WO2024100235A1

WO2024100235A1 - Group a streptococcus vaccine antigen

Info

Publication number: WO2024100235A1
Application number: PCT/EP2023/081380
Authority: WO
Inventors: Pierre Ladislas Edith Marie Robert SMEESTERS; Anne Monique Roberte BOTTEAUX
Original assignee: Université Libre de Bruxelles
Priority date: 2022-11-10
Filing date: 2023-11-10
Publication date: 2024-05-16

Abstract

The invention provides immunogenic peptides, polypeptides, and compositions based on group A Streptococcus (GAS) Enn protein, and their use in the therapy, particularly prophylaxis, of GAS infections.

Description

GROUP A STREPTOCOCCUS VACCINE ANTIGEN

FIEED

The invention is broadly in the medical field, particularly in the field of treating infectious diseases, such as specifically bacterial infections, and more particularly concerns substances and compositions useful for preventing group A Streptococcus (GAS) infections, as well as methods for preventing GAS infections which employ said substances or compositions.

BACKGROUND

Group A Streptococcus (Strep A or GAS) is a major cause of morbidity globally and causes more than 500,000 deaths per year. Strep A causes a broad spectrum of diseases ranging from uncomplicated pharyngitis and skin infections to life-threatening invasive illnesses and nonsuppurative immune-mediated sequelae like acute rheumatic fever and glomerulonephritis.

There is a clear need for vaccines to prevent serious Strep A disease, particularly in developing countries.

Strep A vaccine candidates can be broadly divided into M protein-based and non-M protein-based vaccine candidates. None of the non-M protein-based candidates has entered clinical trials so far. Three M protein-based candidate vaccines have reached the phase 1 clinical trial and only one has reached the phase II clinical trial.

The well-known M protein, encoded by the emm gene, is a major virulence factor acting notably through the binding of several host proteins. The N-terminal part of the M protein has a variable sequence of around 50 amino acids (also known as HVR), resulting in antigenic diversity which is the basis for serotyping and the widely used nucleotide based cmm-typing scheme defining more than 230 different cmm-typcs.

Based on available epidemiological data showing that limited cmm-typcs were circulating in US and Europe, a 26 and a 30 valent vaccine candidate were developed and shown to be protective in animals and humans (McNeil et al. Clin Infect Dis 2005, vol. 41, 1114-1122). However, as the distribution of cmm-typcs was quite different (more diverse) in developing countries settings, these vaccine candidates might not be efficacious against GAS infections globally (Smeesters et al. Expert Rev Vaccines 2009, vol. 8(12), 1705-20). Despite the observation of cross-opsonising immunity between closely related cmm-typcs (grouped in cmm-clustcrs) (Frost et al. Clin Infect Dis. 2017, vol. 65, 1523- 1531), which might induce broader protection, said cross-opsonisation was found to vary by emm- cluster. M-based multivalent vaccines may therefore not be protective against some emm-c\ usters. WO 2012/174455 discloses immunogenic compositions comprising immunogenic peptides derived from an M protein or Spa protein, which induce an immune response against certain GAS serotypes. WO 2014/124446 discloses GAS Mrp polypeptides and peptides that evoke cross-opsonic and cross- protective anti-GAS and anti-Streptococcus dysgcilcicticie subspecies equisimilus antibodies in animals. There remains a need for alternative, complementary, and/or improved immunogenic compositions useful in vaccination against Group A Strepotococcus , in particular to allow for affordable vaccination efforts in low-income settings.

SUMMARY

The present invention is at least in part based on the inventors’ discovery that peptides derived from group A Streptococcus (GAS) Enn protein can induce a meaningful immune-protective response against GAS bacteria in test subjects. Further, the inventors have also uncovered novel ways of classifying GAS based on their Enn protein sequence, which facilitates Enn antigen design, such as in particular the design of multivalent Enn vaccines.

Accordingly, an aspect of the invention provides an immunogenic peptide or polypeptide from a group A Streptococcus (GAS) Enn protein, for use in the prevention of a GAS infection. A related aspect provides a method for treating a GAS infection in a subject in need thereof, comprising administering to the subject an effective amount of an immunogenic peptide or polypeptide from a GAS Enn protein. Also provided is use of an immunogenic peptide or polypeptide from a GAS Enn protein for the manufacture of a medicament for the prevention of a GAS infection.

A further aspect provides an immunogenic peptide or polypeptide for use in the prevention of a GAS infection, wherein the immunogenic peptide or polypeptide is from an amino acid sequence at least 70% identical to the amino acid sequence of any one of SEQ ID NO: 1 to 101. A related aspect provides a method for treating a GAS infection in a subject in need thereof, comprising administering to the subject an effective amount of an immunogenic peptide or polypeptide is from an amino acid sequence at least 70% identical to the amino acid sequence of any one of SEQ ID NO: 1 to 101. Also provided is use of an immunogenic peptide or polypeptide is from an amino acid sequence at least 70% identical to the amino acid sequence of any one of SEQ ID NO: 1 to 101, for the manufacture of a medicament for the prevention of a GAS infection.

Another aspect provides a hybrid peptide or polypeptide, or an immunogenic composition, comprising at least two immunogenic peptides or polypeptides selected from the group consisting of: an immunogenic peptide or polypeptide from a GAS Enn protein; an immunogenic peptide or polypeptide from an amino acid sequence at least 70% identical to the amino acid sequence of any one of SEQ ID NO: 1 to 101; and mixtures thereof; wherein the hybrid peptide or polypeptide, orthe immunogenic composition, is capable of conferring host immunity to an infection by a GAS. A further aspect provides a hybrid peptide or polypeptide, or an immunogenic composition, comprising at least one immunogenic peptide or polypeptide selected from: an immunogenic peptide or polypeptide from a GAS Enn protein; an immunogenic peptide or polypeptide which is from an amino acid sequence at least 70% identical to the amino acid sequence of any one of SEQ ID NO: 1 to 101; and mixtures thereof; said hybrid peptide or polypeptide or immunogenic composition further comprising at least one other GAS immunogen; wherein the hybrid peptide or polypeptide, or the immunogenic composition, is capable of conferring host immunity to an infection by a GAS.

Also provided is a nucleic acid molecule encoding said hybrid peptide or polypeptide, wherein the hybrid polypeptide is a fusion polypeptide.

A further aspect provides a vaccine composition comprising a pharmaceutically acceptable adjuvant and one or more of: (i) an immunogenic peptide or polypeptide from a GAS Enn protein; (ii) an immunogenic peptide or polypeptide which is from an amino acid sequence at least 70% identical to the amino acid sequence of any one of SEQ ID NO: 1 to 101; (iii) the aforementioned hybrid peptide or polypeptide or immunogenic composition; or (iv) the aforementioned nucleic acid molecule; wherein the vaccine composition is capable of conferring host immunity to an infection by a GAS.

Another aspect relates to the aforementioned hybrid polypeptide, nucleic acid, or vaccine composition, for use in the prevention of a GAS infection. A related aspect provides a method for treating a GAS infection in a subject in need thereof, comprising administering to the subject an effective amount of the aforementioned hybrid polypeptide, nucleic acid, or vaccine composition. Also provided is use of the aforementioned hybrid polypeptide, nucleic acid, or vaccine composition, for the manufacture of a medicament for the prevention of a GAS infection.

These and further aspects and preferred embodiments of the invention are described in the following sections and in the appended claims. The subject-matter of the appended claims is hereby specifically incorporated in this specification.

BRIEF DESCRIPTION OF DRAWINGS

The following description of the figures of specific embodiments of the invention is merely exemplary in nature and is not intended to limit the present teachings, their application or uses.

Fig. 1 illustrates anti -Enn antibody titres in rabbits injected with peptides EA1-10 (identified by reference to their respective Enn vaccine clusters, VE1 to VE10, see Table 1), as assessed by ELISA with synthetic Enn peptides. Antibody titres were expressed as the inverse of the last dilution giving a signal > 0.1 OD. N=3.

Fig. 2 illustrates the ability of the antibodies raised against the HVR of Enn from Enn vaccine cluster VE1 to VE10 to induce antibodies-dependent cellular phagocytosis, expressed in ‘phagoscore’. PI : pre-immune sera used as a negative control. One-way ANOVA analysis with Dunnett's multiple comparison. * : p-value < 0,05 ; **** : p-value = 0,0001.

Fig. 3 illustrates multivalent Enn HVR antigens.

Fig. 4 illustrates interaction of 8 representative Enn proteins with human serum proteins, in an SDS- PAGE analysis followed by Coomassie blue staining (A) or by western blot with specific antibodies (B) directed against human IgG, albumin, vitronectin and C4BP (N=3). M28 was used as a positive control of binding.

Fig. 5 illustrates relative expression of mga, mrp, emm, and enn genes in presence of pharyngeal cells (left bar) or human blood (right bar) compared with their expression in rich medium (THY). recA was used as a housekeeping gene.

Fig. 6 illustrates (A) whole cell binding in human serum using wild-type strain (M25, Streptococcus pyogenes, emm-type 25, Rosenbach 12204, ATCC # NCTC 8306) and its knock-out mutants (Senn, Semm, and mrp), (B) co-purification of Enn314 and its point mutants with recombinant C4BP (rC4BP) or with human serum (sC4BP).

Fig. 7 illustrates survival in whole blood of the wild-type strain (M98) and its knock-out mutants (Senn314). N=2 (same non-immune donor).

