least partly derived from, or corresponding to, a GAS M protein C-region peptide. An exemplary amino acid sequence of a p*17 peptide is set forth in SEQ ID NO: 4. An exemplary amino acid sequence of a minimal epitope of the p*17 peptide is set forth in SEQ ID NO: 5. The minimal epitope of the p*17 peptide or peptide is or comprises a variant of the minimal epitope of the pl45 protein (i.e., a lysine to asparagine substitution at position 6 and a lysine to arginine substitution at position 10 of SEQ ID NO: 3). As used herein, a “J8 peptide” comprises an amino acid sequence at least partly derived from, or corresponding to, a GAS M protein C-region peptide. In this regard, J8 is a chimeric peptide that comprises the pl45 minimal epitope (i.e., SEQ ID NO: 3) and flanking GCN4 DNA-binding protein sequences which assist maintaining the correct helical folding and conformational structure of the J8 peptide. An exemplary amino acid sequence of the J8 peptide is set forth in SEQ ID NO: 6. Exemplary amino acid sequences of J8 peptide variants are set forth in SEQ ID NOs: 7 to 10.

[0123] As used herein, a “ J14 peptide” may comprise the amino acid sequence set forth in SEQ ID NO: 11, or a fragment, variant or derivative thereof. In this regard, J14 is a chimeric peptide that contains 14 amino acids from an M protein C-region, including the pl45 minimal epitope (i.e., SEQ ID NO: 3), and is flanked by yeast-derived GCN4 sequences which maintain the correct helical folding and conformational structure of the peptide.

[0124] The term "minimal epitope" refers to a portion of an epitope predicted to bind to MHC I or MHC II molecules. To this end, the minimal epitope in isolation may be immunogenic, and thereby capable of eliciting an immune response. The term minimal epitope thus may refer to short sequence or portion of an epitope, which are predicted to bind to MHC I or MHC II. Streptococcus pyogenes cell envelope proteinase (SpyCEP) protein is a 170-kDa multidomain serine protease expressed on the surface of the human pathogen Streptococcus pyogenes. The SpyCEP protein plays an important role in infection by catalysing cleavage and inactivation of the neutrophil chemoattractant interleukin-8. Non-limiting examples of SpyCEP protein amino acid sequences may be found under accession numbers YP597949.1 and (5. pyogenes MGAS 10270) and YP596076.1 (5. pyogenes MGAS9429). An exemplary amino acid sequence of a full length SpyCEP protein is set forth in SEQ ID NO: 14. An exemplary amino acid sequence of a SpyCEP protein fragment (i.e., S2 peptide) is set forth in SEQ ID NO: 12. An exemplary amino acid sequence of a derivative of the SpyCEP protein fragment of SEQ ID NO: 12 is set forth in SEQ ID NO: 13.

[0125] Thus, the present disclosure relates to a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3 (i.e., a pl45 minimal epitope), or a fragment, variant or derivative thereof. In particular examples, the first immunogenic protein comprises, consists of or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3 and 6 to 11 or a fragment, variant or derivative thereof. More particularly, the first immunogenic protein suitably comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 6 or a fragment, variant or derivative thereof. In other examples, the first immunogenic protein comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 2 or a fragment, variant or derivative thereof. For certain examples, the first immunogenic protein comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 7 or a fragment, variant or derivative thereof. Referring to various examples, the first immunogenic protein comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 8 or a fragment, variant or derivative thereof. In some examples, the first immunogenic protein comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 9 or a fragment, variant or derivative thereof. In some examples, the first immunogenic protein comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 10 or a fragment, variant or derivative thereof. In some examples, the first immunogenic protein comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 11 or a fragment, variant or derivative thereof. Suitably, the first immunogenic protein suitably does not comprise a full-length or wildtype M protein, such as that provided in SEQ ID NO: 1.

[0126] The present disclosure also relates to a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5 (i.e., a p*17 minimal epitope) or a fragment, variant or derivative thereof. In particular examples, the second immunogenic protein comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 4 or a fragment, variant or derivative thereof. Suitably, the second immunogenic protein suitably does not comprise a full-length or wildtype M protein, such as that provided in SEQ ID NO: 1.

[0127] The present disclosure also relates to a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14 or a fragment, variant or derivative thereof. In particular examples, the third immunogenic protein comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 12 or a fragment, variant or derivative thereof. For other examples, the third immunogenic protein comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 13 or a fragment, variant or derivative thereof. In various examples, the third immunogenic protein comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 14 or a fragment, variant or derivative thereof. In alternative examples, however, the third immunogenic protein suitably does not comprise a full-length or wildtype SpyCEP protein, such as that provided in SEQ ID NO: 14. As used herein, the term “immunogenic” will be understood to refer to a compound or composition that induces or generates an immune response, such as GAS bacteria or one or more molecular components thereof, such as M protein or SpyCEP. A compound or composition that induces or generates an immune response is typically one that induces a humoral and / or cell-mediated response in a subject to which the compound or composition is administered. The humoral and / or cell-mediated response may be specific to the compound or composition. Such a response may be detected and / or quantified by determining the induction of antibodies in the subject and / or cellular responses. Methods for assessing and / or measuring an immune response are well known in the art. Non-limiting examples of such methodologies are provided in Example 5. A quantitative antibody measurement may be determined. Alternatively, or in addition, a qualitative antibody measurement may be determined. For example, but without limitation, a measure of one or more functional features of antibodies elicited in a subject to which the compound or composition is administered, may be determined. Suitable functional features which can be measured are known in the art and include, without limitation, p*17 or J8 inhibition. A quantitative method for the determination of expression or secretion of cytokines or alteration in the phenotype of immune cells may also be used.

[0128] The immune response may be a protective immune response. By “protective immune response” it is meant an immune response that is sufficient to prevent or at least reduce the severity or extent of one or more symptoms of an infection, disease or condition associated with group A streptococcal (GAS) bacteria in a subject. A protective immune response may be determined directly through experimentation (such as by performing challenge studies to a subject vaccinated with a particular compound or composition). Alternatively, or in addition, a protective immune response may be determined via a measurement of one or more suitable correlates of protection. Suitable correlates of protection will be appreciated by one of skill in the art, and may include, for example but without limitation, antibody binding titre, microneutralisation (MN) titre, cytokine assays, cellular responses, antibody dependent cellular cytotoxicity assays.

[0129] By “protein” is meant an amino acid polymer. The amino acids may be natural or nonnatural amino acids, D- or L-amino acids as are well understood in the art. The term “protein” includes and encompasses “peptide”, which is typically used to describe a protein having no more than fifty amino acids and “polypeptide”, which is typically used to describe a protein having more than fifty amino acids.

[0130] Suitably, the immunogenic proteins described herein comprise up to or no more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acid residues or any range therein. In particular examples, the first immunogenic protein comprises about 10 to about 45 amino acid residues, about 15 to about 40 amino acid residues, about 15 to about 30 amino acid residues or about 20 to about 35 amino acid residues. For certain examples, the second immunogenic protein comprises about 10 to about 40 amino acid residues, about 12 to about 35 amino acid residues, about 15 to about 30 amino acid residues or about 15 to about 25 amino acid residues. Referring to other examples, the third immunogenic protein comprises about 10 to about 40 amino acid residues, about 12 to about 35 amino acid residues, about 15 to about 30 amino acid residues or about 15 to about 25 amino acid residues.

[0131] A “fragment” is a segment, domain, portion or region of a protein, which constitutes less than 100% of the amino acid sequence of the protein. In general, fragments may comprise up to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625 or 650 amino acids of an amino acid sequence, such as of any of the immunogenic proteins set forth in any one of SEQ ID NOs: 1 to 16.

[0132] A protein fragment provided herein may be a functional fragment and thereby retains, at least partly, functionality of a full-length protein, such as a full-length protein set forth in any one of SEQ ID NOs: 1 to 16. The fragment described herein can be immunogenic. Immunogenic protein fragments may have comparable immunogenic activity to the immunogenic protein from which they are derived (e.g., that of any one of SEQ ID NOs: 1 to 16). An immunogenic protein fragment described herein may elicit a comparable immune response, such as a protective immune response, to GAS bacteria. An immunogenic protein fragment described herein may be utilised in methods of preventing, ameliorating or treating an infection, disease or condition associated with GAS bacteria, such as those described herein.

[0133] As used herein, a protein “variant” shares a definable amino acid sequence relationship with a reference amino acid sequence. The reference amino acid sequence may be the amino acid sequence of any one of SEQ ID NOs: 1 to 16, for example, or a fragment or derivative thereof. The “variant” protein may have one or a plurality of amino acids of the reference amino acid sequence deleted or substituted by different amino acids. It is well understood in the art that some amino acids may be substituted or deleted without changing the activity of the immunogenic protein fragment and / or immunogenic protein (conservative substitutions). One or more of the other residues of SEQ ID NOs: 1 to 16, or a fragment or derivative thereof, may be conservatively modified (e.g., by amino acid substitution or deletion) so that the variant substantially retains the immunogenicity of the immunogenic protein from which it has been derived. Protein variants may share at least 70% or 75%, more suitably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89% or even more suitably at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% sequence identity with a reference amino acid sequence, such as one of those set forth in SEQ ID NOs: 1 to 16.

[0134] Suitably, such modifications do not substantially alter the immunogenicity of the immunogenic protein, such as those set forth in SEQ ID NOs: 1 to 16, or a fragment or derivative thereof. It is also envisaged, however, that modification of a “wild-type” or “unmodified” immunogenic protein, such as that of SEQ ID NOs: 1 to 16, may substantially improve or enhance the immunogenicity of the immunogenic fragment.

[0135] The term “variant” also includes isolated proteins or fragments thereof disclosed herein, produced from, or comprising amino acid sequences of, naturally occurring (e.g., allelic) variants, orthologs and synthetic variants, such as produced in vitro using mutagenesis techniques. Typically, modification includes substitution of one or more amino acids of an immunogenic protein provided herein. Variants may retain the biological activity of a corresponding wild-type protein (e.g., allelic or serotype variants, paralogs and orthologs) or may lack, or have a substantially reduced, biological activity compared to a corresponding wild-type protein.

[0136] Terms used generally herein to describe sequence relationships between respective proteins and nucleic acids include “comparison window”, “sequence identity”, “percentage of sequence identity” and “substantial identity”. Because respective nucleic acids / proteins may each comprise (i) only one or more portions of a complete nucleic acid / protein sequence that are shared by the nucleic acids / proteins, and (ii) one or more portions which are divergent between the nucleic acids / proteins, sequence comparisons are typically performed by comparing sequences over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” refers to a conceptual segment of typically 6, 9 or 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence for optimal alignment of the respective sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (Geneworks program by Intelligenetics; GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA, incorporated herein by reference) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids Res. 25 3389, which is incorporated herein by reference. A detailed discussion of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons Inc NY, 1995-1999).

[0137] The term “sequence identity” is used herein in its broadest sense to include the number of exact nucleotide or amino acid matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison. Thus, a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U) or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For example, “sequence identity” may be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA).

[0138] The present disclosure also contemplates derivatives of the immunogenic proteins disclosed herein. As used herein, “derivatives” are molecules such as proteins, fragments or variants thereof that have been altered, for example by conjugation, complexing, coupling or otherwise being linked with other chemical moieties, by post-translational modification (e.g., phosphorylation, acetylation and the like), modification of glycosylation (e.g., adding, removing or altering glycosylation), lipidation and / or inclusion of additional amino acid sequences as would be understood in the art.

[0139] Additional amino acid sequences may include fusion partner amino acid sequences which create a fusion protein. By way of example, fusion partner amino acid sequences may assist in detection and / or purification of the isolated fusion protein. Non-limiting examples include metal-binding (e.g., polyhistidine) fusion partners, maltose binding protein (MBP), Protein A, glutathione S-transferase (GST), fluorescent protein sequences (e.g., GFP), epitope tags such as myc, FLAG, HEX and haemagglutinin tags.

[0140] The additional amino acid sequences may comprise carrier proteins. For example, an immunogenic protein or peptide described herein may be conjugated, coupled or otherwise linked to a carrier protein. Examples of carrier proteins include a thyroglobulin or a fragment, variant or derivative thereof, an albumin or a fragment, variant or derivative thereof, a toxin or toxoid or a fragment, variant or derivative thereof or a mutant cross-reactive material (CRM) or a fragment, variant or derivative thereof.

[0141] The carrier protein may be a thyroglobulin or a fragment, variant or derivative thereof. The carrier protein may be an albumin or a fragment, variant or derivative thereof. The carrier protein may be a toxin or a fragment, variant or derivative thereof. The carrier protein may be a toxoid or a fragment, variant or derivative thereof. The carrier protein may be a mutant cross- reactive material (CRM) or a fragment, variant or derivative thereof.

[0142] The toxin or fragment, variant or derivative thereof may be a tetanus toxin or toxoid or a fragment, variant or derivative thereof. The toxin or fragment, variant or derivative thereof may be a diphtheria toxin or toxoid or a fragment, variant or derivative thereof. The toxin or fragment, variant or derivative thereof may be a pertussis toxin or toxoid or a fragment, variant or derivative thereof. The toxin or fragment, variant or derivative thereof may be a Pseudomonas toxin or toxoid or a fragment, variant or derivative thereof. The toxin or fragment, variant or derivative thereof may be an E. coli toxin or toxoid or a fragment, variant or derivative thereof The toxin or fragment, variant or derivative thereof may be a Staphylococcus toxin or toxoid or a fragment, variant or derivative thereof. The toxin or fragment, variant or derivative thereof may be a Streptococcus toxin or toxoid or a fragment, variant or derivative thereof.

[0143] The mutant cross-reactive material (CRM) protein or a fragment, variant or derivative thereof may be a CRM197 protein or a fragment, variant or derivative thereof, for example, as described in International Publication W02017 / 070735, which is incorporated herein by way of reference. The CRM197 protein is a nontoxic mutant version of the diphtheria toxin, wherein the single amino acid exchange of a glycine (Gly, G) in position 52 to a glutamic acid (Glu, E) renders the protein non-toxic. An exemplary amino acid sequence of a CRM197 protein is set forth in SEQ ID NO: 15.

[0144] An exemplary amino acid sequence of a diphtheria toxin is set forth in SEQ ID NO: 16. In some examples, the diphtheria toxin is set forth in SEQ ID NO: 16 is a detoxified form of diphtheria toxin (e.g., a diphtheria toxoid).

[0145] The immunogenic protein or peptide described herein may be conjugated to a carrier protein at the C terminus or the N terminus thereof. Moreover, the immunogenic protein may be conjugated to a carrier protein directly or alternatively by way of a heterologous amino acid sequence, such as a linker sequence. For example, the immunogenic protein or peptide described herein may be conjugated to a carrier protein by a C terminus cysteine using 6- maleimido-caproyl N-hydroxysuccinimide (MCS). One method of conjugation is described in Batzloff et al. 2003, which is incorporated herein by way of reference.

[0146] Thus, the present disclosure relates to a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3 (i.e., a pl45 minimal epitope), or a fragment, variant or derivative thereof conjugated to a CRM197 protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 15. The first immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3 and 6 to 11 or a fragment, variant or derivative thereof conjugated to a CRM197 protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 15. The first immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence set forth in SEQ ID NO: 6 or a fragment, variant or derivative thereof conjugated to a CRM197 protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 15. The first immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence set forth in SEQ ID NO: 2 or a fragment, variant or derivative thereof conjugated to a CRM197 protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 15. The first immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence set forth in SEQ ID NO: 7 or a fragment, variant or derivative thereof conjugated to a CRM197 protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 15. The first immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence set forth in SEQ ID NO: 8 or a fragment, variant or derivative thereof conjugated to a CRM197 protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 15. The first immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence set forth in SEQ ID NO: 9 or a fragment, variant or derivative thereof conjugated to a CRM197 protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 15. The first immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence set forth in SEQ ID NO: 10 or a fragment, variant or derivative thereof conjugated to a CRM197 protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 15. The first immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence set forth in SEQ ID NO: 11 or a fragment, variant or derivative thereof conjugated to a CRM197 protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 15. The present disclosure also relates to a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5 (i.e., a p*17 minimal epitope) or a fragment, variant or derivative thereof conjugated to a CRM197 protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 15. The second immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence set forth in SEQ ID NO: 4 or a fragment, variant or derivative thereof conjugated to a CRM 197 protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 15.