DESCRIPTION OF EMBODIMENTS

As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

The terms “comprising”, “comprises” and “comprised of’ as used herein are synonymous with “including”, “includes”, “containing”, or “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms also encompass “constituted of’, “consists in”, “consisting of’, and “consists of’, and also the terms “consisting essentially of’, “consisting essentially in” and “consists essentially of’, which enjoy well-established meanings in patent terminology.

The recitation of numerical ranges by endpoints includes all integer numbers and, where appropriate, fractions subsumed within the respective ranges, as well as the recited endpoints. This applies to numerical ranges irrespective of whether they are introduced by the expression “from ... to ... ” or the expression “between. . . and. . . ” or another expression. Any numerical range recited herein is intended to include all sub-ranges subsumed therein.

The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of +/-10% or less, preferably +/-5% or less, more preferably +/-1% or less, and still more preferably +/-0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

Whereas the terms “one or more” or “at least one”, such as one or more members or at least one member of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members. In another example, “one or more” or “at least one” may refer to 1, 2, 3, 4, 5, 6, 7 or more.

As used herein, the term “and/or” when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a list is described as comprising group A, B, and/or C, the list can comprise A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B, and C in combination.

The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge in any country as of the priority date of any of the claims.

Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents herein specifically referred to are incorporated by reference.

Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the invention. When specific terms are defined in connection with a particular aspect of the invention or a particular embodiment of the invention, such connotation or meaning is meant to apply throughout this specification, i.e., also in the context of other aspects or embodiments of the invention, unless otherwise defined.

In the following passages, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

Reference throughout this specification to “one embodiment”, “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

Similarly, it should be appreciated that in the description of illustrative embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects.

Human pathogens expressing the P-haemolytic phenotype are a heterogeneous group of organisms that includes groups A, C, G, and L streptococci. Group A streptococci (GAS) are ubiquitous human pathogens and Group A streptococcal infections result in approximately 500,000 deaths per year worldwide, with invasive infections and rheumatic heart disease as the major contributors to mortality. The acute infections range from uncomplicated pharyngitis, cellulitis, and pyoderma to life-threatening infections that include necrotizing fasciitis, sepsis, pneumonia, and streptococcal toxic shock syndrome. Mild and even asymptomatic infections can be followed by serious autoimmune diseases, among which acute rheumatic fever (ARF) and rheumatic heart disease (RHD) are the most significant.

Serological classification of streptococci in groups is based upon expression of unique carbohydrate antigens in the bacterial cell wall (Lancefield. Exp Med. 1928, vol. 47, 91-103). All GAS serotypes express the Lancefield group A carbohydrate (GAC), comprising a polyrhamnose backbone with an immunodominant N-acetylglucosamine (GlcNAc) side chain, which is the basis of rapid diagnostic tests (McCarty. J Exp Med. 1952, vol. 96, 569-580; McCarty. J Exp Med. 1956, vol. 104, 629-643). Streptococcus pyogenes is the predominant species harbouring the Lancefield group A antigen, and is often referred to as group A Streptococcus (GAS). However, both Streptococcus dysgalcicticie and the Streptococcus anginosus group can possess group A antigen as well. Hence, in certain embodiments, GAS refers to a bacterium, particularly a pathogenic bacterium, belonging to the species .S', pyogenes, S. dysgalaciiae. or .S', anginosus. In certain preferred embodiments, GAS refers to a bacterium, particularly a pathogenic bacterium, belonging to the species .S', pyogenes.

In an aspect, the invention provides an immunogenic peptide or polypeptide from a group A Streptococcus (GAS) Enn protein, for use in the prevention of a GAS infection. A related aspect provides a method for treating a GAS infection in a subject in need thereof, comprising administering to the subject an effective amount of an immunogenic peptide or polypeptide from a GAS Enn protein. Also provided is use of an immunogenic peptide or polypeptide from a GAS Enn protein for the manufacture of a medicament for the prevention of a GAS infection.

The terms “protein”, “polypeptide”, and “peptide” as used throughout this specification generally encompass macromolecules comprising one or more polypeptide chains, i.e., polymeric chains of amino acid residues linked by peptide bonds. The terms are not limited to any minimum length, for example, an amino acid chain may be composed of two or more amino acids, such as about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more amino acids. The terms may encompass naturally, recombinantly, semi- synthetically or synthetically produced proteins, polypeptides, or peptides. The terms also encompass proteins, polypeptides, or peptides that carry one or more co- or post-expression-type modifications of (poly)peptide chain(s), such as, without limitation, glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-terminal Met removal, conversion of pro-enzymes or pre-hormones into active forms, etc.

Amino acids may be referred to herein according to the single letter and three letter codes, which are understood according to common textbook knowledge in the art, and as such a person skilled in the art is familiar with the meanings of the abbreviations. In the art, the number of amino acids covalently bonded to form a “peptide” and the number of amino acids that form a “polypeptide” varies. A peptide may be defined as composed of about 50 amino acids or less or may be defined as composed of about 100 amino acids or less. For example, a peptide may be about 95 or 90 or less amino acids long, such as about 85 or 80 or less amino acids long, about 75 or 70 or less amino acids long, about 65 or 60 or less amino acids long, about 55 or 50 or less amino acids long, about 45 or 40 or less amino acids long, about 35 or 30 or less amino acids long, about 25 or 20 or less amino acids long, or about 15 or 10 or less amino acids long. A polypeptide would more commonly have greater than about 50 amino acids or greater than about 100 amino acids. As used herein, the term “immunogenic” refers to the ability of a substance, such as an antigen (or an epitope of the antigen), such as a peptide or polypeptide, to elicit, provoke or produce an immune response in a host animal, such as a mammal or particularly human. The immune response is in particular a specific immune response directed to said substance, and may be either humorally- mediated (antibody-mediated), or cellularly-mediated, or both. For example, an immunogenic peptide or polypeptide may comprise a sufficient number of contiguous amino acids of a cognate protein, such as Enn, that form at least one epitope that induces an immune response specific for the peptide or polypeptide, for the full- length mature cognate protein, and for GAS bacteria that express the full-length mature cognate protein, and potentially other proteins comprising the immunogenic peptide or polypeptide.

It is particularly intended that the immunogenic peptides or polypeptides taught herein when administered to a subject - optionally comprised by an immunogenic conjugate and/or coadministered with one or more adjuvants - are capable of inducing an immune response that is immunoprotective or therapeutic against one or more group A Streptococcus (GAS).

The term “Enn protein” as used interchangeably herein with the term “Enn polypeptide” has its well- established meaning in the field. Enn proteins are so-called M-like proteins, which show structural and functional similarities with the well-characterised M proteins. The M-like proteins (Enn and Mrp) and the M protein are expressed from the mga (multiple gene activator) regulon (containing the emm, enn and mrp genes), which further contains genes coding for Mga and C5a peptidase. Enn proteins are expressed on the cell surface of GAS. They have similar structures to M proteins, sharing several structural domains including a C-terminal proline-glycine-threonine-serine (PGTS)-rich region, a cell wall-associated region, a central heptad-repeat region (C-repeats), and a variable length N-terminal region (also referred to herein as hyper-variable region or “HVR”).

Frost et al. (mSphere 2020, vol. 5(1), e00806-19) reported an extensive sequence analysis of GAS M and M-like proteins. An enn gene was present in 87.1% of genomes, and of these there were 352 unique enn alleles. The genes encoded 276 unique mature protein sequences following removal of signal sequences and cleaved regions. Mature proteins ranged in length from 199 to 346 amino acids and had an average pairwise identity of 68.6%. Enn proteins therefore presented genetic diversity that is intermediate between the high diversity of M and the low diversity of Mrp and were generally the smaller of this trio of protein families.

The repeat region of Enn proteins contained C-repeats which are predicted to form alpha-helices disrupted by small regions of random coil and divided by linker regions of 7, 14, or 28 amino acids. The Cl repeat was present in 100% of sequences and had 94% sequence identity, the C2 repeat was present in 94% of sequences and had 94% sequence identity, and the C3 repeat was present in 37% of sequences and had 93% sequence identity. The number of repeats present and the combination of linker regions had a large effect on the protein lengths.

Following the variable region at the N terminus, Enn proteins had either an EQ-rich central core (55% of sequences) with significant similarity to the analogous region in M proteins or, in 39% of sequences, an 18-amino-acid consensus sequence (EKEKEDLKTTLAKTTKEN, SEQ ID NO: 102). There was greater sequence similarity between the N-terminal 50 first amino acids from Enn proteins with the 18-amino-acid core than EQ-rich cores, with 58 and 34% pairwise identities, respectively.

The most C-terminal part of the proteins contained the LPXTG sortase motif, which allows attachment of the protein to the bacterial cell wall. This was also the region of the most sequence homogeneity in the mature Enn proteins, and the proteins became increasingly heterogeneous more distally. Average amino acid sequence identities in the different regions of the analysed Enn proteins were 96.2%, 27.3%, 56.2%, and 93.7% for the signal peptide, the first 50 N-terminal amino acids (the hypervariable region, HVR), the 51st amino acid to repeat region, and for repeat region to LPXTG, respectively.

The terms “Enn protein” or “Enn polypeptide” thus in particular refer to proteins or polypeptides the amino acid sequence of which is identical or substantially identical (i.e., largely but not wholly identical) to the amino acid sequence of a naturally occurring Enn protein of GAS. In particular, the terms may denote proteins identical to or substantially identical to mature Enn proteins lacking the signal peptide sequence. The terms do not imply any particular source of such proteins or polypeptides - for example, they may have been isolated from a GAS strain, or produced recombinantly or synthetically.