[0147] The present disclosure also relates to a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14 or a fragment, variant or derivative thereof conjugated to a CRM197 protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 15. The third immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence set forth in SEQ ID NO: 12 or a fragment, variant or derivative thereof conjugated to a CRM 197 protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 15. The third immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence set forth in SEQ ID NO: 13 or a fragment, variant or derivative thereof conjugated to a CRM197 protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 15. The third immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence set forth in SEQ ID NO: 14 or a fragment, variant or derivative thereof conjugated to a CRM197 protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 15.

[0148] Thus, the present disclosure relates to a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3 (i.e., a pl45 minimal epitope), or a fragment, variant or derivative thereof conjugated to a diphtheria toxin comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 16. The first immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3 and 6 to 11 or a fragment, variant or derivative thereof conjugated to a diphtheria toxin comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 16. The first immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence set forth in SEQ ID NO: 6 or a fragment, variant or derivative thereof conjugated to a diphtheria toxin comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 16. The first immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence set forth in SEQ ID NO: 2 or a fragment, variant or derivative thereof conjugated to a diphtheria toxin comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 16. The first immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence set forth in SEQ ID NO: 7 or a fragment, variant or derivative thereof conjugated to a diphtheria toxin comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 16. The first immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence set forth in SEQ ID NO: 8 or a fragment, variant or derivative thereof conjugated to a diphtheria toxin comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 16. The first immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence set forth in SEQ ID NO: 9 or a fragment, variant or derivative thereof conjugated to a diphtheria toxin comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 16. The first immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence set forth in SEQ ID NO: 10 or a fragment, variant or derivative thereof conjugated to a diphtheria toxin comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 16. The first immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence set forth in SEQ ID NO: 11 or a fragment, variant or derivative thereof conjugated to a diphtheria toxin comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 16.

[0149] The present disclosure also relates to a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5 (i.e., a p*17 minimal epitope) or a fragment, variant or derivative thereof conjugated to a diphtheria toxin comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 16. The second immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence set forth in SEQ ID NO: 4 or a fragment, variant or derivative thereof conjugated to a diphtheria toxin comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 16.

[0150] The present disclosure also relates to a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14 or a fragment, variant or derivative thereof conjugated to a diphtheria toxin comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 16. The third immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence set forth in SEQ ID NO: 12 or a fragment, variant or derivative thereof conjugated to a diphtheria toxin comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 16. The third immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence set forth in SEQ ID NO: 13 or a fragment, variant or derivative thereof conjugated to a diphtheria toxin comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 16. The third immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence set forth in SEQ ID NO: 14 or a fragment, variant or derivative thereof conjugated to a diphtheria toxin comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 16.

[0151] Other derivatives contemplated by the invention include, but are not limited to, modification to side chains, incorporation of unnatural amino acids and / or their derivatives during protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the immunogenic proteins, fragments and variants of the invention.

[0152] In this regard, the skilled person is referred to Chapter 15 of CURRENT PROTOCOLS IN PROTEIN SCIENCE, Eds. Coligan et al. (John Wiley & Sons NY 1995-2008) for more extensive methodology relating to chemical modification of proteins.

[0153] The present disclosure also envisages an isolated protein comprising a plurality of immunogenic proteins described herein, such as in the form of a “polytope” protein (e.g., a single protein comprising an amino acid sequence of a J8 peptide, or a fragment, variant or derivative thereof, an amino acid sequence of a p*17 peptide, or a fragment, variant or derivative thereof, and optionally an amino acid sequence of a SpyCEP protein, or a fragment, variant or derivative thereof). For example, said immunogenic proteins may be present singly or as repeats, which also includes tandemly repeated proteins or “mixtures” of different proteins. Heterologous amino acid sequences (e.g., “spacer” amino acids) may also be included between one or a plurality of the immunogenic proteins present in said isolated protein.

[0154] Additionally, nucleic acid molecules encoding such an isolated protein (i.e., a single nucleic acid molecule encoding a plurality of the immunogenic proteins described herein, such as a first immunogenic protein, a second immunogenic protein and optionally a third immunogenic protein) are contemplated herein.

[0155] The present disclosure also provides an isolated protein that consists of: (i) one or more of the immunogenic proteins described herein or a segment, domain, portion or region thereof (e.g., an epitope or antigenic determinant thereof), and inclusive of fragments, variants or derivatives thereof; and (ii) optionally one or more additional amino acid sequences. In this regard, the additional amino acid sequences are suitably heterologous amino acid sequences that can be at the N- and / or C-termini of the recited amino acid sequence of the aforementioned proteins, although without limitation thereto. In particular examples, a variant protein or peptide may comprise one or a plurality (e.g., 1, 2, 3, 4 etc) of lysine residues at an N and / or C -terminus thereof. For some examples, the variant protein includes 1, 2, 3 or 4 lysine residues at an N and / or C-terminus thereof. More particularly, the variant protein includes 1, 2, 3 or 4 lysine residues at an N-terminus thereof. Even more particularly, the variant protein includes 1, 2, 3 or 4 lysine residues at a C-terminus thereof. The plurality of lysine residues (e.g., polylysine sequence) may be a linear sequence of lysine residues or may be branched chain sequences of lysine residues. These additional lysine residues may facilitate increased peptide solubility. A non-limiting example of such a variant is designated “K4S2” (i.e., SEQ ID NO: 13).

[0156] The immunogenic protein fragments and / or isolated immunogenic proteins described herein, inclusive of fragments, variants and derivatives thereof, may be produced by any means known in the art, including but not limited to, chemical synthesis, recombinant DNA technology and proteolytic cleavage to produce protein fragments.

[0157] Chemical synthesis is inclusive of solid phase and solution phase synthesis. Such methods are well known in the art, although reference is made to examples of chemical synthesis techniques as provided in Chapter 9 of SYNTHETIC VACCINES Ed. Nicholson (Blackwell Scientific Publications) and Chapter 15 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al., (John Wiley & Sons, Inc. NY USA 1995-2008). In this regard, reference is also made to International Publication WO 99 / 02550 and International Publication WO 97 / 45444.

[0158] Recombinant peptides or proteins may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook et al., MOLECULAR CLONING. A Laboratory Manual (Cold Spring Harbor Press, 1989), in particular Sections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al., (John Wiley & Sons, Inc. NY USA 1995-2008), in particular Chapters 10 and 16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan etal., (John Wiley & Sons, Inc. NY USA 1995-2008), in particular Chapters 1, 5 and 6. Typically, recombinant protein preparation includes expression of a nucleic acid encoding the protein in a suitable host cell, such as those described herein.

[0159] The immunogenic proteins and / or nucleic acid molecules described herein may be considered to be isolated or otherwise purified. For the purposes of this disclosure, by “isolated” or “purified” is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in native, chemical synthetic or recombinant form.

[0160] Nucleic acid molecules

[0161] The present disclosure further relates to nucleic acid molecules that encode an immunogenic protein described herein, inclusive of fragments, variants and derivatives thereof.

[0162] The present disclosure further relates to one or more nucleic acid molecules that encode an immunogenic protein described herein, inclusive of fragments, variants and derivatives thereof.

[0163] The present disclosure also relates to one or more nucleic acid molecules that encode one or more of the immunogenic proteins described herein (e.g., a first immunogenic protein, a second immunogenic protein and optionally a third immunogenic protein and / or one or more carrier proteins), inclusive of fragments, variants and derivatives thereof.

[0164] The present disclosure also provides a first nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto. In particular examples, the first nucleic acid encodes a first immunogenic protein that comprises, consists of or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3 and 6 to 11 or a fragment, variant or derivative thereof. More particularly, the first nucleic acid encodes a first immunogenic protein that suitably comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 6 or a fragment, variant or derivative thereof. In other examples, the first nucleic acid encodes a first immunogenic protein that comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 2 or a fragment, variant or derivative thereof. For certain examples, the first nucleic acid encodes a first immunogenic protein that comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 7 or a fragment, variant or derivative thereof. Referring to various examples, the first nucleic acid encodes a first immunogenic protein that comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 8 or a fragment, variant or derivative thereof. In some examples, the first nucleic acid encodes a first immunogenic protein that comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 9 or a fragment, variant or derivative thereof. In some examples, the first nucleic acid encodes a first immunogenic protein that comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 10 or a fragment, variant or derivative thereof. In some examples, the first nucleic acid encodes a first immunogenic protein that comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 11 or a fragment, variant or derivative thereof.

[0165] The present disclosure also provides a first nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a first immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 17, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto. In particular examples, the first nucleic acid molecule comprises, consists of or consists essentially of a nucleotide sequence encoding a first immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence selected from the group consisting of SEQ ID NO: 31 and 32, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto.

[0166] The first nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a first immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence selected from the group consisting of SEQ ID NO: 17, 31 and 32, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding a carrier protein is selected from the group consisting of: a thyroglobulin or a fragment, variant or derivative thereof; an albumin or a fragment, variant or derivative thereof; a toxin or a fragment, variant or derivative thereof; a toxoid or a fragment, variant or derivative thereof; and a mutant cross-reactive material (CRM) or a fragment, variant or derivative thereof.

[0167] The first nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a first immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 17, or a nucleotide sequence complementary thereto. The first nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a first immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 17, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding CRM197 protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 39, or a nucleotide sequence complementary thereto. The first nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a first immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 17, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding diphtheria toxin or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 40, or a nucleotide sequence complementary thereto.

[0168] The first nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a first immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 31, or a nucleotide sequence complementary thereto. The first nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a first immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 31, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding CRM197 protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 39, or a nucleotide sequence complementary thereto. The first nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a first immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 31, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding diphtheria toxin or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 40, or a nucleotide sequence complementary thereto. The first nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a first immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 32, or a nucleotide sequence complementary thereto. The first nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a first immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 32, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding CRM197 protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 39, or a nucleotide sequence complementary thereto. The first nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a first immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 32, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding diphtheria toxin or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 40, or a nucleotide sequence complementary thereto.

[0169] The first nucleic acid may be one or more nucleic acid molecules.

[0170] The present disclosure also provides a second nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto. In particular examples, the second nucleic acid molecule encodes a second immunogenic protein that comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 4 or a fragment, variant or derivative thereof.

[0171] The present disclosure also provides a second nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 41, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto. In particular examples, the second nucleic acid molecule comprises, consists of or consists essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence selected from the group consisting of SEQ ID NO: 18 to 30, or a nucleotide sequence complementary thereto.

[0172] The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 18, or a nucleotide sequence complementary thereto. The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 18, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding CRM197 protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 39, or a nucleotide sequence complementary thereto. The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 18, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding diphtheria toxin or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 40, or a nucleotide sequence complementary thereto.

[0173] The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 19, or a nucleotide sequence complementary thereto. The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 19, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding CRM197 protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 39, or a nucleotide sequence complementary thereto. The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 19, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding diphtheria toxin or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 40, or a nucleotide sequence complementary thereto.

[0174] The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 20, or a nucleotide sequence complementary thereto. The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 20, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding CRM197 protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 39, or a nucleotide sequence complementary thereto. The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 20, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding diphtheria toxin or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 40, or a nucleotide sequence complementary thereto.

[0175] The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 21, or a nucleotide sequence complementary thereto. The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 21, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding CRM197 protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 39, or a nucleotide sequence complementary thereto. The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 21, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding diphtheria toxin or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 40, or a nucleotide sequence complementary thereto.

[0176] The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 22, or a nucleotide sequence complementary thereto. The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 22, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding CRM197 protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 39, or a nucleotide sequence complementary thereto. The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 22, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding diphtheria toxin or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 40, or a nucleotide sequence complementary thereto.

[0177] The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 23, or a nucleotide sequence complementary thereto. The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 23, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding CRM197 protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 39, or a nucleotide sequence complementary thereto. The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 23, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding diphtheria toxin or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 40, or a nucleotide sequence complementary thereto.

[0178] The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 24, or a nucleotide sequence complementary thereto. The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 24, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding CRM197 protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 39, or a nucleotide sequence complementary thereto. The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 24, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding diphtheria toxin or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 40, or a nucleotide sequence complementary thereto. The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 25, or a nucleotide sequence complementary thereto. The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 25, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding CRM197 protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 39, or a nucleotide sequence complementary thereto. The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 25, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding diphtheria toxin or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 40, or a nucleotide sequence complementary thereto.

[0179] The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 26, or a nucleotide sequence complementary thereto. The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 26, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding CRM197 protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 39, or a nucleotide sequence complementary thereto. The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 26, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding diphtheria toxin or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 40, or a nucleotide sequence complementary thereto.

[0180] The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 27, or a nucleotide sequence complementary thereto. The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 27, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding CRM197 protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 39, or a nucleotide sequence complementary thereto. The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 27, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding diphtheria toxin or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 40, or a nucleotide sequence complementary thereto.

[0181] The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 28, or a nucleotide sequence complementary thereto. The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 28, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding CRM197 protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 39, or a nucleotide sequence complementary thereto. The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 28, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding diphtheria toxin or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 40, or a nucleotide sequence complementary thereto.

[0182] The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 29, or a nucleotide sequence complementary thereto. The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 29, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding CRM197 protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 39, or a nucleotide sequence complementary thereto. The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 29, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding diphtheria toxin or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 40, or a nucleotide sequence complementary thereto.

[0183] The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 30, or a nucleotide sequence complementary thereto. The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 30, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding CRM197 protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 39, or a nucleotide sequence complementary thereto. The second nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 30, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding diphtheria toxin or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 40, or a nucleotide sequence complementary thereto.

[0184] The second nucleic acid may be one or more nucleic acid molecules.

[0185] The present disclosure also provides a third nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14 or a fragment, variant or derivative thereof or a nucleotide sequence complementary thereto. In particular examples, the third nucleic acid molecule encodes a third immunogenic protein that comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 12 or a fragment, variant or derivative thereof. For other examples, the third nucleic acid molecule encodes a third immunogenic protein that comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 13 or a fragment, variant or derivative thereof. In various examples, the third nucleic acid molecule encodes a third immunogenic protein that comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 14 or a fragment, variant or derivative thereof.

[0186] The present disclosure also provides a third nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a third immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence selected from the group consisting of SEQ ID NO: 33 to 38, or a nucleotide sequence complementary thereto.

[0187] The third nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a third immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 33, or a nucleotide sequence complementary thereto. The third nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a third immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 33, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding CRM197 protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 39, or a nucleotide sequence complementary thereto. The third nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a third immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 33, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding diphtheria toxin or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 40, or a nucleotide sequence complementary thereto.

[0188] The third nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a third immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 34, or a nucleotide sequence complementary thereto. The third nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a third immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 34, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding CRM197 protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 39, or a nucleotide sequence complementary thereto. The third nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a third immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 34, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding diphtheria toxin or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 40, or a nucleotide sequence complementary thereto.

[0189] The third nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a third immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 35, or a nucleotide sequence complementary thereto. The third nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a third immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 35, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding CRM197 protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 39, or a nucleotide sequence complementary thereto. The third nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a third immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 35, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding diphtheria toxin or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 40, or a nucleotide sequence complementary thereto.

[0190] The third nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a third immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 36, or a nucleotide sequence complementary thereto. The third nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a third immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 36, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding CRM197 protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 39, or a nucleotide sequence complementary thereto. The third nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a third immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 36, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding diphtheria toxin or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 40, or a nucleotide sequence complementary thereto.

[0191] The third nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a third immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 37, or a nucleotide sequence complementary thereto. The third nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a third immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 37, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding CRM197 protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 39, or a nucleotide sequence complementary thereto. The third nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a third immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 37, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding diphtheria toxin or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 40, or a nucleotide sequence complementary thereto.

[0192] The third nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a third immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 38, or a nucleotide sequence complementary thereto. The third nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a third immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 38, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding CRM197 protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 39, or a nucleotide sequence complementary thereto. The third nucleic acid molecule may comprise, consist of or consist essentially of a nucleotide sequence encoding a third immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 38, or a nucleotide sequence complementary thereto; conjugated to a nucleotide sequence encoding diphtheria toxin or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 40, or a nucleotide sequence complementary thereto.

[0193] The third nucleic acid may be one or more nucleic acid molecules.

[0194] It will be appreciated that any of the one or more nucleic acid molecules described herein may encode the first immunogenic protein, the second immunogenic protein and optionally the third immunogenic protein, as individual or separate proteins or as a polytope protein, such as those described herein. For example, the one or more nucleic acid molecules described herein may encode the first immunogenic protein, the second immunogenic protein and optionally the third immunogenic protein as individual or separate proteins. Alternatively, the one or more nucleic acid molecules described herein may encode the first immunogenic protein, the second immunogenic protein and optionally the third immunogenic protein as a polytope protein (e.g., a fusion protein comprising multiple epitopes).

[0195] The first nucleic acid molecule, the second nucleic acid molecule, and optionally the third nucleic acid molecule may be each separate nucleic acid molecules. Alternatively, the first nucleic acid molecule, the second nucleic acid molecule, and optionally the third nucleic acid molecule may be combined on a single nucleic acid molecule.

[0196] Thus, the present disclosure also provides one or more nucleic acid molecules comprising: a first nucleotide sequence encoding a first immunogenic protein described herein and a second nucleotide sequence encoding a second immunogenic protein described herein. Optionally, the one or more nucleic acid molecules may further encode a third immunogenic protein described herein. Also optionally, the one or more nucleic acid molecules may further encode one or more carrier proteins such as those described herein as conjugated to the first, second and / or third immunogenic proteins.

[0197] The one or more nucleic acid molecules comprise a first nucleotide sequence encoding a first immunogenic protein that comprises, consists of or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3 and 6 to 11, or a fragment, variant or derivative thereof, and a second nucleotide sequence encoding a second immunogenic protein that comprises, consists of or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 5, or a fragment, variant or derivative thereof. The one or more nucleic acid molecules comprise a first nucleotide sequence encoding a first immunogenic protein that comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof, and a second nucleotide sequence encoding a second immunogenic protein that comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof.

[0198] The one or more nucleic molecules comprise may further comprise a third nucleotide sequence encoding a third immunogenic protein that comprises, consists of or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14, or a fragment, variant or derivative thereof. The one or more nucleic molecules may further comprise a third nucleotide sequence encoding a third immunogenic protein that comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NOs: 12. The one or more nucleic molecules may further comprise a third nucleotide sequence encoding a third immunogenic protein that comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NOs: 13. The one or more nucleic molecules may further comprise a third nucleotide sequence encoding a third immunogenic protein that comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NOs: 14.

[0199] The one or more nucleic acid molecules comprise a first nucleotide sequence encoding a first immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence selected from the group consisting of SEQ ID NO: 31 and 32, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, and a second nucleotide sequence encoding a second immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence selected from the group consisting of SEQ ID NO: 18 to 30 and 41, or a nucleotide sequence complementary thereto.

[0200] The one or more nucleic molecules comprise may further comprise a third nucleotide sequence encoding a third immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence selected from the group consisting of SEQ ID NO: 33 to 38, or a nucleotide sequence complementary thereto. In such examples it will be clear that the first nucleic acid molecule, the second nucleic acid molecule, and optionally the third nucleic acid molecule may be combined on a single nucleic acid molecule or each be separate nucleic acid molecules.

[0201] Nucleotide sequences, such as codon-optimised nucleotide sequence, encoding the immunogenic proteins of the present disclosure may be readily deduced from the complete amino acid sequences set forth in SEQ ID NOs: 1 to 16. Suitably, the nucleic acid molecule comprises, consists of or consists essentially of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 17 to 41, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto. Codon optimisation may be conveniently performed using standard protocols, such as those described in PCTZEP2017 / 081685, herein incorporated by reference in its entirety.

[0202] The term “nucleic acid” as used herein designates single- or double-stranded deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA includes genomic DNA and coding DNA (cDNA). RNA includes RNA, messenger RNA (mRNA), RNA interference (RNAi), small interfering RNA (siRNA), complementary RNA (cRNA) and autocatalytic RNA. Nucleic acids may also be DNA-RNA hybrids. A nucleic acid comprises a nucleotide sequence which typically includes nucleotides that comprise an A, G, C, T or U base. However, nucleotide sequences may include other bases such as inosine, methyly cytosine, methylinosine, methyladenosine and / or thiouridine, although without limitation thereto.

[0203] The nucleic acid molecule may be or comprise DNA. The nucleic acid molecule may be or comprise cDNA. In this regard, the nucleic acid molecule may be a DNA plasmid suitable for DNA vaccination.

[0204] The nucleic acid molecule encoding an immunogenic protein described herein may be in the form of RNA, such as mRNA, suitable for administration to a mammal, such as a human. The nucleic acid molecule may be or comprise an mRNA having an open reading frame encoding one or more of the immunogenic proteins provided herein (e.g., one or more of a J8 peptide, a p*17 peptide and optionally a SpyCEP protein, inclusive of fragment, variants or derivatives thereof). mRNA vaccines are described, for example, in International Patent Application Nos. PCT / US2015 / 027400 and PCT / US2016 / 044918, herein incorporated by reference in their entirety.

[0205] It will be appreciated that nucleic acid, and more particularly mRNA, delivery of immunogenic proteins provides a unique therapeutic alternative to peptide-based or DNA- based methods of administering such agents or vaccines. When the mRNA is delivered to a cell, the mRNA will be processed into a polypeptide or peptide by the intracellular machinery which can then process the polypeptide or peptide into the immunogenic proteins capable of eliciting an immune response within the subject.

[0206] Immunogenic compositions or vaccines comprising one or more isolated nucleic acids or polynucleotides that encode the immunogenic proteins described herein, or fragments, variants or derivatives thereof that may be used for such methods, are also contemplated by the present disclosure. For such examples, the composition suitably comprises a delivery agent or system, such as a nanoparticle, a lipid nanoparticle or a liposome, as are known in the art. In various examples, the nanoparticle has a mean diameter of 50-200 nm. In some examples, the composition comprising the mRNA polynucleotide (e.g., an mRNA polynucleotide having an open reading frame that encodes an immunogenic protein described herein) is formulated in a lipid nanoparticle.

[0207] A “polynucleotide” is a nucleic acid typically having 80 or more contiguous nucleotides, while an “oligonucleotide” generally has less than 80 contiguous nucleotides.

[0208] A “probe” may be a single or double-stranded oligonucleotide or polynucleotide, suitably labelled for the purpose of detecting complementary sequences in Northern or Southern blotting, for example.

[0209] A “primer” is usually a single-stranded oligonucleotide, typically having 15-50 contiguous nucleotides, which is capable of annealing to a complementary nucleic acid “template” and being extended in a template-dependent fashion by the action of a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or Sequenase™.

[0210] Suitably, the present disclosure provides a variant of an isolated nucleic acid that encodes an isolated immunogenic protein described herein, such as a variant of the nucleotide sequences set forth in any one of SEQ ID NOs: 17 to 41. Nucleic acid variants may encode a variant of an immunogenic protein described herein (e.g., a variant of any one of SEQ ID NOs: 1 to 16).

[0211] As used herein, a nucleic acid “variant” shares a definable nucleotide sequence relationship with a reference nucleic acid sequence. The “variant” nucleic acid may have one or a plurality of nucleic acids of the reference nucleic acid sequence deleted or substituted by different nucleic acids. Suitably, nucleic acid variants share at least about 35%, 40%, 45%, 50%, 55%, 60% or 65%, 66%, 67%, 68%, 69%, preferably at least about 70%, 71%, 72%, 73%, 74% or 75%, more preferably at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89% and even more preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% nucleotide sequence identity with an isolated nucleic acid of the present disclosure (e.g., any one of SEQ ID NOs: 17 to 41). The present disclosure also contemplates nucleic acids that have been modified such as by taking advantage of codon sequence redundancy. In such examples, codon usage may be modified to optimise expression of a nucleic acid in a particular organism or cell type.

[0212] Further contemplated herein are nucleic acid derivatives. By way of example, the use of modified purines (for example, inosine, methylinosine and methyladenosine), modified pyrimidines (for example, thiouridine and methylcytosine) and / or modified intemucleotide linkages or backbones is also envisaged for the nucleic acid molecules disclosed herein.

[0213] Nucleic acid conjugates, inclusive of the genetic constructs provided herein, are also envisaged. By way of example, the nucleic acid molecule or the genetic construct disclosed herein may be conjugated to one or more moieties or groups which enhance the activity, cell / tissue targeting, cellular distribution or cellular uptake thereof. These moieties or groups may be covalently bound to functional groups such as primary or secondary hydroxyl groups. Exemplary moieties or groups include intercalators, receptor ligands, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols (e.g., lithocholic acid or lauric acid), carbohydrates (e.g., GalNAc) lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins and dyes / labels (e.g., Cy5 dye to determine cellular uptake and / or localisation of the nucleic acid molecule).

[0214] Also contemplated herein are nucleic acid fragments, such as those encoding fragments or truncated versions of the immunogenic proteins described herein. A “fragment” is a segment, domain, portion or region of a nucleic acid, which respectively constitutes less than 100% of the respective nucleotide sequence. In particular examples, a nucleic acid fragment may comprise, for example, at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 4800, 4900, 4930 (inclusive of any range therein) contiguous nucleotides of said nucleic acid.

[0215] The isolated nucleic acids of the present disclosure can be conveniently prepared using standard protocols such as those described in Chapter 2 and Chapter 3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Eds. Ausubel et al. John Wiley & Sons NY, 1995-2008). Nucleic acid molecules may also be isolated, detected and / or subjected to recombinant DNA technology using nucleic acid sequence amplification techniques. Suitable nucleic acid amplification techniques covering both thermal and isothermal methods are well known to the skilled addressee and include polymerase chain reaction (PCR); strand displacement amplification (SDA); rolling circle replication (RCR); nucleic acid sequence-based amplification (NASBA), Q-P replicase amplification, recombinase polymerase amplification (RPA) and helicase-dependent amplification, although without limitation thereto.

[0216] As used herein, an “amplification product” refers to a nucleic acid product generated by nucleic acid amplification. Nucleic acid amplification techniques may include particular quantitative and semi-quantitative techniques such as qPCR, real-time PCR and competitive PCR, as are well known in the art.

[0217] Genetic constructs

[0218] It is further envisaged that the nucleic acid molecules of the present disclosure may be included in or be in the form of a genetic construct or a vector (e.g., a viral vector), as are known in the art. To this end, it is contemplated that the genetic construct may include nucleotide sequences that encode one or more (e.g., one, two or three) of the immunogenic proteins described herein (e.g., one or more, two or more or all of a p*17 peptide, a J8 peptide and a SpyCEP peptide, inclusive of fragments, variants and derivatives thereof).

[0219] The present disclosure also provides a genetic construct comprising, consisting of or consisting essentially of a first nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto. For example, the nucleotide sequence of the genetic construct can be SEQ ID NO: 17, a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto.

[0220] The present disclosure also provides a genetic construct comprising, consisting of or consisting essentially of a second nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto. For example, the nucleotide sequence of the genetic construct can be SEQ ID NO: 41, a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto. The present disclosure also provides a genetic construct comprising, consisting of or consisting essentially of a third nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14 or a fragment, variant or derivative thereof or a nucleotide sequence complementary thereto. For example, the nucleotide sequence of the genetic construct can be selected from the group consisting of SEQ ID NO: 33 to 38, a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto.

[0221] The present disclosure also provides a genetic construct comprising, consisting of or consisting essentially of: a first nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto; and a second nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto. For such examples, the genetic construct may further include a third nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14 or a fragment, variant or derivative thereof or a nucleotide sequence complementary thereto.

[0222] In particular examples, the genetic construct comprises: a first nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 6; and a second nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 4. For example, the first nucleic acid molecule of the genetic construct can comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 17, 31 and 32, a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto; and the second nucleic acid molecule of the genetic construct can comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 18 to 30 and 41, a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto. In other examples, the genetic construct comprises: a first nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 6; a second nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 4; and a third nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NOs: 12 or 13. In some examples, the first nucleic acid molecule of the genetic construct can comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 17, 31 and 32, a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto; the second nucleic acid molecule of the genetic construct can comprise a nucleotide sequence selected from the group consisting of SEQ ID NO: 18 to 30 and 41, a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto; and the third nucleic acid molecule of the genetic construct can comprise a nucleotide sequence selected from the group consisting of SEQ ID NO: 33 to 38, a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto.

[0223] In addition to the encoding nucleotide sequences of one or more of the immunogenic proteins provided herein, the genetic construct may include one or more additional or heterologous sequences, as are known in the art. Suitably, the genetic construct is in the form of, or comprises one or more genetic components of, a plasmid, a bacteriophage, a cosmid, a yeast, a viral vector or a bacterial artificial chromosome, as are well understood in the art. Genetic constructs may be suitable for maintenance and propagation of the isolated nucleic acid in bacteria or other host cells, for manipulation by recombinant DNA technology and / or expression of the nucleic acid or an encoded protein of the present disclosure.

[0224] For the purposes of host cell expression, the genetic construct is suitably an expression construct. According to some examples, the expression construct comprises the nucleic acid molecule of the present disclosure operably linked to one or more additional or heterologous sequences in an expression vector. An “expression vector” may be either a self-replicating extra-chromosomal vector such as a plasmid, or a vector that integrates into a host genome. By “operably linked” is meant that said additional nucleotide sequence(s) is / are positioned relative to the nucleic acid suitably to initiate, regulate or otherwise control transcription thereof. Regulatory nucleotide sequences will generally be appropriate for the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells.

[0225] Typically, said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters as known in the art are contemplated by the invention.

[0226] The present disclosure also provides a host cell transformed with nucleic acid molecules or genetic constructs described herein.

[0227] Suitable host cells for expression may be prokaryotic or eukaryotic. For example, suitable host cells may include but are not limited to mammalian cells (e.g. EleLa, HEK293T, Jurkat cells), yeast cells (e.g. Saccharomyces cerevisiae), insect cells (e.g. Sf9, Trichoplusia ni) utilized with or without a baculovirus expression system, plant cells (e.g. Chlamydomonas reinhardtii, Phaeodactylum tricornutum) or bacterial cells, such as E. coll. Introduction of genetic constructs into host cells (whether prokaryotic or eukaryotic) is well known in the art, as for example described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel etal., (John Wiley & Sons, Inc. 1995-2009), in particular Chapters 9 and 16.

[0228] The present disclosure also provides a method of producing immunogenic proteins described herein, comprising; (i) culturing the previously transformed host cell hereinbefore described; and (ii) isolating the immunogenic protein(s) from the host cell cultured in step (i).

[0229] The present disclosure also provides a method of producing a nucleic acid molecule, inclusive of a genetic construct (e.g., a DNA plasmid), described herein, comprising; (i) culturing the previously transformed host cell hereinbefore described; and (ii) isolating the nucleic acid molecule from said host cell cultured in step (i).

[0230] The recombinant protein may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook, et al., MOLECULAR CLONING. A Laboratory Manual (Cold Spring Harbor Press, 1989), in particular Sections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al., (John Wiley & Sons, Inc. 1995-2009), in particular Chapters 10 and 16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al., (John Wiley & Sons, Inc. 1995- 2009), in particular Chapters 1, 5 and 6.

[0231] It is envisaged that the genetic construct can alternatively be suitable for administration to a subject, such as a human. The genetic construct may be suitable for use in the methods described herein. The genetic construct may be suitable for DNA vaccination of a subject, such as a human. DNA vaccination may be by way of one or more DNA plasmid or viral vector expression constructs. A useful reference describing DNA vaccinology is DNA Vaccines, Methods and Protocols, Second Edition (Volume 127 of Methods in Molecular Medicine series, Humana Press, 2006).