The amino acid sequence of an Enn protein or polypeptide may for example be at least about 70% identical or at least about 73% identical or at least about 75% identical, e.g., preferably at least about 80% identical or at least about 83% identical or at least about 85% identical, e.g., more preferably at least about 90% identical, e.g., at least 91% identical, at least 92% identical, more preferably at least about 93% identical, e.g., at least 94% identical, even more preferably at least about 95% identical, e.g., at least 96% identical, yet more preferably at least about 97% identical, e.g., at least 98% identical, and most preferably at least 99% identical to a naturally occurring Enn protein, especially a mature naturally occurring Enn protein (Enn proteins or polypeptides the sequence of which diverges from that of a naturally occurring Enn protein may be denoted as “Enn variants”). In certain embodiments, such degree of sequence identity to a native Enn protein may be obtained when the whole or entire Enn sequences are queried in the sequence alignment (i.e., overall sequence identity). In certain embodiments, such degree of sequence identity to a native Enn protein may be obtained when only select portions of the whole or entire Enn sequences are queried in the sequence alignment, such as for example, the hypervariable regions, the 51st amino acid to repeat region, or the repeat region to LPXTG.

By means of an example and without limitation, the sequence of mature naturally occurring Enn74 protein is as below:

DQAASGTSVNNGASKSVESFKYDALRGENADLRNVNAKYLEKINAEEEKNKKLEATNKE LNENYYKLQDGIDALEKEKEDLKTTLAKTTKENEISEASRKGLSRDLEASRTAKKELEAKH QKLEAENKKLTEANQISEASRKGLSNDLEASRAAKKELEAKHQKLAEEHQVSETSRKGLS RDLEASREANKKVTSELTQAKAQLSALEESKKLSEKEKAELQAKLDAQGKALKEQLAKQ TEELAKLRAEKAAGSKIPATKPANKERSGRAAQTATRPSQNKGMRSQLPSA (SEQ ID NO: 103)

By means of an example and without limitation, the sequence of mature naturally occurring Enn201 protein is as below:

DSNNSVSVNNEAKEKSSEDVERHYLRKLDQEHKEHQKEKQQQQQEQEERQKNQEQLERK YQREVEKRYQEQQQKQQQLEAENQKLTEANKVSEASRKGLSNDLEASRAAKKELEAKHQ KLEADHQALEAKHQKLEADHQALEAKHQKLEADHQVSEASRRGLSRDLEASREANKKVT SELTQAKAQLSALEESKKLSEKEKAELQAKLDAQGKALKEQLAKQAEELAKLRAEKAAG SKTPATKPANKERSGRAAQTATRPIQNKGMRSQLPST (SEQ ID NO: 104)

The term “sequence identity” with regard to amino acid sequences denotes the extent of sequence identity expressed in % between the amino acid sequences read from N-terminus to C-terminus. Sequence identity may be determined using suitable algorithms for performing sequence alignments and determination of sequence identity as know per se. Exemplary but non-limiting algorithms include those based on the Basic Local Alignment Search Tool (BLAST) originally described by Altschul et al. 1990 (J Mol Biol 215: 403-10), such as the “Blast 2 sequences” algorithm described by Tatusova and Madden 1999 (FEMS Microbiol Lett 174: 247-250), for example using the published default settings or other suitable settings (such as, e.g., the BLASTP algorithm: matrix = Blosum62 (Henikoff et al., 1992, Proc. Natl. Acad. Sci., 89: 10915-10919), cost to open a gap = 11, cost to extend a gap = 1, expectation value = 10.0, word size = 3).

An example procedure to determine the percent identity between a particular amino acid sequence and a query amino acid sequence will entail aligning the two amino acid sequences each read from N-terminus to C-terminus using the Blast 2 sequences (B12seq) algorithm, available as a web application

(https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&BLAST _SPEC=blast2seq&LINK_LOC=blasttab) or as a standalone executable programme (BLAST version 2.11.0+) at the NCBI web site (htps://ftp.ncbi.nlm.nih.gov/blasVexecutables/blastH7LATEST/), using suitable algorithm parameters. An example of suitable algorithm parameters includes: matrix = Blosum62, cost to open a gap = 11, cost to extend a gap = 1, expectation value = 10.0, word size = 3). If the two compared sequences share identity, then the output will present those regions of identity as aligned sequences. If the two compared sequences do not share identity, then the output will not present aligned sequences. Once aligned, the number of matches will be determined by counting the number of positions where an identical amino acid residue is presented in both sequences. The percent identity is determined by dividing the number of matches by the length of the query sequence, followed by multiplying the resulting value by 100. The percent identity value may, but need not, be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 may be rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 may be rounded up to 78.2. It is further noted that the detailed view for each segment of /alignment as outputed by B12seq already conveniently includes the percentage of identities.

Where an amino acid sequence differs, varies or diverges from a certain other amino acid sequence - for example, where the former amino acid sequence is said to display a certain degree or percentage of sequence identity to the later amino acid sequence, or where the former amino acid sequence is said to differ by a certain number of amino acids from the later amino acid sequence - such sequence variation may be constituted by one or more amino acid additions (e.g., a single amino acid or a stretch of two or more contiguous amino acids may be added at one position of an amino acid sequence or each independently at two or more positions of an amino acid sequence), deletions (e.g., a single amino acid or a stretch of two or more contiguous amino acids may be deleted at one position of an amino acid sequence or each independently at two or more positions of an amino acid sequence), and/or or substitutions (e.g., a single amino acid or a stretch of two or more contiguous amino acids may substitute a single one or a stretch of two or more contiguous amino acids at one position of an amino acid sequence or each independently at two or more positions of an amino acid sequence).

Preferably, the one or more amino acid substitutions, in particular one or more single amino acid substitutions, may be conservative amino acid substitutions. A conservative amino acid substitution is a substitution of one amino acid for another with similar characteristics. Conservative amino acid substitutions include substitutions within the following groups: valine, alanine and glycine; leucine, valine, and isoleucine; aspartic acid and glutamic acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and phenylalanine and tyrosine. The nonpolar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (i.e., basic) amino acids include arginine, lysine and histidine. The negatively charged (i.e., acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the above-mentioned polar, basic, or acidic groups by another member of the same group can be deemed a conservative substitution. By contrast, a non-conservative substitution is a substitution of one amino acid for another with dissimilar characteristics.

Reference to a peptide or polypeptide “from a GAS Enn protein” in particular relates to a peptide or polypeptide comprising or consisting of a certain number of contiguous amino acids as found in an Enn protein as set forth above. This therefore denotes a correspondence between the amino acid sequences, rather than any act of obtainment of the peptide or polypeptide starting from an actual Enn protein encompassing it. Hence, the peptide or polypeptide may be for example prepared recombinantly or synthetically, based on the knowledge of the amino acid sequence of an Enn protein, such that the peptide’s or polypeptide’s amino sequence corresponds to (is identical to) a contiguous amino acid stretch in the Enn protein.

In certain embodiments, the immunogenic peptide or polypeptide from a GAS Enn protein may comprise or consist of at least, or precisely, 5, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 or more contiguous amino acids of an Enn protein. In certain embodiments, the immunogenic peptide or polypeptide from a GAS Enn protein may comprise or consist of at least, or precisely, any integer between 5-25, 5- 50, 5-10, 10-15, 15-20, 20-25, 25-30, 25-50, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 85-90, 90-95, 95-100, 100-105, 105-110, or 110-120 amino acids of an Enn protein. In certain embodiments, the immunogenic peptide or polypeptide from a GAS Enn protein may comprise or consist of at least, or precisely, between 20 and 80, such as between 30 and 70, or between 40 and 60, or between 45 and 55, such as 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 contiguous amino acids of an Enn protein.

The amino acid sequence of a peptide or polypeptide from an Enn protein may for example be at least about 70% identical or at least about 73% identical or at least about 75% identical, e.g., preferably at least about 80% identical or at least about 83% identical or at least about 85% identical, e.g., more preferably at least about 90% identical, e.g., at least 91% identical, at least 92% identical, more preferably at least about 93% identical, e.g., at least 94% identical, even more preferably at least about 95% identical, e.g., at least 96% identical, yet more preferably at least about 97% identical, e.g., at least 98% identical, and most preferably at least 99% identical to the corresponding amino acid stretch in a naturally occurring Enn protein.

A person skilled in the vaccine and immunology arts readily appreciates that certain epitopes may comprise two or more non-contiguous regions of a polypeptide, which epitopes form when the polypeptide folds into a three-dimensional structure. An immunogenic peptide or polypeptide from an Enn protein comprising such a three-dimensional epitope may thus comprise a sufficient number of contiguous amino acids of the Enn protein to form the epitope.

In certain preferred embodiments, the immunogenic peptide or polypeptide is from the hypervariable region (HVR) of an Enn protein. Protein sequence alignment tools as described herein can be used to delineate the HVR in any given Enn protein (for example by multiple sequence alignment with one or more distinct naturally occurring Enn protein sequences). In particular embodiments, the HVR may be constituted by the first at least 30 contiguous N-terminal amino acids of a mature Enn protein (i.e., in which the signal peptide has been removed), such as by the first at least 35, 40, 45, or 50 contiguous N-terminal amino acids of a mature Enn protein; such as more particularly by amino acid 1 to 30, 1 to 35, 1 to 40, 1 to 45, or 1 to 50 (counting from the N-terminus) of a mature Enn protein. For example, the C-terminal boundary ofthe HVRmay be between amino acid 30 and 35, or between amino acid 35 and 40, or between amino acid 40 and 45, or between amino acid 45 and 50 of a mature Enn protein. The HVR of Enn is relatively distal from the bacterial cell wall and thus advantageously exposed and potentially more readily targetable by the immune system.

In certain embodiments, the immunogenic peptide or polypeptide from a GAS Enn protein may comprise or consist of at least, or precisely, 5, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, or 50 contiguous amino acids of an HVR of an Enn protein. In certain embodiments, the immunogenic peptide or polypeptide from a GAS Enn protein may comprise or consist of at least, or precisely, any integer between 5-25, 5- 50, 5-10, 10-15, 15-20, 20-25, 25-30, 25-50, 30-35, 35-40, 40-45, or 45-50, amino acids of an HVR of an Enn protein. In certain embodiments, the immunogenic peptide or polypeptide from a GAS Enn protein may comprise or consist of at least, or precisely, between 20 and 50, such as between 30 and 50, or between 40 and 50, or between 45 and 50, such as 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 contiguous amino acids of an HVR of an Enn protein. Hence, in certain preferred embodiments, the immunogenic peptide or polypeptide comprises or consists of at least 25, or at least 30, or at least 35, or at least 40, or at least 45, or at least 50 contiguous amino acids of an HVR of an Enn protein.

In certain embodiments, the immunogenic peptide or polypeptide comprises or consists of the hypervariable region (HVR), namely the entirely HVR, of an Enn protein. Hence, depending on the length of the HVR, the immunogenic peptide or polypeptide may comprise or consist of the first at least 30 contiguous N-terminal amino acids of a mature Enn protein (i.e., in which the signal peptide has been removed), such as the first at least 35, 40, 45, or 50 contiguous N-terminal amino acids of a mature Enn protein; such as more particularly amino acid 1 to 30, 1 to 35, 1 to 40, 1 to 45, or 1 to 50 (counting from the N-terminus) of a mature Enn protein. The Enn protein may be a naturally occurring Enn protein. Table 1 elsewhere in this specification lists exemplary sequences (SEQ ID NO: 1 to 101) of the hypervariable region of a number of naturally occurring Enn proteins.

Accordingly, a further aspect provides an immunogenic peptide or polypeptide for use in the prevention of a GAS infection, wherein the immunogenic peptide or polypeptide is from an amino acid sequence at least 70% identical to the amino acid sequence of any one of SEQ ID NO: 1 to 101. A related aspect provides a method for treating a GAS infection in a subject in need thereof, comprising administering to the subject an effective amount of an immunogenic peptide or polypeptide is from an amino acid sequence at least 70% identical to the amino acid sequence of any one of SEQ ID NO: 1 to 101. Also provided is use of an immunogenic peptide or polypeptide which is from an amino acid sequence at least 70% identical to the amino acid sequence of any one of SEQ ID NO: 1 to 101, for the manufacture of a medicament for the prevention of a GAS infection.

In certain embodiments, the immunogenic peptide or polypeptide may comprise or consist of at least, or precisely, 5, 6, 8, 10, 15, 20, more preferably at least, or precisely, 25, 30, 35, 40, 45, or 50 contiguous amino acids of an amino acid sequence at least 70% identical to the amino acid sequence of any one of SEQ ID NO: 1 to 101. In certain embodiments, the immunogenic peptide or polypeptide may comprise or consist of at least, or precisely, any integer between 5-25, 5- 50, 5-10, 10-15, 15- 20, 20-25, 25-30, 25-50, 30-35, 35-40, 40-45, or 45-50, contiguous amino acids of an amino acid sequence at least 70% identical to the amino acid sequence of any one of SEQ ID NO: 1 to 101 . In certain embodiments, the immunogenic peptide or polypeptide may comprise or consist of at least, or precisely, between 20 and 50, such as between 30 and 50, or between 40 and 50, or between 45 and 50, such as 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 contiguous amino acids of an amino acid sequence at least 70% identical to the amino acid sequence of any one of SEQ ID NO: 1 to 101. Hence, in certain preferred embodiments, the immunogenic peptide or polypeptide comprises or consists of at least 25, or at least 30, or at least 35, or at least 40, or at least 45, or at least 50 contiguous amino acids of an amino acid sequence at least 70% identical to the amino acid sequence of any one of SEQ ID NO: I to 101.

In certain embodiments, in increasing order of preference, the amino acid sequence may be at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, such as 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NO: 1 to 101. In certain embodiments, in increasing order of preference, the amino acid sequence may be at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, such as 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96. In certain particularly preferred embodiments, the amino acid sequence may be any one of SEQ ID NO: 1 to 101. In certain particularly preferred embodiments, the amino acid sequence may be any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96. In certain particularly preferred embodiments, the immunogenic peptide or polypeptide may comprise or consist of the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96. In certain particularly preferred embodiments, the immunogenic peptide or polypeptide is selected from the group consisting of the peptides as set forth in SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96.

Given the description herein, persons skilled in the art can readily determine which amino acids in the full-length and in the amino terminal portions of Enn, such as in particular naturally occurring Enn, may be more amenable to alteration (i.e., substitution, deletion, or addition of one or more amino acids) and which amino acids may not be amenable to change. Also given the description herein and given the many molecular biology, protein expression, and protein isolation techniques and methods routinely practiced in the art for introducing mutations in peptide or polypeptides, isolating said peptides and polypeptides and variants thereof, and analysing the same, a polypeptide or peptide variant having the desired immunogenicity can be made readily and without undue experimentation.

A further aspect provides a hybrid peptide or polypeptide, or an immunogenic composition, comprising at least two immunogenic peptides or polypeptides selected from the group consisting of: an immunogenic peptide or polypeptide from a GAS Enn protein, such as for example as described in the preferred embodiments set forth above; an immunogenic peptide or polypeptide which is from an amino acid sequence at least 70% identical to the amino acid sequence of any one of SEQ ID NO: 1 to 101, such as for example as described in the preferred embodiments set forth above; and mixtures thereof; wherein the hybrid peptide or polypeptide, or the immunogenic composition, is capable of conferring host immunity to an infection by a GAS. In certain embodiments, the hybrid peptide or polypeptide, or the immunogenic composition, may comprise at least, or precisely, 3, 4, 5, 6, 7, 10, or between 10 and 20, or between 20 and 30 such immunogenic peptides or polypeptides.

Hence, certain embodiments provide, for example, a hybrid peptide or polypeptide, or an immunogenic composition, comprising at least two immunogenic peptides or polypeptides selected from the group consisting of: an immunogenic peptide or polypeptide from a GAS Enn protein; an immunogenic peptide or polypeptide which is from, or preferably comprises at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95% identical to the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96, or comprising the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96; and mixtures thereof; wherein the hybrid peptide or polypeptide, or the immunogenic composition, is capable of conferring host immunity to an infection by a group A Streptococcus (GAS).

A further aspect provides a hybrid peptide or polypeptide, or an immunogenic composition, comprising at least one immunogenic peptide or polypeptide selected from: an immunogenic peptide or polypeptide from a GAS Enn protein, such as for example as described in the preferred embodiments set forth above; an immunogenic peptide or polypeptide which is from an amino acid sequence at least 70% identical to the amino acid sequence of any one of SEQ ID NO: 1 to 101, such as for example as described in the preferred embodiments set forth above; and mixtures thereof; and further comprising at least one other GAS immunogen; wherein the hybrid peptide or polypeptide, or the immunogenic composition, is capable of conferring host immunity to an infection by a group A Streptococcus (GAS). In certain embodiments, the hybrid peptide or polypeptide, or the immunogenic composition, may comprise at least, or precisely, 3, 4, 5, 6, 7, 10, or between 10 and 20, or between 20 and 30 such immunogenic peptides or polypeptides.

Hence, certain embodiments provide, for example, a hybrid peptide or polypeptide, or an immunogenic composition, comprising at least one immunogenic peptide or polypeptide selected from: an immunogenic peptide or polypeptide from a GAS Enn protein; an immunogenic peptide or polypeptide which is from, or preferably comprises at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95% identical to the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96, or comprising the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96; and mixtures thereof; and further comprising at least one other GAS immunogen; wherein the hybrid peptide or polypeptide, or the immunogenic composition, is capable of conferring host immunity to an infection by a group A Streptococcus (GAS).

In certain embodiments of any such hybrid peptides or polypeptides, or such immunogenic compositions, any one of the following may preferably apply: the immunogenic peptide or polypeptide, or the at least two immunogenic peptides or polypeptides each independently, is from, or preferably comprises at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of the hypervariable region (HVR) of the Enn protein; the immunogenic peptide or polypeptide, or the at least two immunogenic peptides or polypeptides each independently, comprises the hypervariable region (HVR) of the Enn protein; the immunogenic peptide or polypeptide, or the at least two immunogenic peptides or polypeptides each independently, is from, or preferably comprises at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96; or the immunogenic peptide or polypeptide, or the at least two immunogenic peptides or polypeptides each independently, comprises the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96.