[0232] The genetic construct may comprise a DNA plasmid. DNA plasmids typically comprise a viral promoter (such as SV40, RSV or CMV promoters). The DNA plasmid may include a tissue specific promoter (e.g., a liver-specific promoter), such as those described herein. Intron A may be included to improve mRNA stability and thereby increase protein expression. Plasmids may further include a multiple cloning site, a strong polyadenylation / transcription termination signal, such as bovine growth hormone or rabbit beta-globulin polyadenylation sequences. Secretion signals, such as that of tissue plasminogen activator, may also be included. The plasmid may further comprise Mason-Pfizer monkey virus c / .s-acting transcriptional elements (MPV-CTE) with or without HIV rev increased envelope expression. Additional modifications that may improve expression include the insertion of enhancer sequences, synthetic introns, adenovirus tripartite leader (TPL) sequences and / or modifications to polyadenylation and / or transcription termination sequences. Non-limiting examples of DNA vaccine plasmids include pVAC (Invivogen) and pVR1020 (Vical).

[0233] The genetic construct may comprise a viral vector, in which nucleotide sequences described herein may be ligated into a viral genome. Examples of viral vectors include, but are not limited to, vaccinia, RNA viruses (replicons), adeno-associated virus and replicationdefective viruses, like avipox, fowlpox, canarypox, MV A, and adenovirus.

[0234] The viral vector may include any regulatory sequence required to establish expression or induce expression of the encoded immunogenic protein in the target tissue, organ or cell of the subject. The viral vector may comprise an expression cassette comprising the nucleic acid encoding the immunogenic protein operably linked to a promoter and a polyadenylation recognition site. The expression cassette may be flanked at 5’ and 3’ ends thereof by viral cisacting regulatory sequences, such as inverted terminal repeats (ITRs). The viral vector may also include other regulatory sequences to establish expression of the encoded immunogenic protein, such as a ribosome binding element, a terminator, an enhancer, a selection marker, an intron, a polyA signal, and / or an origin of replication. The regulatory sequences may impart tissue-specific (e.g., liver-specific) gene expression capabilities. In certain instances, the tissuespecific regulatory sequences bind tissue specific transcription factors that induce transcription in a tissue specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are well known in the art. The regulatory sequences may include one or more target sequences or motifs that bind a regulatory molecule, such as an miRNA molecule, with a cell type, tissue or organ-specific expression profile to modulate (e.g., increase or decrease) expression of the encoded immunogenic protein in the cell type, tissue or organ in question (e.g., the liver).

[0235] Administration of the nucleic acid molecule, the genetic construct or the viral vector, provided herein can induce expression of the immunogenic protein(s) in the subject. Administration of the nucleic acid molecule, the genetic construct or the viral vector provided herein may specifically or selectively induce expression of the immunogenic protein(s) in a particular cell type, tissue or organ of the subject.

[0236] The terms “selective expression”, “selectively express” and grammatical equivalents thereof refer to a comparison of relative levels of expression in two or more regions (e.g., tissues, organs etc) of interest. For example, "selective expression" when used in connection with tissues refers to a substantially greater level of expression of a gene of interest in a particular tissue, or to a substantially greater number of cells which express the gene within that tissue, as compared, respectively, to the level of expression of, and the number of cells expressing, the same gene in another tissue (i.e., selectivity need not be absolute). Selective expression does not require, although it may include expression of a gene of interest in a particular tissue and a total absence of expression of the same gene in another tissue. Similarly, “selective expression” as used herein in reference to cell types refers to a substantially greater level of expression of, or a substantially greater number of cells which express, a gene of interest in a particular cell type, when compared, respectively, to the expression levels of the gene and to the number of cells expressing the gene in another cell type.

[0237] Exemplary viral vectors suitable for use according to the present disclosure include viral vectors derived from retroviruses, lentiviruses, herpes simplex- 1 viruses (HSV-1), adenoviruses, and adeno-associated viruses (AAVs).

[0238] Immunogenic compositions

[0239] The inventors have surprisingly shown that an immunogenic composition comprising the combination of a p* 17 peptide and a J8 peptide is capable of eliciting the production of a broader population of anti-M protein antibodies that may provide cross-serotype reactivity and broader protection against GAS bacteria than either immunogenic protein administered alone.

[0240] Accordingly, the present disclosure provides an immunogenic composition comprising: a first immunogenic protein described herein and a second immunogenic protein described herein; or one or more nucleic acid molecules encoding the first and second immunogenic proteins. Optionally, the immunogenic composition may further comprise a third immunogenic protein described herein or the one or more nucleic acid molecules may further encode the third immunogenic protein. To this end, the immunogenic composition may include one or more nucleic acid molecules described herein.

[0241] Accordingly, the present disclosure provides an immunogenic composition comprising:

[0242] (a) a first immunogenic protein comprising an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof, and / or a first nucleic acid molecule comprising a nucleotide sequence encoding the first immunogenic protein or a nucleotide sequence complementary thereto; and

[0243] (b) a second immunogenic protein comprising an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, and / or a second nucleic acid molecule comprising a nucleotide sequence encoding the second immunogenic protein or a nucleotide sequence complementary thereto.

[0244] Accordingly, the present disclosure also provides an immunogenic composition comprising:

[0245] (i)(a) a first immunogenic protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3 and 6 to 11, or a fragment, variant or derivative thereof; and

[0246] (b) a second immunogenic protein comprising an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, and / or

[0247] (ii) one or more nucleic acid molecules comprising a nucleotide sequence encoding the first and the second immunogenic protein or a nucleotide sequence complementary thereto.

[0248] The present disclosure also provides an immunogenic composition comprising:

[0249] (i)(a) a first immunogenic protein comprising an amino acid sequence set forth in SEQ ID NO: 2, or a fragment, variant or derivative thereof; and

[0250] (b) a second immunogenic protein comprising an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, and / or

[0251] (ii) one or more nucleic acid molecules comprising a nucleotide sequence encoding the first and the second immunogenic protein or a nucleotide sequence complementary thereto.

[0252] The present disclosure also provides an immunogenic composition comprising:

[0253] (i)(a) a first immunogenic protein comprising an amino acid sequence set forth in SEQ ID NO: 6, or a fragment, variant or derivative thereof; and (b) a second immunogenic protein comprising an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, and / or

[0254] (ii) one or more nucleic acid molecules comprising a nucleotide sequence encoding the first and the second immunogenic protein or a nucleotide sequence complementary thereto.

[0255] The present disclosure also provides an immunogenic composition comprising:

[0256] (i)(a) a first immunogenic protein comprising an amino acid sequence set forth in SEQ ID NO: 7, or a fragment, variant or derivative thereof; and

[0257] (b) a second immunogenic protein comprising an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, and / or

[0258] (ii) one or more nucleic acid molecules comprising a nucleotide sequence encoding the first and the second immunogenic protein or a nucleotide sequence complementary thereto.

[0259] The present disclosure also provides an immunogenic composition comprising:

[0260] (i)(a) a first immunogenic protein comprising an amino acid sequence set forth in SEQ

[0261] ID NO: 8, or a fragment, variant or derivative thereof; and

[0262] (b) a second immunogenic protein comprising an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, and / or

[0263] (ii) one or more nucleic acid molecules comprising a nucleotide sequence encoding the first and the second immunogenic protein or a nucleotide sequence complementary thereto.

[0264] The present disclosure also provides an immunogenic composition comprising:

[0265] (i)(a) a first immunogenic protein comprising an amino acid sequence set forth in SEQ ID NO: 9, or a fragment, variant or derivative thereof; and

[0266] (b) a second immunogenic protein comprising an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, and / or

[0267] (ii) one or more nucleic acid molecules comprising a nucleotide sequence encoding the first and the second immunogenic protein or a nucleotide sequence complementary thereto.

[0268] The present disclosure also provides an immunogenic composition comprising:

[0269] (i)(a) a first immunogenic protein comprising an amino acid sequence set forth in SEQ ID NO: 2, or a fragment, variant or derivative thereof; and

[0270] (b) a second immunogenic protein comprising an amino acid sequence set forth in SEQ ID NO: 10, or a fragment, variant or derivative thereof, and / or

[0271] (ii) one or more nucleic acid molecules comprising a nucleotide sequence encoding the first and the second immunogenic protein or a nucleotide sequence complementary thereto.

[0272] The present disclosure also provides an immunogenic composition comprising: (i)(a) a first immunogenic protein comprising an amino acid sequence set forth in SEQ ID NO: 11, or a fragment, variant or derivative thereof; and

[0273] (b) a second immunogenic protein comprising an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, and / or

[0274] (ii) one or more nucleic acid molecules comprising a nucleotide sequence encoding the first and the second immunogenic protein or a nucleotide sequence complementary thereto.

[0275] It is contemplated that the immunogenic composition described herein may further include a SpyCEP protein, such as an S2 fragment thereof, or an encoding nucleic acid molecule thereof. As such, the immunogenic composition may further include a third immunogenic protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14 (or more particularly SEQ ID NOs: 12 or 13), or a fragment, variant or derivative thereof, and / or a third nucleic acid molecule comprising a nucleotide sequence encoding the third immunogenic protein or a nucleotide sequence complementary thereto.

[0276] It is envisaged that the immunogenic composition may comprise mixtures of immunogenic proteins and encoding nucleic acids. By way of example, the immunogenic composition may comprise the first immunogenic protein and the second nucleic acid molecule. Alternatively, the immunogenic composition may comprise the first nucleic acid molecule and the second immunogenic protein. Such examples may then further comprise the third immunogenic protein or the third nucleic acid molecule.

[0277] The present disclosure provides an immunogenic composition comprising:

[0278] (a) a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3 or a fragment, variant or derivative thereof; and

[0279] (b) a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5 or a fragment, variant or derivative thereof.

[0280] Such immunogenic compositions may further include a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14 or a fragment, variant or derivative thereof. The immunogenic compositions may further include a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NOs: 12 or 13, or a fragment, variant or derivative thereof.

[0281] The present disclosure also provides an immunogenic composition comprising: (a) a first nucleic acid molecule comprising a nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto; and

[0282] (b) a second nucleic acid molecule comprising a nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto.

[0283] The present disclosure also provides an immunogenic composition comprising a nucleic acid molecule that comprises:

[0284] (a) a first nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto; and

[0285] (b) a second nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto.

[0286] The present disclosure also provides an immunogenic composition comprising:

[0287] (a) a first nucleic acid molecule comprising a nucleotide sequence encoding a first immunogenic protein, or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence selected from the group consisting of SEQ ID NO: 17, 31 and 32, or a nucleotide sequence complementary thereto; and

[0288] (b) a second nucleic acid molecule comprising a nucleotide sequence encoding a second immunogenic protein, or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence selected from the group consisting of SEQ ID NO: 18 to 30 and 41, or a nucleotide sequence complementary thereto.

[0289] The present disclosure also provides an immunogenic composition comprising a nucleic acid molecule that comprises:

[0290] (a) a first nucleotide sequence encoding a first immunogenic protein, or a fragment, variant or derivative thereof, the first nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence selected from the group consisting of SEQ ID NO: 17, 31 and 32, or a nucleotide sequence complementary thereto; and (b) a second nucleotide sequence encoding a second immunogenic protein, or a fragment, variant or derivative thereof, the second nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence selected from the group consisting of SEQ ID NO: 18 to 30 and 41, or a nucleotide sequence complementary thereto.

[0291] The immunogenic composition described herein may further comprise a third nucleic acid molecule comprising a nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14 or a fragment, variant or derivative thereof or a nucleotide sequence complementary thereto. The immunogenic composition may further comprise a third nucleic acid molecule comprising a nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NOs: 12 or 13, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto. The immunogenic composition may further comprise a third nucleic acid molecule comprising a nucleotide sequence encoding a third immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence selected from the group consisting of SEQ ID NO: 33 to 38, or a nucleotide sequence complementary thereto.

[0292] The nucleic acid molecule of the immunogenic composition described herein may further comprise a third nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14 or a fragment, variant or derivative thereof or a nucleotide sequence complementary thereto. The nucleic acid molecule may further comprise a third nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NOs: 12 or 13, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto. The nucleic acid molecule may further comprise a third nucleotide sequence encoding a third immunogenic protein or a fragment, variant or derivative thereof, the nucleotide sequence comprising, consisting of or consisting essentially of a nucleotide sequence selected from the group consisting of SEQ ID NO: 33 to 38, or a nucleotide sequence complementary thereto.

[0293] It will be understood that the first nucleotide sequence, the second nucleotide sequence and optionally the third nucleotide sequence may be on separate nucleic acid molecules or combined on a single nucleic acid molecule.

[0294] The present disclosure also envisages that the immunogenic composition may be utilised in vaccine compositions. An immunogenic composition or a vaccine may comprise, consist of or consist essentially of a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein, a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein, and optionally a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein.

[0295] An immunogenic composition or a vaccine may comprise, consist of or consist essentially of a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3 and 6 to 11, or a fragment, variant or derivative thereof, a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 4, or a fragment, variant or derivative thereof, and optionally a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14 or a fragment, variant or derivative thereof.

[0296] An immunogenic composition or a vaccine may comprise, consist of or consist essentially of a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, and 6 to 11, or a fragment, variant or derivative thereof conjugated, coupled or otherwise linked to a carrier protein, a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 4, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein, and optionally a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14 or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein.

[0297] An immunogenic composition or a vaccine may comprise, consist of or consist essentially of a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, and 6 to 11, or a fragment, variant or derivative thereof, a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 4, or a fragment, variant or derivative thereof, and optionally a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 13 or a fragment, variant or derivative thereof.

[0298] An immunogenic composition or a vaccine may comprise, consist of or consist essentially of a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3 and 6 to 11, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein, a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 4, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein, and optionally a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 13 or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein.

[0299] An immunogenic composition or a vaccine may comprise, consist of or consist essentially of a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 6 or a fragment, variant or derivative thereof, a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 4 or a fragment, variant or derivative thereof, and optionally a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 13 or a fragment, variant or derivative thereof.

[0300] An immunogenic composition or a vaccine may comprise, consist of or consist essentially of a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 6, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein, a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 4, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein, and optionally a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 13, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein.

[0301] An immunogenic composition or a vaccine may comprise, consist of or consist essentially of a first immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence set forth in SEQ ID NO: 6 or a fragment, variant or derivative thereof conjugated to a CRM197 protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 15, a second immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence set forth in SEQ ID NO: 4 or a fragment, variant or derivative thereof conjugated to a CRM197 protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 15, and optionally a third immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence set forth in SEQ ID NO: 13 or a fragment, variant or derivative thereof conjugated to a CRM197 protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 15.

[0302] An immunogenic composition or a vaccine may comprise, consist of or consist essentially of a first immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence set forth in SEQ ID NO: 6 or a fragment, variant or derivative thereof conjugated to a diphtheria toxin comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 16, a second immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence set forth in SEQ ID NO: 4 or a fragment, variant or derivative thereof conjugated to a diphtheria toxin comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 16, and optionally a third immunogenic protein may comprise, consist of or consist essentially of an amino acid sequence set forth in SEQ ID NO: 13 or a fragment, variant or derivative thereof conjugated to a diphtheria toxin comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 16.

[0303] An immunogenic composition or a vaccine may comprise, consist of or consist essentially of a first nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein or a nucleotide sequence complementary thereto, a second nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein or a nucleotide sequence complementary thereto, and optionally a third nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein or a fragment, variant or derivative thereof or a nucleotide sequence complementary thereto.

[0304] An immunogenic composition or a vaccine may comprise, consist of or consist essentially of a nucleic acid molecule comprising a first nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein or a nucleotide sequence complementary thereto, a second nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein or a nucleotide sequence complementary thereto, and optionally a third nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein or a fragment, variant or derivative thereof or a nucleotide sequence complementary thereto.