Individual immunogenic peptides or polypeptides included in or constituting such hybrid peptides may for example be linked or conjugated chemically as generally known in the art (any art recognised chemistry can be employed for such chemical linkage) to create a covalent connection between them (optionally via a suitable non-immunogenic peptide or non-peptide spacer or linker), or may be conveniently fused (optionally interposed by a suitable non-immunogenic spacer or linker peptide) into a common polypeptide chain by well-established recombinant technologies or peptide synthesis methods. In an immunogenic composition, the individual immunogenic peptides or polypeptides may remain unconjugated, or some of the individual immunogenic peptides may be conjugated into hybrid peptide(s) or polypeptide(s), while others may remain unconjugated.

Where the hybrid peptide or polypeptide, or the immunogenic composition, comprises at least two immunogenic peptides or polypeptides based on the Enn protein, the at least two immunogenic peptides or polypeptides may preferably be from different GAS Enn proteins. For example, in certain embodiments, the hybrid peptide or polypeptide, or the immunogenic composition, may comprise at least immunogenic peptides or polypeptides from at least, or precisely, 3, 4, 5, 6, 7, 10, or between 10 and 20, or between 20 and 30, different GAS Enn proteins.

In certain embodiments, the at least two Enn-based immunogenic peptides or polypeptides may preferably be from Enn proteins belonging to different GAS Enn vaccine clusters, more preferably selected from Vaccine clusters VE1, VE2, VE3, VE4, VE5, VE6, VE7, VE8, VE9, and VE10 (see Table 1).

In certain preferred embodiments, with reference to the naturally occurring Enn HVR sequences shown in Table 1, where the hybrid peptide or polypeptide, or the immunogenic composition, comprises at least two immunogenic peptides or polypeptides based on the Enn protein, the at least two immunogenic peptides or polypeptides may be from different subgroups a) to j), wherein: subgroup a) consists of immunogenic peptides and polypeptides which are from, or preferably comprise at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95%, identical to the amino acid sequence of any one of SEQ ID NO: 1 to 15, preferably SEQ ID NO: 4 or 9; subgroup b) consists of immunogenic peptides and polypeptides, which are from, or preferably comprise at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95%, identical to the amino acid sequence of any one of SEQ ID NO: 16 to 20, preferably SEQ ID NO: 17 (including any preferred lengths and/or sequence identities of such peptides or polypeptides, as described in the preferred embodiments set forth above); subgroup c) consists of immunogenic peptides and polypeptides, which are from, or preferably comprise at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95%, identical to the amino acid sequence of any one of SEQ ID NO: 21 to 37, preferably SEQ ID NO: 35 (including any preferred lengths and/or sequence identities of such peptides or polypeptides, as described in the preferred embodiments set forth above); subgroup d) consists of immunogenic peptides and polypeptides, which are from, or preferably comprise at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95%, identical to the amino acid sequence of any one of SEQ ID NO: 38 to 39, preferably SEQ ID NO: 38 (including any preferred lengths and/or sequence identities of such peptides or polypeptides, as described in the preferred embodiments set forth above); subgroup e) consists of immunogenic peptides and polypeptides, which are from, or preferably comprise at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95%, identical to the amino acid sequence of any one of SEQ ID NO: 40 to 43, preferably SEQ ID NO: 40 (including any preferred lengths and/or sequence identities of such peptides or polypeptides, as described in the preferred embodiments set forth above); subgroup f) consists of immunogenic peptides and polypeptides, which are from, or preferably comprise at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95%, identical to the amino acid sequence of any one of SEQ ID NO: 44 to 63, preferably SEQ ID NO: 53 or 60 (including any preferred lengths and/or sequence identities of such peptides or polypeptides, as described in the preferred embodiments set forth above); subgroup g) consists of immunogenic peptides and polypeptides, which are from, or preferably comprise at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95%, identical to the amino acid sequence of any one of SEQ ID NO: 64 to 65, preferably SEQ ID NO: 64 (including any preferred lengths and/or sequence identities of such peptides or polypeptides, as described in the preferred embodiments set forth above); subgroup h) consists of immunogenic peptides and polypeptides, which are from, or preferably comprise at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95%, identical to the amino acid sequence of any one of SEQ ID NO: 66 to 78, preferably SEQ ID NO: 76 (including any preferred lengths and/or sequence identities of such peptides or polypeptides, as described in the preferred embodiments set forth above); subgroup i) consists of immunogenic peptides and polypeptides, which are from, or preferably comprise at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95%, identical to the amino acid sequence of any one of SEQ ID NO: 79 to 89, preferably SEQ ID NO: 86 (including any preferred lengths and/or sequence identities of such peptides or polypeptides, as described in the preferred embodiments set forth above); and subgroup j) consists of immunogenic peptides and polypeptides, which are from, or preferably comprise at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95%, identical to the amino acid sequence of any one of SEQ ID NO: 90 to 101, preferably SEQ ID NO: 96.

In certain preferred embodiments, with reference to the naturally occurring Enn HVR sequences shown in Table 1, where the hybrid peptide or polypeptide, or the immunogenic composition, comprises at least two immunogenic peptides or polypeptides based on the Enn protein, the at least two immunogenic peptides or polypeptides may be from different subgroups a*) to j*), wherein: subgroup a*) consists of immunogenic peptides and polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 1 to 15, preferably SEQ ID NO: 4 or 9; subgroup b*) consists of immunogenic peptides and polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 16 to 20, preferably SEQ ID NO: 17; subgroup c*) consists of immunogenic peptides and polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 21 to 37, preferably SEQ ID NO: 35; subgroup d*) consists of immunogenic peptides and polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 38 to 39, preferably SEQ ID NO: 38; subgroup e*) consists of immunogenic peptides and polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 40 to 43, preferably SEQ ID NO: 40; subgroup f*) consists of immunogenic peptides and polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 44 to 63, preferably SEQ ID NO: 53 or 60; subgroup g*) consists of immunogenic peptides and polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 64 to 65, preferably SEQ ID NO: 64; subgroup h*) consists of immunogenic peptides and polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 66 to 78; subgroup i*) consists of immunogenic peptides and polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 79 to 89, preferably SEQ ID NO: 76; and subgroup j*) consists of immunogenic peptides and polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 90 to 101, preferably SEQ ID NO: 96.

In certain particularly preferred embodiments, with reference to the naturally occurring Enn HVR sequences shown in Table 1, where the hybrid peptide or polypeptide, or the immunogenic composition, comprises at least two immunogenic peptides or polypeptides based on the Enn protein, the at least two immunogenic peptides or polypeptides may be from the group consisting of the peptides as set forth in SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96.

In certain embodiments, the hybrid peptide or polypeptide, or the immunogenic composition, may preferably comprise or consist of at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE1 Enn protein; at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE2 Enn protein; and at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE3 Enn protein.

In certain embodiments, the hybrid peptide or polypeptide, or the immunogenic composition, may preferably comprise or consist of at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE4 Enn protein; at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE5 Enn protein; at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE6 Enn protein; at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE7 Enn protein; at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE8 Enn protein; at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE9 Enn protein; and at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE10 Enn protein.

In certain embodiments, the hybrid peptide or polypeptide, or the immunogenic composition, may preferably comprise or consist of at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE1 Enn protein; at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE2 Enn protein; at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE3 Enn protein; at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE4 Enn protein; at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE5 Enn protein; at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE6 Enn protein; at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE7 Enn protein; at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE8 Enn protein; at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE9 Enn protein; and at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE10 Enn protein.

In certain embodiments, the hybrid peptide or polypeptide, or the immunogenic composition, may preferably comprise or consist of at least two, 3, 4, 5, 6, 7, 8, 9, or 10 elements selected from the group consisting of the following elements xl-xlO: xl) at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE1 Enn protein; x2) at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE2 Enn protein; x3) at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE3 Enn protein; x4) at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE4 Enn protein; x5) at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE5 Enn protein; x6) at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE6 Enn protein; x7) at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE7 Enn protein; x8) at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE8 Enn protein; x9) at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE9 Enn protein; and xlO) at least one, such as precisely one, two, or three, immunogenic peptide or polypeptide, from vaccine cluster VE10 Enn protein.

In certain embodiments, where the hybrid peptide or polypeptide, or the immunogenic composition, comprises at least one “other” GAS immunogen, i.e., a GAS immunogen not based on or derived from the Enn protein, the at least one other GAS immunogen may be selected from the group consisting of an immunogenic peptide from a GAS M protein, an immunogenic peptide from a GAS Mrp protein, and mixtures thereof.

The cell surface M protein of GAS is a major virulence factor for this pathogen. The M protein, which is encoded by the emm gene, extends from the cell surface as an a-helical coiled-coil dimer that appears as a fibril on the surface of a GAS bacterium. The M proteins form an antigenically diverse group, and GAS strains have been serologically categorized according to M protein serotypes. More than 120 M protein serotypes have been identified, and within some serotypes, subtypes have also been identified. The amino acid sequences of M proteins and subtypes of M proteins are available in protein databases, such as GenBank, GenEMBL, and Swiss Prot databases. In further embodiments, said at least one M protein is selected from the M protein of group A streptococcus (GAS) serotype 1, 2, 3, 4, 5, 6, 11, 12, 14, 18, 19, 22, 24, 28, 29, 44, 49, 58, 73, 75, 77, 78, 81, 82, 83, 87, 89, 92, 114, and 118. Selection of M protein serotypes to include in the hybrid polypeptides may be based on available epidemiology and serotype prevalence of GAS infections. In certain embodiments, the hybrid peptides or polypeptides may comprise two or more immunogenic peptides or polypeptides from an M protein of different GAS serotypes. In certain embodiments, the hybrid peptides or polypeptides may comprise two or more immunogenic peptides or polypeptides each from an M protein of a different GAS serotype. Antibodies to the M protein can facilitate opsonophagocytosis by phagocytic cells present in blood. The amino terminal regions such as the hypervariable region of M proteins have been shown to evoke antibodies with the greatest bactericidal (protective) activity. Immunogenic peptides or polypeptides of a GAS M protein may comprise at least 20, 25, 30, 35, 40, 45, 50, 55, or 60 or more contiguous amino acids (or any number of amino acids between 20-45, 20-50, 25-45, 25-50, 25-60, 25-30, 30-35, 35-40, 40-45, 45-50, 40- 55, or 55-60, or more than 60 contiguous amino acids) of the mature M protein (i.e., the M polypeptide from which the signal peptide sequence has been removed). In certain particular embodiments, the immunogenic peptide or polypeptide of a GAS M protein may comprise at least 25, 30, 35, 40, 45, 50, 55, or 60 or more contiguous amino acids from the amino terminal portion such as the hypervariable region of the M protein (or any number of amino acids between 25-45, 25- 50, 25-60, 25-30, 30-35, 35- 40, 40-45, 45-50, 40-55, or 55-60, or more than 60 contiguous amino acids from the amino terminal portion of the protein). The preferred features explained above for Enn-based immunogenic peptides or polypeptides, such as with respect of their length and/or degree of sequence identity to naturally occurring Enn proteins or parts thereof (e.g., HVR), can be applied mutatis mutandis to M-protein-based immunogenic peptides or polypeptides. Many non-limiting M- protein-based antigens and technical principles useful for their application in the context of the present invention have also been described in WO 2012/174455, incorporated by reference herein.

Immunogenic peptides or polypeptides of a GAS Mrp protein may comprise at least 20, 25, 30, 35, 40, 45, 50, 55, or 60 or more contiguous amino acids (or any number of amino acids between 20-45, 20-50, 25-45, 25-50, 25-60, 25-30, 30-35, 35-40, 40-45, 45-50, 40-55, or 55-60, or more than 60 contiguous amino acids) of the mature Mrp protein (i.e., the Mrp polypeptide from which the signal peptide sequence has been removed). In certain particular embodiments, the immunogenic peptide or polypeptide of a GAS Mrp protein may comprise at least 25, 30, 35, 40, 45, 50, 55, or 60 or more contiguous amino acids from the amino terminal portion such as the hypervariable region of the Mrp protein (or any number of amino acids between 25-45, 25-50, 25-60, 25-30, 30-35, 35- 40, 40-45, 45-50, 40-55, or 55-60, or more than 60 contiguous amino acids from the amino terminal portion of the protein). The preferred features explained above for Enn-based immunogenic peptides or polypeptides, such as with respect of their length and/or degree of sequence identity to naturally occurring Enn proteins or parts thereof (e.g., HVR), can be applied mutatis mutandis to Mrp protein- based immunogenic peptides or polypeptides. Many non-limiting Mrp-protein-based antigens and technical principles useful for their application in the context of the present invention have also been described in WO2014124446, incorporated by reference herein.

In connection with M proteins of group A Streptococcus, such as .S', pyogenes, Frost et al. Clin Infect Dis 2017, vol. 65, 1523-1531 investigated human serological response to GAS impetigo in Fijian schoolchildren, focusing on 3 major emm clusters (E4, E6, and D4), and reported that the M proteins included in D4 cluster were not as immunogenic as those of other clusters (E4, E6) and that D4 did not induce cross-protection within its cluster unlike E4 and E6. Yet, the GAS emm cluster D is of high interest. The cluster contains many different M proteins (32 different cmm-typcs. see Sanderson- Smith et al. J Infect Dis. 2014, vol. 210, 1325-38) that are particularly relevant in many low-income settings. In addition, the present inventors performed a systematic review of the global distribution of .S', pyogenes emm -types and emm -clusters from 1990 to 2023, including 203 articles and reports from 55 countries encompassing 74,668 bacterial isolates belonging to 211 emm-types, and found that D4 was associated with skin infection and accounted for 8.8% of all GAS strains worldwide. Considering the lower immunogenicity of D4 M proteins, an M-protein based vaccine will likely miss a significant disease burden globally.

In this light the present Enn-based immunogenic peptides or polypeptides and vaccine compositions comprising the same may in certain embodiments be used to prevent an infection with GAS, such as .S', pyogenes, belonging to emm cluster D4. In certain embodiments, the GAS may be any one or more of emm-types 33, 41, 43, 52, 53, 56, 56.2 (st3850), 64, 70, 72, 80, 83, 86, 91, 93, 98, 101, 108, 116, 119, 120, 121, 178 (st22), 186 (st2940), 192 (st3757), 194 (st38), 208 (st62), 223 (stD432), 224 (stD631), 225 (stD633), 230 (stNS1033), 242 (st2926), as reported in Table 1 of Sanderson-Smith et al. J Infect Dis. 2014, vol. 210, 1325-38).

In certain embodiments, the operative part of the present molecules, i.e., the part eliciting immune response to GAS, such as in particular any immunogenic peptide or polypeptide, optionally a hybrid one, as taught herein, may be connected to one or more further moieties, groups, components or parts, which may serve other functions or perform other roles and activities, preferably covalently connected, bound, linked or fused, directly or through a linker, which may be a peptide or a nonpeptide linker. Where such further moiety, group, component or part is a peptide, polypeptide or protein, the connection to the operative part of the molecule may preferably involve a peptide bond, direct one or through a peptide linker. Without limitation, suitable peptide linkers may comprise from 5 to 100 amino acid residues, and include for example various proline or glycine-serine spacers, as known in the art, such as for example PP, PPP, GS, SG, SGG, SSG, GSS, GGS, GSGS (SEQ ID NO: 105), or (Gly4Ser)_n, wherein n = 1-12. Without limitation, non-peptide linkers may comprise, consist essentially of or consist of a non-peptide polymer. The term “non-peptide polymer” as used herein refers to a biocompatible polymer including two or more repeating units linked to each other by a covalent bond excluding the peptide bond. For example, the non-peptide polymer may be 2 to 200 units long or 2 to 100 units long or 2 to 50 units long or 2 to 45 units long or 2 to 40 units long or 2 to 35 units long or 2 to 30 units long or 5 to 25 units long or 5 to 20 units long or 5 to 15 units long. The non-peptide polymer may be selected from the group consisting of polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers such as PLA (poly(lactic acid) and PLGA (polylactic-glycolic acid), lipid polymers, chitins, hyaluronic acid, and combinations thereof. Particularly preferred is polyethylene glycol) (PEG). Another particularly envisaged chemical linker is Ttds (4,7,10-trioxatridecan-13-succinamic acid). The non-peptide polymer may have reactive group(s) capable of binding to the elements which are to be coupled by the linker, such as reactive group(s) selected from the group consisting of a reactive aldehyde group, a propione aldehyde group, a butyl aldehyde group, a maleimide group, and a succinimide derivative, and combinations thereof. The succinimide derivative may be succinimidyl propionate, hydroxy succinimidyl, succinimidyl carboxymethyl or succinimidyl carbonate.

Such functions, roles or activities may be useful or desired for example in connection with the production, synthesis, isolation, purification or formulation of the molecule, or in connection with its experimental or therapeutic uses. By means of an example and without limitation, the immunogenic peptide or polypeptide(s) taught herein may be connected to one or more detectable moiety, an immunogenicity enhancing moiety, a tag moiety useful for purification purposes (for example, biotin (isolatable using an affinity purification method utilising streptavidin), his-tag (isolatable using an affinity purification method utilising metal ion, e.g., Ni²⁺), maltose (isolatable using an affinity purification method utilising maltose binding protein), glutathione S-transferase (GST) (isolatable using an affinity purification method utilising glutathione), or myc or FLAG tag (isolatable using an affinity purification method utilising anti-myc or anti-FLAG antibody, respectively)), or any combination thereof. For example, the immunogenic peptide or polypeptide(s) taught herein may be associated or conjugated with tetanus toxoid, cholera toxoid, other bacterial toxoids, keyhole limpet hemocyanin, or other protein or protein fragment, used in the art to enhance an immune response to an antigen of interest.

Any one of the immunogenic peptides or polypeptides, optionally hybrid ones as described herein may be produced recombinantly or may be chemically synthesized. By means of an example, nucleic acids (polynucleotides) encoding the peptides or polypeptides, optionally fused to one or more heterologous polypeptide moieties, may be constructed by recombinant methods or chemically synthesized, and the constructed or synthesized nucleic acids may be incorporated into expression vectors for production of the respective peptides, polypeptides, or fusions in a host cell into which the expression vector has been introduced.

Peptides and polypeptides may be chemically synthesized by manual techniques or by automated procedures. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as, e.g., Perkin-Elmer, Inc. and Applied BioSystems Division, and may be operated according to the manufacturer's instructions. If required, synthesized peptides or polypeptides may be purified using techniques and methods well know in the art, including, for example, preparative reverse phase chromatography, partition chromatography, gel filtration, gel electrophoresis, or ionexchange chromatography.