[0305] An immunogenic composition or a vaccine may comprise, consist of or consist essentially of a first nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NOs: 2, 3 and 6 to 11, or a fragment, variant or derivative thereof or a nucleotide sequence complementary thereto, a second nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 4, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, and optionally a third nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14 or a fragment, variant or derivative thereof or a nucleotide sequence complementary thereto.

[0306] An immunogenic composition or a vaccine may comprise, consist of or consist essentially of a nucleic acid molecule comprising a first nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NOs: 2, 3 and 6 to 11, or a fragment, variant or derivative thereof or a nucleotide sequence complementary thereto, a second nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 4, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, and optionally a third nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14 or a fragment, variant or derivative thereof or a nucleotide sequence complementary thereto.

[0307] An immunogenic composition or a vaccine may comprise, consist of or consist essentially of a first nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, and 6 to 11, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein, or a nucleotide sequence complementary thereto, a second nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 4, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein, or a nucleotide sequence complementary thereto, and optionally a third nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein, or a nucleotide sequence complementary thereto.

[0308] An immunogenic composition or a vaccine may comprise, consist of or consist essentially of a nucleic acid molecule comprising a first nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, and 6 to 11, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein, or a nucleotide sequence complementary thereto, a second nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 4, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein, or a nucleotide sequence complementary thereto, and optionally a third nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein, or a nucleotide sequence complementary thereto.

[0309] An immunogenic composition or a vaccine may comprise, consist of or consist essentially of a first nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, and 6 to 11, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, a second nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 4, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, and optionally a third nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 13, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto.

[0310] An immunogenic composition or a vaccine may comprise, consist of or consist essentially of a nucleic acid molecule comprising a first nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, and 6 to 11, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, a second nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 4, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, and optionally a third nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 13, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto.

[0311] An immunogenic composition or a vaccine may comprise, consist of or consist essentially of a first nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3 and 6 to 11, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein, or a nucleotide sequence complementary thereto, a second nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 4, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein, or a nucleotide sequence complementary thereto, and optionally a third nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 13, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein, or a nucleotide sequence complementary thereto.

[0312] An immunogenic composition or a vaccine may comprise, consist of or consist essentially of a nucleic acid molecule comprising a first nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3 and 6 to 11, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein, or a nucleotide sequence complementary thereto, a second nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 4, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein, or a nucleotide sequence complementary thereto, and optionally a third nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 13, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein, or a nucleotide sequence complementary thereto.

[0313] An immunogenic composition or a vaccine may comprise, consist of or consist essentially of a first nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 6, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, a second nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 4, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, and optionally a third nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 13, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto. An immunogenic composition or a vaccine may comprise, consist of or consist essentially of a nucleic acid molecule comprising a first nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 6, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, a second nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 4, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, and optionally a third nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 13, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto.

[0314] An immunogenic composition or a vaccine may comprise, consist of or consist essentially of a first nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 6, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein, or a nucleotide sequence complementary thereto, a second nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 4, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein, or a nucleotide sequence complementary thereto, and optionally a third nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 13, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein, or a nucleotide sequence complementary thereto.

[0315] An immunogenic composition or a vaccine may comprise, consist of or consist essentially of a nucleic acid molecule comprising a first nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 6, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein, or a nucleotide sequence complementary thereto, a second nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 4, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein, or a nucleotide sequence complementary thereto, and optionally a third nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 13, or a fragment, variant or derivative thereof, conjugated, coupled or otherwise linked to a carrier protein, or a nucleotide sequence complementary thereto.

[0316] Suitably, the immunogenic composition or vaccine described herein comprises an adjuvant or an immunostimulatory agent, as are known in the art. The term “adjuvant” refers to a compound or mixture that enhances the immune response to an antigen (e.g., the immunogenic protein or an encoding nucleic acid molecule). Adjuvants may act primarily as a delivery system, primarily as an immune modulator or have features of both. Suitable adjuvants include those suitable for use in mammals, including humans, cattle and dogs.

[0317] Examples of known adjuvants that can be used in mammals include, but are not limited to, calcium phosphate; squalane and squalene (or other oils of plant or animal origin); block copolymers; detergents such as Tween®-80; Quil® A, mineral oils such as Montanide ISA-50, Montanide ISA 61 VG (Seppic SA), anhydro mannitol ether octodecenoate, carbopol, Amphigen®, Amphigen® Mark II (Hydronics, USA), Alhydrogel® (BSA2; Accurate Scientific, Westbury, NY), Drakeol or Marcol, vegetable oils such as peanut oil; Corynebacterium-^oxw' Q^ adjuvants such as Corynebacterium parvum Propionibacterium- derived adjuvants such as Propionibacterium acne: Mycobacterium bovis (Bacille Calmette and Guerin or BCG); Bordetella pertussis antigens; tetanus toxoid; diphtheria toxoid; a pertussis toxin or toxoid; a Pseudomonas toxin or toxoid; an E. coli toxin or toxoid; a Staphylococcus toxin or toxoid; a Streptococcus toxin or toxoid; a CRM protein, such as CRM197; surface active substances such as hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyldioctadecylammonium bromide, N,N-dicoctadecyl-N', N'bis(2- hydroxyethyl-propanediamine), methoxyhexadecylglycerol, and pluronic polyols; polyamines such as pyran, dextransulfate, poly IC carbopol; peptides such as muramyl dipeptide and derivatives, dimethylglycine, tuftsin; oil emulsions such as BayolF / Arlacel A and water, or an emulsion of vegetable oil, water and an emulsifier such as lecithin; alum; bovine cytokines; cholesterol; additional oil emulsions, including a water in oil emulsion, and a water in oil in water emulsion; and mineral gels such as aluminium phosphate, alum (e.g., aluminium phosphate, aluminium sulfate or aluminium hydroxide); interleukins such as interleukin 2 and interleukin 12; monokines such as interleukin 1; tumour necrosis factor; interferons such as gamma interferon; immunostimulatory DNA such as CpG DNA; combinations such as saponin-aluminium hydroxide or Quil-A aluminium hydroxide; liposomes (e.g., see International Publication W02017 / 070735); ISCOM® and ISCOMATRIX® adjuvant; mycobacterial cell wall extract; synthetic glycopeptides such as muramyl dipeptides or other derivatives; Avridine; Lipid A derivatives; dextran sulfate; DEAE-Dextran alone or with aluminium phosphate; carboxypolymethylene such as Carbopol EMA; acrylic copolymer emulsions such as Neocryl A640 (e.g., U.S. Pat. No. 5,047,238); water in oil emulsions, such as Montanide ISA 720, Montanide ISA 61 VG; oil-in-water emulsions, such as MF59 (4.3% w / v squalene, 0.5% w / v polysorbate 80 (Tween 80), 0.5% w / v sorbitan trioleate (Span 85)); poly(D,L-laetide-co-glycolide) (PLG) microparticles or nanoparticles (e.g. , the Next Adjuvant or NexaVAC™ System from the NA Vaccine Institute); ballistic particles; ceramic particles; polymeric particles; poliovirus, vaccinia or animal poxvirus proteins; a mixture of 3-O-desacyl-4'-monophosphoryl lipid A (MPL) and the saponin QS-21 (e.g., AS01); a Matrix-M adjuvant (i.e., two distinct fractions of saponins purified from the Quillaja saponaria Molina tree, combined with cholesterol and phospholipids to form 40- nm open cage-like nanoparticles), such as R21; or mixtures thereof.

[0318] The adjuvant may be aluminium hydroxide (Alum).

[0319] The adjuvant may be a liposomal adjuvant (e.g., formulated as a liposomal formulation). The liposomal adjuvant may comprise dimethyldioctadecylammonium and trehalose 6,6'-dibehenate.

[0320] The proportion of antigen and adjuvant can be varied over a broad range so long as both are present in effective amounts. The concentration of adjuvants useful in the context of the present disclosure can readily be determined by the skilled artisan.

[0321] Suitably, the adjuvant is present in an amount of about 10% to about 90% (e.g., about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or any range therein) by weight of the composition. The adjuvant may be present in an amount of about 20% to about 90% by weight of the composition. The adjuvant may be present in an amount of about 50% to about 90% by weight of the composition. The adjuvant may be present in an amount of about 80% to about 90% by weight of the composition. The adjuvant may be present in an amount of about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85% or about 90% by weight of the composition.

[0322] The immunogenic compositions or vaccines described herein may comprise one or more pharmaceutically acceptable carriers, diluents or excipients.

[0323] By “acceptable carrier, diluent or excipient” is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of carriers, diluent and excipients well known in the art may be used. These may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates, water and pyrogen-free water.

[0324] A useful reference describing acceptable carriers, diluents and excipients is Remington’s Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991), which is incorporated herein by reference.

[0325] Any suitable procedure is contemplated for producing immunogenic compositions or vaccine compositions described herein. Exemplary procedures include, for example, those described in New Generation Vaccines (1997, Levine et al.. Marcel Dekker, Inc. New York, Basel, Hong Kong), which is incorporated herein by reference.

[0326] Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, nasal sprays, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release may be effected by coating with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, the controlled release may be effected by using other polymer matrices, liposomes and / or microspheres.

[0327] Immunogenic compositions or vaccines may be presented as discrete units such as capsules, sachets, functional foods / feeds or tablets each containing a pre-determined amount of one or more therapeutic agents of the disclosure, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water- in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy, but all methods include the step of bringing into association one or more agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the agents of the disclosure with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.

[0328] Suitably, the immunogenic composition, the vaccine, the immunogenic protein, the nucleic acid molecule, the genetic construct and / or the viral vector of the present disclosure are at least partly capable of inducing cross-serotype protection against GAS bacteria. In this regard, the immunogenic composition, the vaccine, the immunogenic protein, the nucleic acid molecule, the genetic construct and / or the viral vector described herein are suitably capable of eliciting an immune response in a subject that is specific for or at least partly protective against a GAS bacterial infection with two or more GAS serotypes or strains, such as a first GAS serotype or strain capable of a skin-based infection and a second GAS serotype or strain capable of a mucosal-based infection. To this end, the immune response may be at least partly protective against skin- and mucosal -based GAS infections in the subject.

[0329] Also disclosed herein is a container comprising the immunogenic compositions or vaccines described herein. Any suitable container known in the art may be used. For example, the container may be selected from the group consisting of a vial, a syringe, an ampoule, a flask, a fermenter, a bioreactor, a bag, a jar, an ampoule, a cartridge and a disposable pen. In one example, the container is a vial, an ampoule or a syringe. The container may be made of glass, metals (e.g., steel, stainless steel, aluminium, etc.) and / or polymers (e.g., thermoplastics, elastomers, thermoplastic-elastomers). The container may be at least partially siliconized.

[0330] Methods of eliciting an immune response, treatment and prevention

[0331] By virtue of their ability to elicit an immune response, the immunogenic proteins described herein and / or their encoding nucleic acids can be used to prevent, ameliorate or treat an infection, disease or condition associated with group A streptococcus (GAS) bacteria in a subject.

[0332] Thus, the present disclosure provides a method of eliciting an immune response to group A streptococcus (GAS) bacteria in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of an immunogenic composition, a vaccine, a nucleic acid molecule, a genetic construct and / or a viral vector described herein, to thereby elicit the immune response.

[0333] For such examples, the method may comprise the step of administering to the subject a therapeutically effective amount of an immunogenic composition or a vaccine comprising, consisting of or consisting essentially of a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof, a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, and optionally a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14, or a fragment, variant or derivative thereof, to thereby elicit the immune response.

[0334] In other examples, such methods may include the step of administering to the subject a therapeutically effective amount of an immunogenic composition or a vaccine that comprises, consists of or consists essentially of a first nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, a second nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, and optionally a third nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14 or a fragment, variant or derivative thereof or a nucleotide sequence complementary thereto, to thereby elicit the immune response.

[0335] In other examples, such methods may include the step of administering to the subject a therapeutically effective amount of an immunogenic composition or a vaccine that comprises, consists of or consists essentially of a nucleic acid molecule comprising a first nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, a second nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, and optionally a third nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14 or a fragment, variant or derivative thereof or a nucleotide sequence complementary thereto, to thereby elicit the immune response.

[0336] In a related form, the present disclosure also provides for the use of:

[0337] (a) an immunogenic composition or a vaccine that comprises, consists of or consists essentially of a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof, a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, and optionally a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14 or a fragment, variant or derivative thereof, to thereby elicit the immune response;

[0338] (b) an immunogenic composition or a vaccine that comprises, consists of or consists essentially of a first nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, a second nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, and optionally a third nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14 or a fragment, variant or derivative thereof or a nucleotide sequence complementary thereto; and / or

[0339] (c) an immunogenic composition or a vaccine that comprises, consists of or consists essentially of a nucleic acid molecule comprising a first nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, a second nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, and optionally a third nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14 or a fragment, variant or derivative thereof or a nucleotide sequence complementary thereto, in the manufacture of a medicament for eliciting an immune response to group A streptococcus (GAS) bacteria in a subject.

[0340] The present disclosure also provides:

[0341] (a) an immunogenic composition or a vaccine that comprises, consists of or consists essentially of a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof, a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, and optionally a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14 or a fragment, variant or derivative thereof, to thereby elicit the immune response;

[0342] (b) an immunogenic composition or a vaccine that comprises, consists of or consists essentially of a first nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, a second nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, and optionally a third nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto; and / or

[0343] (c) an immunogenic composition or a vaccine that comprises, consists of or consists essentially of a nucleic acid molecule comprising a first nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, a second nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, and optionally a third nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14 or a fragment, variant or derivative thereof or a nucleotide sequence complementary thereto, for use in a method of eliciting an immune response to group A streptococcus (GAS) bacteria in a subject.

[0344] With respect to the aspects described herein, the term “subject”, “patient” and “individual” includes, but is not limited to, mammals, inclusive of humans, performance animals (such as horses, camels, greyhounds), livestock (such as cows, sheep, horses) and companion animals (such as cats and dogs). Suitably, the subject is a human.

[0345] As used herein the terms “group A streptococcus”, “group A streptococci”, “group A streptococcal”, “group A strep” and the abbreviation “GAS” refer to streptococcal bacteria of Lancefield serogroup A, which are gram positive P-haemolytic bacteria of the species Streptococcus pyogenes. An important virulence factor of GAS is M protein, which is strongly anti -phagocytic and binds to serum factor H, destroying C3-convertase and preventing opsonization by C3b. These also include virulent “mutants”, such as CovR / S or CovRS mutants such as described in Graham et al., 2002, PNAS USA 99 13855, although without limitation thereto.

[0346] Group A streptococcus (Streptococcus pyogenes) can be classified into various “serotypes” or “strains” based on the antigenic properties of bacterial surface structures, for example, the surface-exposed M protein, as described in Bessen et al., 2022, Streptococcus pyogenes: Basic Biology to Clinical Manifestations, 2nd edition, Chapter 6, which is incorporated herein by reference. Currently, there are more than 250 identified, distinct serotypes. Exemplary serotypes include, but are not limited to, Ml (e.g., 5448AP and 2031), M3, M4, M5, M6, M12 (e.g., NS9), M14, M17, M18, M19, M24, M28, M29, M33, M41, M42, M49, M52, M53, M55, M59, M60, M70, M73, M75 and M89 (e.g., SN1).

[0347] By “elicit an immune response” is meant generate or stimulate the production or activity of one or more elements of the immune system inclusive of the cellular immune system, humoral immune system (i.e., antibodies) and / or the native immune system. Suitably, the immune response described herein includes one or more elements of the immune system, such as T lymphocytes, B lymphocytes, antibodies, neutrophils, dendritic cells inclusive of plasmacytoid dendritic cells, cytokines and / or chemokines. Non-limiting examples of cytokines include pro-inflammatory cytokines such as TNF-a, IL-6, IL-2, IL-6, IL-8, IL-17A and IL-1 (e.g., IL-ip). A non-limiting example of a chemokine is the neutrophil chemoattractant IL-8. In certain examples, the immune response that is elicited by the immunogenic compositions described herein is protective.

[0348] By “protective immune response” is meant an immune response that reduces a subject's propensity to develop a pathogen infection (e.g., a GAS infection), that reduces the severity of the infection, or that ameliorates a symptom of a pathogen infection.

[0349] The methods or uses described herein suitably elicit a mucosal immune response and / or a systemic immune response in the subject. The immune response may be assessed by any means in the art, such as by measuring the level of immune cells (e.g., B cells and / or T cells specific for an immunogenic protein), macrophages, cytokines (e.g., IL-17) or immunoglobulins present in the subject with or without subsequent challenge or stimulation by the immunogenic composition or a corresponding pathogen. Accordingly, the methods or uses described herein suitably elicit an immune response that is at least partly protective against skin-based infections and mucosal -based infections with a GAS bacteria.

[0350] Typically, mucosal immune responses are characterised by the production of IgA, with minimal production of other antibody isotypes such as IgG. For the methods described herein, the mucosal immune response elicited by administration of the immunogenic composition or the vaccine described herein can be characterised by the production of IgA. IgG production may be relatively reduced compared to IgA production, or is substantially absent. To this end, the immune response described herein can be characterised by the presence of IgA+memory B cells specific for the immunogenic proteins described herein in the subject’s respiratory tract, such as in the mucosa of one or more of the subject’s nose, throat, larynx, trachea, bronchi, and lungs.

[0351] The immune response, inclusive of a mucosal immune response, in the subject, may include activated B cells and / or T cells. The activated B cells and / or T cells may be recruited to a desired anatomic location, such as the subject’s respiratory tract. The recruited activated B cells may be IgA+B cells. The recruited activated B cells may be IgG+B cells. The recruited activated B cells may be both IgA+B cells and IgG+B cells. The recruited activated T cells may be CD4+T cells. The recruited activated T cells may be CD8+T cells. The recruited activated T cells may be both CD4+T cells and CD8+T cells.

[0352] As generally used herein, the terms “immunise”, “vaccinate” and “vaccine” refer to methods and / or compositions that elicit a protective immune response against GAS bacteria, whereby subsequent infection by the GAS bacteria is at least partly prevented or minimised.

[0353] The present disclosure also provides methods of preventing, ameliorating or treating a GAS infection, or a disease, disorder or condition associated therewith, in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of the immunogenic composition described herein.

[0354] The present disclosure also provides for a method of ameliorating or treating a GAS infection, or a disease, disorder or condition associated therewith, in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of:

[0355] (a) an immunogenic composition or a vaccine comprising, consisting of or consisting essentially a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof, a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, and optionally a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14, or a fragment, variant or derivative thereof;

[0356] (b) an immunogenic composition or a vaccine comprising, consisting of or consisting essentially of a first nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof , or a nucleotide sequence complementary thereto, a second nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, and optionally a third nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto; and / or

[0357] (c) an immunogenic composition or a vaccine that comprises, consists of or consists essentially of a nucleic acid molecule comprising a first nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, a second nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, and optionally a third nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14 or a fragment, variant or derivative thereof or a nucleotide sequence complementary thereto; to thereby prevent, ameliorate or treat the GAS infection, or the disease, disorder or condition associated therewith.

[0358] The present disclosure also provides for the use of:

[0359] (a) an immunogenic composition or a vaccine comprising, consisting of or consisting essentially of a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof, a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, and optionally a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14, or a fragment, variant or derivative thereof;

[0360] (b) an immunogenic composition or a vaccine comprising, consisting of or consisting essentially of a first nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, a second nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, and optionally a third nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14, or a fragment, variant or derivative thereof or a nucleotide sequence complementary thereto; and / or

[0361] (c) an immunogenic composition or a vaccine that comprises, consists of or consists essentially of a nucleic acid molecule comprising a first nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, a second nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, and optionally a third nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14 or a fragment, variant or derivative thereof or a nucleotide sequence complementary thereto, in the manufacture of a medicament for preventing, ameliorating or treating a GAS infection, or a disease, disorder or condition associated therewith, in a subject.

[0362] The present disclosure also provides: (a) an immunogenic composition or a vaccine comprising, consisting of or consisting essentially of a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof, a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, and optionally a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14 or a fragment, variant or derivative thereof;

[0363] (b) an immunogenic composition or a vaccine comprising, consisting of or consisting essentially of a first nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, a second nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, and optionally a third nucleic acid molecule comprising, consisting of or consisting essentially of a nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14 or a fragment, variant or derivative thereof or a nucleotide sequence complementary thereto; and / or

[0364] (c) an immunogenic composition or a vaccine that comprises, consists of or consists essentially of a nucleic acid molecule comprising a first nucleotide sequence encoding a first immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, a second nucleotide sequence encoding a second immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto, and optionally a third nucleotide sequence encoding a third immunogenic protein comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14 or a fragment, variant or derivative thereof or a nucleotide sequence complementary thereto, for use in a method of preventing, ameliorating or treating a GAS infection, or a disease, disorder or condition associated therewith in a subject. As used herein, “preventing”, “prevent” or “prevention” refers to a course of action initiated prior to infection by, or exposure to, GAS bacteria or molecular components thereof and / or before the onset of a symptom or pathological sign of the infection, disease, disorder or condition associated with GAS bacteria, so as to prevent infection and / or reduce the symptom or pathological sign. It is to be understood that such preventing need not be absolute to be beneficial to a subject. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of the disease, disorder or condition, or exhibits only early signs for the purpose of decreasing the risk of developing a symptom or pathological sign of the disease, disorder or condition.

[0365] The term “ameliorating,” with reference to an infection, disease or condition associated with GAS bacteria, refers to any observable beneficial effect of the treatment. Treatment need not be absolute to be beneficial to the subject. The beneficial effect can be determined using any methods or standards known to the ordinarily skilled artisan.

[0366] As used herein, “treating”, “treat” or “treatment” refers to a therapeutic intervention that at least partly ameliorates, eliminates or reduces a symptom or pathological sign of an infection, disease, disorder or condition associated with GAS bacteria after it has begun to develop. Treatment need not be absolute to be beneficial to the subject. The beneficial effect can be determined using any methods or standards known to the ordinarily skilled artisan.

[0367] Infections, diseases, disorders or conditions associated with GAS bacteria include cellulitis, erysipelas, impetigo, scarlet fever, throat infections such as acute pharyngitis (“strep throat”), bacteraemia, invasive GAS diseases such as streptococcal toxic shock syndrome (STSS), necrotizing fasciitis, acute rheumatic fever and acute glomerulonephritis, although without limitation thereto.

[0368] In various examples, the GAS infection is a GAS autoimmune infection. As used herein, a “GAS autoimmune infection” is meant any clinical pathology resulting from an immune response triggered by GAS bacteria, where the immune system mistakenly attacks the body's own tissues. Autoimmune responses to GAS bacteria can lead to various poststreptococcal disorders, such as acute rheumatic fever, poststreptococcal glomerulonephritis, Poststreptococcal Reactive Arthritis, Sydenham's Chorea, Erythema Nodosum, Scarlet Fever and Paediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal infections, due to the immune system's cross-reactivity with the body's own tissues.

[0369] In some examples, the GAS infection is a GAS bacterial skin infection. As used herein, a “GAS bacterial skin infection” is meant any clinical pathology resulting from when GAS bacteria infects the skin and underlying tissues and includes impetigo, erysipelas, cellulitis and necrotizing fasciitis.

[0370] In other examples, the GAS infection is a mucosal GAS bacterial infection. As used herein, a “mucosal GAS bacterial infection” is meant any clinical pathology resulting from a mucosal infection by GAS bacteria and includes throat infections, such as acute pharyngitis (“strep throat”) and sequelae thereof, including bacteraemia, scarlet fever, streptococcal toxic shock syndrome, acute rheumatic fever and acute glomerulonephritis, although without limitation thereto.

[0371] For certain examples, the GAS infection is a GAS bacterial intranasal infection. As used herein, a “GAS bacterial intranasal infection” is meant any clinical pathology resulting from when GAS bacteria infects the nasal passages and sequelae thereof, including acute bacterial rhinosinusitis, nasal carriage, otitis media, bacteraemia and meningitis.

[0372] Referring to particular examples, the GAS infection is an upper respiratory tract infection. As used herein, an “upper respiratory tract (URT) GAS bacterial infection” meant any clinical pathology resulting from GAS bacterial infection of the nasal passages, throat and / or sinuses, including pharyngitis, sinusitis and tonsillitis.

[0373] In various examples, the GAS infection is an invasive GAS bacterial infection. As used herein, an “invasive GAS bacterial infection” is meant any clinical pathology resulting from when GAS bacteria spread into the body, beyond the skin and throat, including the bloodstream, muscles and internal organs. Invasive GAS bacterial infection may include necrotizing fasciitis, STSS, bacteraemia, pneumonia, osteomyelitis and septic arthritis.

[0374] The term “therapeutically effective amount” describes a quantity of a specified agent, such as the immunogenic composition, the vaccine, the immunogenic protein, the nucleic acid molecule, the genetic construct and / or the viral vector described herein, sufficient to achieve a desired effect in a subject being treated. For example, this can be the amount of the immunogenic composition, the vaccine, the immunogenic protein, the nucleic acid molecule, the genetic construct and / or the viral vector described herein, necessary to reduce, alleviate and / or prevent infections, diseases, disorders or conditions associated with GAS bacteria.

[0375] A “therapeutically effective amount” may be an amount sufficient to reduce or eliminate a symptom of infections, diseases or conditions associated with GAS bacteria. A “therapeutically effective amount” may be an amount sufficient to achieve a desired biological effect, for example, an amount that is sufficient to elicit a protective immune response in a subject so as to inhibit or prevent infections, diseases, disorders or conditions associated with GAS bacteria. Ideally, a therapeutically effective amount of an agent is an amount sufficient to induce the desired result without causing a substantial cytotoxic effect in the subject. The effective amount of an agent useful for reducing, alleviating and / or preventing infections, diseases or conditions associated with GAS bacteria, will be dependent on the subject being treated, the type and severity of the infection (e.g., the type of GAS bacteria and / or serotype and / or strain of Streptococcus pyogenes), and the manner of administration of the therapeutic composition.

[0376] Administration

[0377] The immunogenic composition, the vaccine, the immunogenic protein, the nucleic acid molecule, the genetic construct and / or the viral vector described herein may be administered to the subject in a manner compatible with the dosage formulation and the route of administration, and in such an amount as to be effective (e.g., capable of eliciting a protective immune response). The dose administered to a subject, in the context of the present disclosure, should be sufficient to effect a beneficial response in a subject over an appropriate period of time. The quantity of agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof, factors that will depend on the judgement of the practitioner. Suitably, administration of the immunogenic composition or a booster dose thereof is timed to coincide with times of maximal challenge or infection with the GAS bacteria.

[0378] Any safe route of administration may be employed for providing a patient with the immunogenic composition of the present disclosure or components thereof (e.g., the immunogenic protein, the nucleic acid molecule, the genetic construct, the viral vector and / or the virus particle disclosed herein). For example, oral, rectal, parenteral, sublingual, buccal, intravenous, intraarticular, intramuscular, intradermal, subcutaneous, intranasal, inhalational, intraocular, periocular, retrobulbar, intravitreal, subretinal, intraperitoneal, intracerebroventricular, transdermal and the like may be employed.

[0379] Suitably, the immunogenic composition or a component thereof disclosed herein is administered to the subject via systemic administration, or more particularly via parenteral administration. A skilled person will be familiar with multiple routes by which systemic or parenteral administration may be achieved, and associated techniques. Examples of systemic or parenteral administration include intravenous administration, intraarterial administration, intramuscular administration, intrathecal administration, intracardiac administration, intramyocardial administration, pericardial administration, epicardial administration and subcutaneous administration. The immunogenic composition, the vaccine, the immunogenic protein, the nucleic acid molecule, the genetic construct and / or the viral vector described herein may be administered to the subject via subcutaneous administration. The immunogenic composition, the vaccine, the immunogenic protein, the nucleic acid molecule, the genetic construct and / or the viral vector described herein may be administered to the subject via intramuscular administration. The immunogenic composition, the vaccine, the immunogenic protein, the nucleic acid molecule, the genetic construct and / or the viral vector described herein may be administered to the subject via intranasal administration.

[0380] The data herein further demonstrates a beneficial role for a second dose comprising a therapeutically effective amount of the immunogenic composition or components thereof described herein that is administered at a period of time after the first dose. Such a second dose may be administered at particular intervals of time after the first dose (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 52 weeks apart or any range therein).

[0381] The methods described herein may comprise the steps of: (a) administering a first dose of the immunogenic composition, the vaccine, the immunogenic protein, the nucleic acid molecule, the genetic construct and / or the viral vector of the present disclosure to the subject; and (b) administering a second dose of the immunogenic composition, the vaccine the immunogenic protein, the nucleic acid molecule, the genetic construct and / or the viral vector of the present disclosure to the subject after the first dose.

[0382] To ensure sustained high levels of protection against disease, it may be helpful to readminister a further dose (e.g., a third, fourth, fifth, six, seventh, eighth, ninth dose) to the subject on a periodic basis. This periodic basis may range from monthly, to every six months, to yearly, to multiple years.

[0383] For example, the data herein further demonstrates a beneficial role for a third dose comprising a therapeutically effective amount of the immunogenic composition or components thereof described herein that is administered at a period of time after the second dose. Such a third dose may be administered at particular intervals of time after the second dose (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 52 weeks apart or any range therein).

[0384] The methods described herein may comprise the steps of: (a) administering a first dose of the immunogenic composition, the vaccine, the immunogenic protein, the nucleic acid molecule, the genetic construct and / or the viral vector of the present disclosure to the subject; (b) administering a second dose of the immunogenic composition, the vaccine the immunogenic protein, the nucleic acid molecule, the genetic construct and / or the viral vector of the present disclosure to the subject after the first dose; and (c) administering a third dose of the immunogenic composition, the vaccine the immunogenic protein, the nucleic acid molecule, the genetic construct and / or the viral vector of the present disclosure to the subject after the second dose.

[0385] It is contemplated that the first and second doses may be administered by the same or different routes of administration. The first and second dose may be administered intramuscularly or subcutaneously to the subject. The first dose may be administered intramuscularly or subcutaneously to the subject, whilst the second dose may be administered intranasally to the subject.

[0386] It is contemplated that the first, second and third doses may be administered by the same or different routes of administration. The first, second and third dose may be administered intramuscularly or subcutaneously to the subject. The first dose may be administered intramuscularly or subcutaneously to the subject, whilst the second and the third dose may be administered intranasally to the subject. The first and second dose may be administered intramuscularly or subcutaneously to the subject, whilst the third dose may be administered intranasally to the subject.

[0387] Additionally, it is envisaged that the immunogenic composition, the vaccine, the immunogenic protein, the nucleic acid molecule, the genetic construct and / or the viral vector may be the same or different between the first and second doses. The immunogenic composition, the vaccine, the immunogenic protein, the nucleic acid molecule, the genetic construct and / or the viral vector described herein may be the same or substantially the same between the first and second doses. The immunogenic composition, the vaccine, the immunogenic protein, the nucleic acid molecule, the genetic construct and / or the viral vector of the first dose may be different to that of the second dose. Dosage regimes also contemplate a mixture of nucleic acid and protein doses (e.g., administration of a first dose comprising a first immunogenic protein, a second immunogenic protein and optionally a third immunogenic protein and a second dose comprising a nucleic acid molecule or genetic construct encoding the first immunogenic protein, the second immunogenic protein and optionally the third immunogenic protein).

[0388] For the methods described herein, the immunogenic composition comprising immunogenic proteins described herein may be administered to the subject at a dose of at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 34, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 pg, or any range therein. The immunogenic composition comprising immunogenic proteins described herein may be administered to the subject at a dose of at least about 20 pg to 100 pg.

[0389] A first dose of an immunogenic composition comprising immunogenic proteins described herein may be administered to the subject at a dose of at least about 20 pg to 100 pg. A second dose of an immunogenic composition comprising immunogenic proteins described herein may be administered to the subject at a dose of at least about 20 pg to 100 pg. A third dose of an immunogenic composition comprising immunogenic proteins described herein may be administered to the subject at a dose of at least about 20 pg to 100 pg.