Nucleic acids that encode the peptides and polypeptides, or fusions described herein may be chemically synthesized or may be constructed by recombinant methods familiar to a person skilled in the art. Polynucleotides can also be synthesized using an automatic synthesizer. The nucleotide sequence can be designed with the appropriate codons for the particular amino acid sequence desired. In general, preferred codons may be selected for the intended host in which the nucleotide sequence will be expressed. Polynucleotides that encode a peptide, polypeptide, or fusion polypeptide described herein may be recombinantly expressed in a variety of different host cells. Host cells containing recombinant expression constructs may be genetically engineered (transduced, transformed, or transfected) with the vectors and/or expression constructs (for example, a cloning vector, a shuttle vector, or an expression construct). The vector or construct may be in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying particular genes or encoding-nucleotide sequences. Selection and maintenance of culture conditions for particular host cells, such as temperature, pH and the like, will be readily apparent to the ordinarily skilled artisan. In general, the desired host cell is one that can be adapted to sustained propagation in culture to yield a stable cell line that can express sufficient amount of the immunogenic peptide, polypeptide, or hybrid polypeptide. For example, the cell line may be an immortal cell line, which refers to a cell line that can be repeatedly passaged (at least ten times while remaining viable) in culture following log-phase growth. In another example, the host cell used to generate a cell line is a cell that is capable of unregulated growth, such as a cancer cell, or a transformed cell, or a malignant cell. Useful bacterial expression constructs are prepared by inserting into an expression vector a structural DNA sequence encoding the desired peptide or polypeptide together with suitable translation initiation and termination signals in an operative reading phase with a functional promoter. The construct may comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector construct and, if desirable, to provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice. Any other plasmid or vector may be used as long as the plasmid or vector is replicable and viable in the host. Selection of the appropriate vector and promoter and preparation of certain recombinant expression constructs comprising at least one promoter or regulated promoter operatively linked to a polynucleotide described herein is well within the level of ordinary skill in the art.

Accordingly, also provided are nucleic acid molecules encoding the hybrid peptides or polypeptides, wherein the hybrid polypeptides are fusion polypeptides.

The term “nucleic acid” as used herein typically refers to a polymer (preferably a linear polymer) of any length composed essentially of nucleoside units. A nucleoside unit commonly includes a heterocyclic base and a sugar group. Heterocyclic bases may include inter alia purine and pyrimidine bases such as adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U), which are widespread in naturally-occurring nucleic acids, other naturally-occurring bases (e.g., xanthine, inosine, hypoxanthine), as well as chemically or biochemically modified (e.g., methylated), nonnatural or derivatised bases. Exemplary modified nucleobases include, without limitation, 5- substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2- aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. In particular, 5 -methylcytosine substitutions have been shown to increase nucleic acid duplex stability. Sugar groups may include inter alia pentose (pentofuranose) groups such as preferably ribose and/or 2-deoxyribose common in naturally-occurring nucleic acids, or arabinose, 2-deoxyarabinose, threose or hexose sugar groups, as well as modified or substituted sugar groups (such as, without limitation, 2’-0-alkylated, e.g., 2’- O-methylated or 2’-0-ethylated sugars such as ribose; 2’-O-alkyloxyalkylated, e.g., 2’-0- methoxyethylated sugars such as ribose; or 2’-O,4’-C-alkylene-linked, e.g., 2’-O,4’-C-methylene- linked or 2’-O,4’-C-ethylene-linked sugars such as ribose; 2’-fluoro-arabinose, etc.). Nucleoside units may be linked to one another by any one of numerous known inter-nucleoside linkages, including inter alia phosphodiester linkages common in naturally-occurring nucleic acids, and further modified phosphate- or phosphonate-based linkages such as phosphorothioate, alkyl phosphorothioate such as methyl phosphorothioate, phosphorodithioate, alkylphosphonate such as methylphosphonate, alkylphosphonothioate, phosphotriester such as alkylphosphotriester, phosphoramidate, phosphoropiperazidate, phosphoromorpholidate, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate; and further siloxane, carbonate, sulfamate, carboalkoxy, acetamidate, carbamate such as 3’-N-carbamate, morpholino, borano, thioether, 3 ’-thioacetal, and sulfone intemucleoside linkages. Preferably, inter-nucleoside linkages may be phosphate-based linkages including modified phosphate-based linkages, such as more preferably phosphodiester, phosphorothioate or phosphorodithioate linkages or combinations thereof. The term “nucleic acid” also encompasses any other nucleobase containing polymers such as nucleic acid mimetics, including, without limitation, peptide nucleic acids (PNA), peptide nucleic acids with phosphate groups (PHONA), locked nucleic acids (LNA), morpholino phosphorodiamidate-backbone nucleic acids (PMO), cyclohexene nucleic acids (CeNA), tricyclo- DNA (tcDNA), and nucleic acids having backbone sections with alkyl linkers or amino linkers (see, e.g., Kurreck 2003 (Eur J Biochem 270: 1628-1644)). “Alkyl” as used herein particularly encompasses lower hydrocarbon moieties, e.g., C1-C4 linear or branched, saturated or unsaturated hydrocarbon, such as methyl, ethyl, ethenyl, propyl, 1 -propenyl, 2-propenyl, and isopropyl.

Nucleic acids as intended herein may include naturally occurring nucleosides, modified nucleosides or mixtures thereof. A modified nucleoside may include a modified heterocyclic base, a modified sugar moiety, a modified inter-nucleoside linkage or a combination thereof. The term “nucleic acid” further preferably encompasses DNA, RNA and DNA/RNA hybrid molecules, specifically including hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g., chemically synthesised) DNA, RNA or DNA/RNA hybrids. A nucleic acid can be naturally occurring, e.g., present in or isolated from nature, can be recombinant, i.e., produced by recombinant DNA technology, and/or can be, partly or entirely, chemically or biochemically synthesised. A “nucleic acid” can be double-stranded, partly double stranded, or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.

By “encoding” is meant that a nucleic acid sequence or part(s) thereof corresponds, by virtue of the genetic code of an organism in question to a particular amino acid sequence, e.g., the amino acid sequence of one or more desired proteins or polypeptides, or to another nucleic acid sequence in a template-transcription product (e.g., RNA or RNA analogue) relationship.

For heterologous expression, the nucleic acid may be made part of an expression cassette, which may in turn be comprised by an expression vector. The term “expression cassette” encompasses a nucleic acid molecule, typically DNA, into which a coding sequence for a protein or proteins of interest may be inserted to be expressed, wherein said nucleic acid molecule comprises one or more nucleic acid sequences operably linked to and controlling the expression of the coding sequence (regulatory sequences), non-limiting examples of which include promoter sequences and transcription terminators. An “operable linkage” is a linkage in which regulatory sequences and sequences sought to be expressed are connected in such a way as to permit said expression. For example, sequences, such as, e.g., a promoter and a coding sequence for a protein of interest, may be said to be operably linked if the nature of the linkage between said sequences does not: (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter to direct the transcription of the coding sequence, (3) interfere with the ability of the coding sequence to be transcribed from the promoter sequence. Hence, “operably linked” may mean incorporated into a genetic construct so that expression control sequences, such as a promoter, effectively control transcription / expression of a sequence of interest. The precise nature of transcriptional and translational regulatory sequences or elements required for expression may vary between expression environments, but typically transcriptional regulatory sequences include a promoter, optionally an enhancer, and a transcription terminator.

Reference to a “promoter” is to be taken in its broadest context and includes transcriptional regulatory sequences required for accurate transcription initiation and where applicable accurate spatial and/or temporal control of gene expression or its response to, e.g., internal or external (e.g., exogenous) stimuli. More particularly, “promoter” may depict a region on a nucleic acid molecule, preferably DNA molecule, to which an RNA polymerase binds and initiates transcription. A promoter is preferably, but not necessarily, positioned upstream, i.e., 5’, of the sequence the transcription of which it controls. Typically, in prokaryotes a promoter region may contain both the promoter per se and sequences which, when transcribed into RNA, will signal the initiation of protein synthesis (e.g., Shine-Dalgamo sequence). A promoter sequence can also include “enhancer regions”, which are one or more regions of DNA that can be bound with proteins (namely the trans-acting factors) to enhance transcription levels of genes in a gene-operon. The enhancer, while typically at the 5’ end of a coding region, can also be separate from a promoter sequence, e.g., can be within an intronic region of a gene or 3’ to the coding region of the gene.

The terms “expression vector” or “vector” as used herein refer to nucleic acid molecules, typically

DNA, to which nucleic acid fragments may be inserted and cloned, i.e., propagated. Hence, a vector will typically contain one or more unique restriction sites, and may be capable of autonomous replication in a defined cell or vehicle organism such that the cloned sequence is reproducible. A vector may also preferably contain a selection marker, such as, e.g., an antibiotic resistance gene, to allow selection of recipient cells that contain the vector. Vectors may include, without limitation, plasmids, phagemids, bacteriophages, bacteriophage-derived vectors, PAC, BAC, linear nucleic acids, e.g., linear DNA, transposons, viral vectors, etc., as appropriate (see, e.g., Sambrook et al., 1989; Ausubel 1992). Viral vectors may include inter alia retroviral vectors, lentiviral vectors, adenoviral vectors, or adeno-associated viral vectors, for example, vectors based on HIV, SV40,

EBV, HSV or BPV. Expression vectors are generally configured to allow for and/or effect the expression of nucleic acids or open reading frames introduced thereto in a desired expression system, e.g., in vitro, in a cell, organ and/or organism. For example, expression vectors may advantageously comprise suitable regulatory sequences.