[0390] For example, the immunogenic composition being administered to the subject may comprise a first immunogenic protein described herein at a dose of at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 33, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 pg, or any range therein, a second immunogenic protein described herein at a dose of at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 33, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 pg, or any range therein and optionally a third immunogenic protein described herein at a dose of at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 33, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 pg, or any range therein.

[0391] Suitably, a first dose of the immunogenic composition being administered to the subject may comprise a first immunogenic protein described herein at a dose of at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 33, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 pg, or any range therein, a second immunogenic protein described herein at a dose of at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 33, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 pg, or any range therein and optionally a third immunogenic protein described herein at a dose of at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 33, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 pg, or any range therein.

[0392] A second dose of the immunogenic composition being administered to the subject may comprise a first immunogenic protein described herein at a dose of at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 33, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 pg, or any range therein, a second immunogenic protein described herein at a dose of at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 33, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 pg, or any range therein and optionally a third immunogenic protein described herein at a dose of at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 33, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 pg, or any range therein.

[0393] A third dose of the immunogenic composition being administered to the subject may comprise a first immunogenic protein described herein at a dose of at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 33, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 pg, or any range therein, a second immunogenic protein described herein at a dose of at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 33, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 pg, or any range therein and optionally a third immunogenic protein described herein at a dose of at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 33, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 pg, or any range therein.

[0394] The immunogenic composition may comprise a first immunogenic protein described herein at a dose of at least about 30 pg to 40 pg, a second immunogenic protein described herein at a dose of at least about 30 pg to 40 pg and optionally a third immunogenic protein described herein at a dose of at least about 30 pg to 40 pg.

[0395] A first dose of the immunogenic composition may comprise a first immunogenic protein described herein at a dose of at least about 30 pg to 40 pg, a second immunogenic protein described herein at a dose of at least about 30 pg to 40 pg and optionally a third immunogenic protein described herein at a dose of at least about 30 pg to 40 pg. A second dose of the immunogenic composition may comprise a first immunogenic protein described herein at a dose of at least about 30 pg to 40 pg, a second immunogenic protein described herein at a dose of at least about 30 pg to 40 pg and optionally a third immunogenic protein described herein at a dose of at least about 30 pg to 40 pg. A third dose of the immunogenic composition may comprise a first immunogenic protein described herein at a dose of at least about 30 pg to 40 pg, a second immunogenic protein described herein at a dose of at least about 30 pg to 40 pg and optionally a third immunogenic protein described herein at a dose of at least about 30 pg to 40 pg.

[0396] The immunogenic composition may comprise a first immunogenic protein described herein at a dose of at least about 2 pg to 10 pg, a second immunogenic protein described herein at a dose of at least about 2 pg to 10 pg and optionally a third immunogenic protein described herein at a dose of at least about 2 pg to 10 pg. A first dose of the immunogenic composition may comprise a first immunogenic protein described herein at a dose of at least about 2 pg to 10 pg, a second immunogenic protein described herein at a dose of at least about 2 pg to 10 pg and optionally a third immunogenic protein described herein at a dose of at least about 2 pg to 10 pg. A second dose of the immunogenic composition may comprise a first immunogenic protein described herein at a dose of at least about 2 pg to 10 pg, a second immunogenic protein described herein at a dose of at least about 2 pg to 10 pg and optionally a third immunogenic protein described herein at a dose of at least about 2 pg to 10 pg. A third dose of the immunogenic composition may comprise a first immunogenic protein described herein at a dose of at least about 2 pg to 10 pg, a second immunogenic protein described herein at a dose of at least about 2 pg to 10 pg and optionally a third immunogenic protein described herein at a dose of at least about 2 pg to 10 pg.

[0397] The immunogenic composition may comprise a first immunogenic protein described herein at a dose of at least about 8 pg, a second immunogenic protein described herein at a dose of at least about 8 pg and optionally a third immunogenic protein described herein at a dose of at least about 8 pg. A first dose of the immunogenic composition may comprise a first immunogenic protein described herein at a dose of at least about 8 pg, a second immunogenic protein described herein at a dose of at least about 8 pg and optionally a third immunogenic protein described herein at a dose of at least about 8 pg. A second dose of the immunogenic composition may comprise a first immunogenic protein described herein at a dose of at least about 8 pg, a second immunogenic protein described herein at a dose of at least about 8 pg and optionally a third immunogenic protein described herein at a dose of at least about 8 pg. A third dose of the immunogenic composition may comprise a first immunogenic protein described herein at a dose of at least about 8 pg, a second immunogenic protein described herein at a dose of at least about 8 pg and optionally a third immunogenic protein described herein at a dose of at least about 8 pg.

[0398] The immunogenic composition may comprise a first immunogenic protein described herein at a dose of at least about 33 pg, a second immunogenic protein described herein at a dose of at least about 33 pg and optionally a third immunogenic protein described herein at a dose of at least about 33 pg. A first dose of the immunogenic composition may comprise a first immunogenic protein described herein at a dose of at least about 33 pg, a second immunogenic protein described herein at a dose of at least about 33 pg and optionally a third immunogenic protein described herein at a dose of at least about 33 pg. A second dose of the immunogenic composition may comprise a first immunogenic protein described herein at a dose of at least about 33 pg, a second immunogenic protein described herein at a dose of at least about 33 pg and optionally a third immunogenic protein described herein at a dose of at least about 33 pg. A third dose of the immunogenic composition may comprise a first immunogenic protein described herein at a dose of at least about 33 pg, a second immunogenic protein described herein at a dose of at least about 33 jj.g and optionally a third immunogenic protein described herein at a dose of at least about 33 pg.

[0399] The immunogenic composition may comprise between about 20 to about 30 pg of the immunogenic proteins contained therein. The immunogenic composition may comprise about 24 pg of the immunogenic proteins contained therein. The immunogenic composition may comprise between 96 to 103 pg of the immunogenic proteins contained therein. The immunogenic composition may comprise about 99 pg of the immunogenic proteins contained therein.

[0400] A first dose of the immunogenic composition may comprise between about 20 to about 30 pg of the immunogenic proteins contained therein. A first dose of the immunogenic composition may comprise about 24 pg of the immunogenic proteins contained therein. A first dose of the immunogenic composition may comprise between 96 to 103 pg of the immunogenic proteins contained therein. A first dose of the immunogenic composition may comprise about 99 pg of the immunogenic proteins contained therein.

[0401] A second dose of the immunogenic composition may comprise between about 20 to about 30 pg of the immunogenic proteins contained therein. A second dose of the immunogenic composition may comprise about 24 pg of the immunogenic proteins contained therein. A second dose of the immunogenic composition may comprise between 96 to 103 pg of the immunogenic proteins contained therein. A second dose of the immunogenic composition may comprise about 99 pg of the immunogenic proteins contained therein.

[0402] A third dose of the immunogenic composition may comprise between about 20 to about 30 pg of the immunogenic proteins contained therein. A third dose of the immunogenic composition may comprise about 24 pg of the immunogenic proteins contained therein. A third dose of the immunogenic composition may comprise between 96 to 103 pg of the immunogenic proteins contained therein. A third dose of the immunogenic composition may comprise about 99 pg of the immunogenic proteins contained therein.

[0403] The immunogenic composition may comprise between about 200 to about 300 pg of the adjuvant contained therein. The immunogenic composition may comprise between 240 to 270 pg of the adjuvant contained therein. The immunogenic composition may comprise about 250 pg of the adjuvant contained therein.

[0404] Similarly, the nucleic acid molecule or the genetic construct described herein may be administered to the subject at a dose of at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 34, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 mg, or any range therein. For example, a first dose, a second dose and / or a third dose of the nucleic acid molecule or the genetic construct described herein may be administered to the subject at a dose of at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 34, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 mg, or any range therein.

[0405] Again, the viral vector described herein may be administered to the subject at a dose of at least about 1 x 101, 1 x 102, 1 x 103, 1 x 104, 1 x 105, 1 x 106, 1 x 107, 1 x 108, 1 x 109, 1 x 1010, 1 x 1011, 1 x 1012, 1 x 10°, 1 x 1014, 1 x 1015, 1 x 1016, 1 x 1017, 1 x 1018, 1 x 1019, 1 x IO20infectious units (IFU) thereof, or any range therein. For example, a first dose, a second dose and / or a third dose of viral vector described herein may be administered to the subject at a dose of at least about 1 x 101, 1 x 102, 1 x 103, 1 x 104, 1 x 105, 1 x 106, 1 x 107, 1 x 108, 1 x 109, 1 x IO10, 1 x 1011, 1 x 1012, 1 x 1013, 1 x 1014, 1 x 1015, 1 x 1016, 1 x 1017, 1 x 1018, 1 x 1019, 1 x IO20infectious units (IFU) thereof, or any range therein.

[0406] It is envisaged that the immunogenic composition or vaccine is formulated in dosage unit form to facilitate administration and ensure a uniform dosage thereof. Referring to protein- or peptide-based compositions, the immunogenic composition suitably comprises at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 33, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 pg, or any range therein, of each of the immunogenic proteins described herein per unit dose.

[0407] For nucleic acid-based compositions, the composition suitably comprises at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 mg, or any range therein, of each of the encoding nucleic acid molecules or the genetic construct described herein per unit dose.

[0408] For viral vector-based compositions, the composition suitably comprises at least about 1 x 101, 1 x 102, 1 x 103, 1 x 104, 1 x 105, 1 x 106, 1 x 107, 1 x 108, 1 x 109, 1 x 1010, 1 x 1011, 1 x 1012, 1 x 1013, 1 x 1014, 1 x 1015, 1 x 1016, 1 x 1017, 1 x 1018, 1 x 1019, 1 x IO20IFU, or any range therein, of each of the viral vectors described herein per unit dose.

[0409] So that preferred embodiments of the present disclosure may be fully understood and put into practical effect, reference is made to the following non-limiting examples.

[0410] Table 1. Sequences of the disclosure

[0411]

[0412] Examples

[0413] Example 1 A Phase 1 clinical trial evaluating safety and immunogenicity of two Group A streptococcus peptide-conjugate vaccine candidates.

[0414] Study Design

[0415] Healthy adults aged 18 to 45 years of age were screened according to the study inclusion and exclusion criteria and recruited into the study (Table 2). A 1.0 mL dose of the vaccine candidate (i) J8K4S2-Alum (containing approximately 50 pg J8-CRM197, 6.25 pg K4S2- CRM197 and Alhydrogel® (-250 pg aluminium hydroxide (Alum)), (ii) p*17K4S2-Alum (containing 25 pg p*17-CRM197, 6.25 pg K4S2-CRM197 and Alhydrogel® (-250 pg aluminium hydroxide (Alum)) or placebo (RabAvert®) was intramuscularly administered to the participants on study day 0, day 21 ±7 and day 42 ±7 (Figure 1).

[0416] The study was conducted in two stages. Stage 1 involved administration of test doses to verify immediate safety (n=5 per test dose) and followed with a safety evaluation by the Data and Safety Monitoring Board.

[0417] Stage 2 is a randomised, double-blind, placebo-controlled Phase 1 clinical trial. It involves twenty participants who are randomised to one of the test dose arms (n=5 of J8-K4S2 vaccine or n=5 of p*17-K4S2 vaccine), and 10 who receive comparator / placebo (rabies; RabAvert) vaccine.

[0418] Table 2. Study inclusion and exclusion criteria Methodology: Stage 1 - Assessment of antigen-specific IgG in participant serum

[0419] For immunogenicity assessments, blood was collected from each participant prior to study vaccination (screening), prior to each vaccination and 2 weeks and 6 months after the third vaccination.

[0420] Immunogenicity was measured by ELISA and reported as peptide-specific serum IgG antibody titres (Figures 2-5). Briefly, participant serum was 2-fold diluted and incubated for 90 minutes to allow peptide-specific antibodies to bind to the immobilised peptides. Pooled serum sample extracted from patients in Fiji (a GAS endemic region) was also included as a positive control for the assay. Bound antibodies were detected with goat anti-human IgG conjugated to horseradish peroxidase. Data were captured as the optical density (OD) at absorbance 450nm. The data were adjusted for background by subtracting the OD readings from plates without antigen (no-Ag plates).

[0421] It was confirmed that intramuscular vaccination with peptide-conjugate vaccine candidate J8K4S2-Alum induced vaccine peptide-specific antibodies in all participants (Figure 2), but surprisingly, these antibodies did not recognise a related peptide p*17 (Figure 3). Similarly, the inventors confirmed that intramuscular vaccination with peptide-conjugate vaccine candidate p*17K4S2-Alum induced vaccine peptide-specific antibodies in all participants (Figure 4), but that the antibodies did not recognise a related peptide J8 (Figure 5). This data surprisingly suggests that J8 and p*17 are recognised by the human immune system as distinct epitopes.

[0422] Methodology: Stage 2

[0423] Participants received three doses, with serum samples and PBMCs collected at baseline (TO), following each immunisation (T1 and T2), two weeks after the final dose (T3), and again at the six-month follow-up (T4).

[0424] All vaccinated participants developed vaccine-specific antibodies that recognised multiple strains of Strep A and mediated bacterial killing in an opsonophagocytic killing assay, with the level of killing increasing with time post-vaccination (up to 6 months). These data are presented in Figures 15-18.

[0425] Example 2 - Assessment of vaccine immunogenicity and efficacy against upper respiratory infection (URT) with Streptococcus pyogenes.

[0426] Methods BALB / c mice (n=5 / group) were immunised three times intramuscularly on days 0, 21, and 42 with i) p*17-DT+K4S2-DT / Alum (p*17 bivalent, 50 pg / dose of p*17-DT and 50 pg / dose of K4S2-DT); ii) J8-DT+K4S2-DT / Alum (J8 bivalent, 50 pg / dose of J8-DT and 50 pg / dose of K4S2-DT); iii) p*17-DT+J8-DT+K4S2-DT / Alum (trivalent, 33 pg / dose of p*17- DT, 33 pg / dose of J8-DT and 33 pg / dose of K4S2-DT); iv) p*17-DT+J8-DT+K4S2-DT / Alum (trivalent, 8 pg / dose of p*17-DT, 8 pg / dose of J8-DT and 8 pg / dose of K4S2-DT) or v) PBS / Alum.

[0427] Saliva and sera were collected from the mice on Days 50 and 65, respectively, and antigen-specific IgG levels were measured using ELISA. The endpoint titer was defined as the highest dilution with an absorbance exceeding 3 standard deviations above the mean of negative control wells (BALB / c mice immunised with PBS / Alum). Data are mean ± standard error of the mean (SEM). Statistical comparisons were performed using the One-way ANOVA in GraphPad Prism 10: ns p > 0.05; *p < 0.05.

[0428] Briefly, peptide-specific serum and mucosal IgG were measured using enzyme-linked immunosorbent assay (ELISA). A peptide (p* 17, J8 or K4S2) was coated onto Nunc MaxiSorp ELISA plates at 5 pg / ml. Samples were assessed using 2-fold serial dilutions of 1 : 100 of serum or 1 :2 of saliva. Peptide-specific antibodies were detected with horseradish peroxidase (HRP)- conjugated goat anti-mouse-IgG antibody for murine studies (1 :3,000 for serum and 1 : 1000 for saliva; Bio-Rad Laboratories) Antibody detection was by SIGMAFAST OPD substrate (Sigma- Aldrich), which was added according to manufacturer’s instructions. Absorbance was measured at 450 nm on a Tecan Infinite M200 Pro plate reader (Tecan Group Ltd.). End-point titer was defined as the highest dilution that gave an absorbance of >3 standard deviations above the mean absorbance of the negative-control wells.

[0429] Next, mice were anaesthetised via an intraperitoneal injection of 100 pL ketamine:xylazil: H2O (1 : 1 : 10). Once immobilized the mice were intranasally infected at 5 pL / nare with S. pyogenes at a concentration of 5 * 10A8 CFU / mL (5 x 10A6 CFU / 10 pL). Mice were monitored daily for signs of illness as per the score sheet approved by Griffith University Animal Ethics Committee. Mice were sacrificed on day two post-infection and nasal shedding, throat swabs, nasal-associated lymphoid tissue (NALT) and lungs were excised for bacterial burden enumeration.

[0430] Results Intramuscular vaccination with trivalent vaccine induced antigen-specific IgG in both sera and saliva, significantly reducing bacterial burden compared to PBS / Alum control mice in nasal shedding, throat swabs, NALT, and lung samples (Figures 6 to 8).

[0431] It was also found that following intranasal challenge, the trivalent vaccine was highly effective in protecting mice at the respiratory surfaces (as shown by significantly reduced bacterial burden in nasal shedding and throat swabs; Figures 8A and 8B) as well as within the respiratory organs (NALT and lungs; Figures 8C and 8D).

[0432] Example 3 - Binding of vaccine induces antibodies to multiple S. pyogenes isolates.

[0433] Vaccine-induced antibodies were assessed for their ability to bind live S. pyogenes, a proxy measure of antibody functionality, since bacterial binding represents the first step in opsonophagocytic killing by neutrophils.

[0434] Methods

[0435] BALB / c mice (n=5 / group) were immunised three times intramuscularly on days 0, 21, and 42 with p*17-DT+K4S2-DT / Alum (p*17 bivalent, 50 pg / dose of p*17-DT and 50 pg / dose of K4S2-DT); ii) J8-DT+K4S2-DT / Alum (J8 bivalent, 50 pg / dose of J8-DT and 50 pg / dose of K4S2-DT); iii) p*17-DT+J8-DT+K4S2-DT / Alum (trivalent, 33 pg / dose of p*17-DT, 33 pg / dose of J8-DT and 33 pg / dose of K4S2-DT); iv) p*17-DT+J8-DT+K4S2-DT / Alum (trivalent, 8 pg / dose of p*17-DT, 8 pg / dose of J8-DT and 8 pg / dose of K4S2-DT) or v) PBS / Alum. Sera was collected on day 50 for binding assessment to S. pyogenes isolates.

[0436] Briefly, the Strep A flow binding assay comprised thawing and growing bacterial isolates from -80°C stock on day 1. Day 2 comprised resuspension of the isolates and plating on agar for counting and adjusting to OD 0.5. The GAS isolate was washed twice with PBS, the supernatant discarded, and the pellet resuspend at OD 0.5 in 5% skim milk / PBS. This was incubated for 2 hours at 4°C with end-over mixing, then washed twice with PBS and resuspended in 0.2% skim milk / PBS. 100 pL of the GAS isolated was dispended into corresponding PCR tubes. Then 100 pL of 1 / 100 diluted sera was added to the GAS isolated and incubated overnight at 4°C with end-over mixing. Day 3 comprised washing the GAS isolated twice with PBS (3000 rpm, 10 min, 4°C), adding anti-mouse IgGl (PE Dazzle) secondary antibody (1 :200 dilution in 0.2% skim milk / PBS) and incubating at 4°C for 1 hour. This was washed twice with PBS (3000 rpm, 10 min, 4°C), then 100 pL of 1% PFA was added, before transferring the solution to FACS tubes, and stored at 4°C until analysis. Samples were analysed using LSR Fortessa and FlowJo software. Results

[0437] Sera from mice immunised trivalent vaccine demonstrated significantly stronger binding to immunised (Ml) compared to sera from PBS / Alum or naive mice. Similarly, sera from mice immunized with trivalent vaccines showed significantly enhanced binding to GAS5448AP (Ml) and GASM59 (M59) relative to PBS / Alum or naive controls.

[0438] Figures 9A, 9B, 10A, 10B, 11A and 11C are histogram representations of sera binding to GAS (n=l sample). A rightward shift indicates stronger binding. Figures 9C, 10C and 11C illustrate the fluorescence intensity of the PE Dazzle secondary antibody, where higher intensity reflects improved binding (n=3 datasets). Figures 9D, 10D and 11D demonstrate the frequency of the PE Dazzle secondary antibody, indicating the percentage of GAS cells positive for PE Dazzle, representing the proportion of cells with antibody binding to GAS (n=3 datasets). Clearly, the trivalent vaccine provides broad cross-serotype protection. Furthermore, these observations suggest a potential variability in expression of M-protein epitopes among various S. pyogenes isolates and further validates the need for a trivalent vaccine.

[0439] Example 4 - Assessment of 1,2 and 3 dose immunogenicity and efficacy.

[0440] Methods

[0441] The immunogenicity and protective efficacy of 1-, 2-, and 3-dose regimens of the trivalent vaccine for URT infection was evaluated in a murine model. BALB / c mice (n = 10 / group) were immunised intramuscularly with the trivalent vaccine formulation containing J8-CRM, p*17-CRM, and K4S2-CRM / Alum (33 pg / dose of p*17-DT, 33 pg / dose of J8-DT and 33 pg / dose of K4S2-DT). Mice were vaccinated according to one of the three regimens:

[0442] 1. A single dose administered on day 0

[0443] 2. Two doses administered on days 0 and 21

[0444] 3. Three doses administered on days 0, 21 and 42.

[0445] Saliva was collected after the final immunisation, and J8-specific, p*17-specific, and K4S2-specific IgG levels were measured by ELISA. Serum was collected after each immunisation, and J8-specific, p*17-specific, and K4S2-specific IgG levels were measured by ELISA.

[0446] On day 77, all mice were infected via the URT with S. pyogenes 5448AP, covR / S mt at 5 * 107cfu / mouse. On day 2 post-infection all surviving mice were euthanized and clinical scores, percent weight loss, survival proportions, nasal shedding scores (0=0, 1=1-10, 2=10- 100, 3=100-1000 and 4 = 1000+) throat swabs and tissues (NALT, nasal turbinates, lungs, spleen and blood) were collected to determine S. pyogenes burden, data are represented as the geomean ± geometric SD (cfu / tissue) on a Log 10 scale. Statistical analysis was performed using non-parametric Mann-Whitney t-test corrected for multiple comparisons (Holm-Sidak) on GraphPad Prism 10: ns, P>0.05; *, P < 0.05; **, P < 0.01.

[0447] Results

[0448] Salivary IgG responses were compared following one, two or three immunization regimens. The levels of salivary IgG post single immunisation were lower than those seen with two or three immunisations (Figure 12).

[0449] Mice vaccinated with two or three doses of the vaccine developed higher serum IgG responses to J8 and p* 17 compared with those receiving a single immunisation. This difference became evident after the T2 time point, suggesting that additional booster doses enhanced antigen-specific antibody levels. No significant differences in K4S2-specific responses were observed among the three groups. Interestingly, the cohort that received a single dose still maintained sustained antibody levels up to the T3 time point (Figure 13).

[0450] To evaluate vaccine efficacy, vaccinated BALB / c mice were challenged intranasally via the URT, with 5448AP. During the infection period, mice were monitored for clinical signs (ruffled fur, response to stimuli, and posture), percentage weight loss, and survival. Mice that received PBS / Alum displayed higher clinical scores and greater weight loss compared with vaccinated groups. Mice immunised with three doses of the trivalent vaccine achieved 100% survival, whereas the PBS / Alum control group showed reduced survival (80%). Mice that received two or three doses of the trivalent vaccine showed significantly reduced bacterial burden in nasal shedding, throat swabs, NALT, nasal turbinates, lungs, and spleen, while vaccinated mice also showed reduced bacterial levels in the blood, but this reduction did not reach statistical significance. Together, these findings demonstrate that the trivalent vaccine provides strong mucosal and systemic protection, substantially reducing S. pyogenes colonisation and dissemination following challenge with a hypervirulent Ml strain (Figure 14).

[0451] Thus, clearly the trivalent vaccine provides immunogenicity and protective efficacy comparable to, or better than, the bivalent vaccine formulations. The flow-binding data further demonstrate that this vaccine broadens coverage across strains with varying expression of conserved M-protein epitopes. The findings also indicate that three doses of the vaccine confer the most robust protection, achieving 100% survival. Example 5 - Development and validation of immunological assays.

[0452] Qualitative and Quantitative ELISA

[0453] ELISA protocols were established to qualitatively assess antigen-specific IgG levels in the sera of vaccine recipients. All vaccinated participants developed robust, vaccine antigenspecific IgG responses that were significantly higher than baseline (TO). IgG levels increased progressively over time, and although a decline was observed at six months post -vaccination, antibody titres remained significantly elevated compared with baseline (Figures 15 and 16). Stage 2 of the trial was conducted in a double-blinded manner, including an additional placebo group that received the RabAvert vaccine. As expected, no measurable antigen-specific responses were detected in the placebo group. The data have now been unblinded, and ELISA results have been verified to be 100% accurate.

[0454] In parallel, a highly specialised quantitative ELISA (Q-ELISA) was developed to measure antigen-specific IgG concentrations in the sera of vaccinated participants. The method involved generating a standard curve using purified antigen-specific IgG obtained through sequential Protein G and antigen-affinity purification steps. The affinity-purified antigenspecific IgG was then used to generate a standard curve, enabling accurate quantification of antigen-specific antibody concentrations in serum samples. The quantitative ELISA data closely mirrored the qualitative findings, providing validation of assay performance and deeper insight into the magnitude and durability of vaccine-induced antibody responses (Figure 16).

[0455] Flow-Based Binding Assay

[0456] A flow cytometry -based competition binding assay was developed using a monoclonal antibody to assess the specificity of vaccine-induced antibody for the M-protein epitope on Strep A (Figure 17). Briefly, Strep A strain 2031 (emml) was incubated with sera from vaccinees (GAS006, GAS007, or GAS010) collected at time points TO, T3, or T4 followed by competition with p*17 mAb. The rationale was that if participant sera contained vaccine- induced IgG specific for the M-protein, these antibodies would occupy the relevant binding sites, causing steric hindrance and thereby preventing the pl7 mAb from binding.

[0457] For participant GAS006, p* 17 mAb binding was high at TO but decreased at T3 and T4 (Figure 17B). GAS007 showed comparable p*17 mAb binding at TO and T3, which was reduced by T4 (Figure 17C). For GAS011, strong binding was observed at TO, which was substantially reduced by T3 and T4 (Figure 17D). Collectively, these data demonstrate that at baseline (TO), participant sera showed minimal binding to Strep A Ml, whereas by T4 (six months post-third vaccination), sera binding was markedly increased across all participants tested. This increase in vaccine-specific binding over time likely reflects ongoing affinity maturation of the antibody response, leading to enhanced antigen recognition.

[0458] Opsonophagocytic Killing (OPK) Assay

[0459] The opsonophagocytic killing (OPK) assay was optimised to evaluate the functional activity of vaccine-induced antibodies. Using this platform, antibody-mediated killing was assessed against at least one, and in some cases two, Strep A strains. A readout of OPK titres was also defined.

[0460] The data demonstrate that all vaccinated individuals developed antibodies capable of opsonophagocytic killing, with this functional activity enhancing over time (Figure 18). Enhanced OPK activity following vaccination was sustained and further increased six months after the third dose.

Claims

CLAIMS:

1. An immunogenic composition comprising:(i)(a) a first immunogenic protein comprising an amino acid sequence set forth in SEQ ID NO: 3, or a fragment, variant or derivative thereof; and(b) a second immunogenic protein comprising an amino acid sequence set forth in SEQ ID NO: 5, or a fragment, variant or derivative thereof, and / or(ii) one or more nucleic acid molecules comprising a nucleotide sequence encoding the first and the second immunogenic protein or a nucleotide sequence complementary thereto.

2. The immunogenic composition of claim 1, further comprising:(i) a third immunogenic protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 14 or a fragment, variant or derivative thereof, and / or(ii) one or more nucleic acid molecules comprising a nucleotide sequence encoding the first, the second and the third immunogenic protein or a nucleotide sequence complementary thereto.

3. The immunogenic composition of claim 1, wherein the first and / or second immunogenic proteins are conjugated, coupled or otherwise linked to a carrier protein or a fragment, variant or derivative thereof.

4. The immunogenic composition of claim 2 or claim 3, wherein the third immunogenic protein is conjugated, coupled or otherwise linked to a carrier protein or a fragment, variant or derivative thereof.

5. The immunogenic composition of claim 3 or claim 4, wherein the carrier protein is selected from the group consisting of: a thyroglobulin or a fragment, variant or derivative thereof; an albumin or a fragment, variant or derivative thereof; a toxin or a fragment, variant or derivative thereof; a toxoid or a fragment, variant or derivative thereof; and a mutant cross- reactive material (CRM) or a fragment, variant or derivative thereof.

6. The immunogenic composition of claim 5, wherein the toxin or fragment, variant or derivative thereof is selected from the group consisting of: a tetanus toxin or toxoid or afragment, variant or derivative thereof; a diphtheria toxin or toxoid or a fragment, variant or derivative thereof; a pertussis toxin or toxoid or a fragment, variant or derivative thereof; a Pseudomonas toxin or toxoid or a fragment, variant or derivative thereof; an E. coli toxin or toxoid or a fragment, variant or derivative thereof; a Staphylococcus toxin or toxoid or a fragment, variant or derivative thereof; and a Streptococcus toxin or toxoid or a fragment, variant or derivative thereof.

7. The immunogenic composition of any one of claims 2 to 4, wherein the carrier protein is or comprises a diphtheria toxin or toxoid (DT) or a fragment, variant or derivative thereof.

8. The immunogenic composition of any one of claims 2 to 4, wherein the carrier protein is or comprises a CRM197 protein or a fragment, variant or derivative thereof.

9. The immunogenic composition of any one of claims 1 to 8, wherein the immunogenic composition comprises an adjuvant, preferably wherein the adjuvant is aluminium hydroxide (Alum) or a liposomal adjuvant.

10. The immunogenic composition of any one of claims 1 to 9, wherein the first immunogenic protein comprises an amino acid sequence set forth in SEQ ID NO: 6 or a fragment, variant or derivative thereof.

11. The immunogenic composition of any one of claims 1 to 10, wherein the second immunogenic protein comprises an amino acid sequence set forth in SEQ ID NO: 4 or a fragment, variant or derivative thereof.

12. The immunogenic composition of any one of claims 2 to 11, wherein the third immunogenic protein comprises an amino acid sequence set forth in SEQ ID NO: 13 or a fragment, variant or derivative thereof.

13. The immunogenic composition of any one of claims 1 to 12, wherein the immunogenic composition comprises a pharmaceutically acceptable diluent, carrier or excipient.

14. The immunogenic composition of any one of claims 1 to 13, wherein the immunogenic composition is capable of providing cross-serotype protection to a subject.

15. A method of eliciting an immune response to group A streptococcus (GAS) bacteria in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of the immunogenic composition of any one of claims 1 to 14, to thereby elicit the immune response.

16. A method of preventing, ameliorating or treating a GAS infection, or a disease, disorder or condition associated therewith in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of the immunogenic composition of any one of claims 1 to 14, to thereby prevent, ameliorate or treat the infection, disease, disorder or condition associated with the GAS bacteria in the subject.

17. Use of the immunogenic composition of any one of claims 1 to 14 in the manufacture of a medicament for:(a) eliciting an immune response to group A streptococcus (GAS) bacteria in a subject; and / or(b) preventing, ameliorating or treating an infection, disease or condition associated with group A streptococcus (GAS) bacteria in a subject.

18. The method of claim 15 or 16, wherein the method further comprises the step of administering the immunogenic composition to the subject parenterally, subcutaneously, intramuscularly or intranasally, or the use of claim 17, wherein the medicament is formulated to be parenterally, subcutaneously, intramuscularly or intranasally administered to the subject.

19. The method of claim 16 or 18 or the use of claim 17 or 18, wherein the GAS infection, or the disease, disorder or condition associated therewith is selected from the group consisting of: a GAS bacterial skin infection, a GAS bacterial intranasal infection, an upper respiratory tract (URT) GAS bacterial infection, a mucosal GAS bacterial infection, a GAS autoimmune infection and an invasive GAS bacterial infection.

20. The method of any one of claims 16, 18 and 19, wherein the immunogenic composition is administered to the subject at a dose of about 20 pg to 100 pg, or the use of any one of claims 17 to 19, wherein the medicament is formulated to be administered to the subject at a dose of about 20 pg to 100 pg.

Images