Recombinant nucleic acid technology may allow not only for heterologous expression and isolation of immunogenic peptides or polypeptides, but may even allow to administer such immunogenic peptides or polypeptides as transgenes, i.e., to administer nucleic acids (such as, for example, DNA- based or RNA-based cassettes, vectors or constructs) encoding the respective immunogenic peptides or polypeptides and capable of effecting the expression of the respective immunogenic peptides or polypeptides, optionally hybrid ones, when introduced into a cell. For example, in a DNA construct an immunogenic peptide or polypeptide coding sequence may be operably linked to regulatory sequence(s) configured to drive the transcription and translation of the immunogenic peptide or polypeptide from the DNA construct, such as a promoter and a transcription terminator. In an RNA or mRNA construct an immunogenic peptide or polypeptide coding sequence may be included such that it can be translated by the cellular protein translation machinery (e.g., mRNA vaccine technologies as known in the art, such as developed and widely commercialized in connection with the Covid- 19 pandemic). In aforementioned constructs an immunogenic peptide or polypeptide coding sequence will be typically preceded by an in-frame translation initiation codon and followed by a translation termination codon, to facilitate proper translation. Accordingly, wherever administration of / therapy with immunogenic peptides or polypeptides as taught herein is envisaged in this specification, the administration of nucleic acids encoding those immunogenic peptides or polypeptides is encompassed by the disclosure. Such administration / therapy may commonly be referred to as gene therapy or in case of mRNA constructs as mRNA therapy or mRNA vaccination. Thus all methods and uses involving the molecules of the application thus also encompass methods and uses where the molecules are provided as the nucleic acid sequence encoding them, and the molecules are expressed from the nucleic acid sequence.

The immunogenic peptides or polypeptides, optionally hybrid ones, as well as various conjugated or fusion polypeptides, nucleic acids, and any combinations thereof, as taught herein, are generally useful for the treatment of GAS infections, and more particularly for prophylactic treatment of GAS infections. As used herein, the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures. The terms “treatment”, “treating”, and the like, as used herein include amelioration or elimination of a developed disease or condition once it has been established or alleviation of the characteristic symptoms of such disease or condition. As used herein these terms preferably encompass, depending on the condition of the subject, preventing the onset of a disease or condition or of symptoms associated with a disease or condition, including reducing the severity of a disease or condition or symptoms associated therewith prior to affliction with said disease or condition. Such prevention or reduction prior to affliction refers to administration of the compound or composition of the invention to a subject that is not at the time of administration afflicted with the disease or condition. “Preventing” also encompasses preventing the recurrence or relapse-prevention of a disease or condition or of symptoms associated therewith, for instance after a period of improvement. The term “subject”, “individual” or “patient”, used interchangeably herein, relates to any organism such as a vertebrate, particularly any mammal, including both a human and other mammals, for whom diagnosis, therapy or prophylaxis is desired, e.g., an animal such as a rodent, a rabbit, a cow, a sheep, a horse, a dog, a cat, a lama, a pig, or a non-human primate (e.g., a monkey). The rodent may be a mouse, rat, hamster, guinea pig, or chinchilla. In one embodiment, the subject is a human, a rat or a non-human primate. Preferably, the subject is a human.

Accordingly, also provided herein are vaccine compositions comprising any one or more of the immunogenic agents or substances as disclosed herein, wherein the vaccine compositions are capable of conferring host immunity to an infection by a group A Streptococcus (GAS). By means of an example, in preferred embodiments, a vaccine composition may comprise one or more of: (i) an immunogenic peptide or polypeptide from a GAS Enn protein; (ii) an immunogenic peptide or polypeptide which is from, or preferably comprises at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95% identical to the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96, or comprising the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96; (iii) the hybrid peptide or polypeptide, or the immunogenic composition, as taught herein; or (iv) the nucleic acid molecule according as taught herein, wherein the vaccine composition is capable of conferring host immunity to an infection by a GAS. In embodiments, the vaccine composition may comprise at least 2, 3, 4, 5, 6, 7, 8 or 9 different immunogenic peptides or polypeptides of a GAS Enn protein, preferably at least 10 such as at least 11, 12, 13, 14, 15, 16, 17, 18 or 19 different immunogenic peptides or polypeptides of a GAS Enn protein, more preferably at least 20, 25, 30 or 35 different immunogenic peptides or polypeptides of a GAS Enn protein as described herein.

14. The vaccine composition according to claim 13, wherein: the immunogenic peptide or polypeptide is from, or preferably comprises at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of the hypervariable region (HVR) of the Enn protein;

By means of an example, in further preferred embodiments, the immunogenic peptide or polypeptide comprises the hypervariable region (HVR) of the Enn protein; the immunogenic peptide or polypeptide is from, or preferably comprises at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96; orthe immunogenic peptide or polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96. In embodiments, the immunogenic peptides and/or polypeptides are each individually combined in the vaccine composition. Each of the immunogenic peptides and/or polypeptides may be chemically synthesized or may be recombinantly produced according to techniques and methods described in greater detail herein and in the art, and formulated in a vaccine composition according to techniques and method described herein and in the art. In other embodiments of the vaccine composition, the immunogenic peptides and/or polypeptides are comprised in one or more, such as two, three, four, five or more, hybrid polypeptides as described herein. Accordingly, in embodiments, the vaccine composition comprises one or more hybrid polypeptides as described herein, wherein each hybrid polypeptide may comprise four, five, six, seven, eight or more immunogenic peptides and/or polypeptides. In yet other embodiments of the vaccine composition, one or more immunogenic peptides and/or polypeptides as described herein may be combined with one or more hybrid polypeptides as described herein.

The vaccine compositions may be formulated such that the compositions are pharmaceutically or physiologically acceptable or suitable compositions or formulations for administration to a human or non-human animal. Such compositions may further comprise one or more pharmaceutically suitable excipients, and may further comprise one or more pharmaceutically suitable adjuvants.

In particular, the vaccine compositions described herein also comprise a suitable adjuvant. An adjuvant is intended to enhance (or improve, augment) the immune response to the immunogens included in the composition (i.e., increase the level of the specific immune response to the immunogens in a statistically, biologically, or clinically significant manner compared with the level of the specific immune response in the absence of administering the adjuvant). For administration in humans, a pharmaceutically acceptable adjuvant is one that has been approved or is approvable for human administration by pertinent regulatory bodies. For example, as discussed herein and known in the art, Complete Freund’s adjuvant is not suitable for human administration. Desired adjuvants augment the response to the immunogens without causing conformational changes in the immunogen that might adversely affect the qualitative immune response. Non-limiting examples of suitable adjuvants include aluminum salts, such as alum (potassium aluminum sulfate), or other aluminum containing adjuvants such as aluminum hydroxide, aluminum phosphate, or aluminum sulfate. Other pharmaceutically suitable adjuvants include nontoxic lipid A-related adjuvants such as, by way of non-limiting example, nontoxic monophosphoryl lipid A (see, e.g., Persing et al, Trends Microbiol. 10:s32-s37 (2002)), for example, 3 De-O-acylated monophosphoryl lipid A (MPL) (see, e.g., United Kingdom Patent Application No. GB 2220211). Other useful adjuvants include QS21 and QuilA that comprise a triterpene glycoside or saponin isolated from the bark of the Quillaja saponaria Molina tree found in South America (see, e.g., Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell and Newman, Plenum Press, NY, 1995); U.S. Patent No. 5,057,540). Other suitable adjuvants include oil in water emulsions, optionally in combination with immune stimulants, such as monophosphoryl lipid A (see, e.g., Stoute et al, N. Engl. J. Med. 336, 86-91 (1997)). Other suitable adjuvants include polymeric or monomeric amino acids such as polyglutamic acid or polylysine, liposomes, and CpG (see, e.g., Klinman, Int. Rev. Immunol. 25(3-4): 135-54 (2006); U.S. Patent No. 7,402,572; European Patent No. 772 619).

Vaccine compositions described herein may also comprise a pharmaceutically acceptable (i.e., physiologically suitable or acceptable) excipient(s). Any physiological or pharmaceutically suitable excipient or carrier (i.e., a non-toxic material that does not interfere with the activity of the active ingredient) known to those of ordinary skill in the art for use in pharmaceutical compositions comprising protein-related immunogens may be employed in the vaccine compositions described herein. Exemplary excipients include diluents and carriers that maintain stability and integrity of the component(s) of the composition. Exemplary excipients for inclusion in the compositions described herein include diluents and carriers that maintain stability and integrity of proteins. Excipients for therapeutic use are well known, and are described, for example, in Remington: The Science and Practice of Pharmacy (Gennaro, 21st Ed. Mack Pub. Co., Easton, PA (2005)). The choice of an excipient depends on several factors, including the stability of the immunogenic peptides, polypeptides, or hybrid polypeptides; the route of administration; and the dosing schedule. For example, saline and phosphate buffered saline at physiological pH may be used. The vaccine compositions may also contain other components, which may be biologically active or inactive. Such components include, but are not limited to, carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins (such as albumin), polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, stabilizers, dyes, flavoring agents, and suspending agents and/or preservatives.

The vaccine compositions described herein may be formulated by combining a plurality of immunogenic peptides and/or polypeptides, and/or a plurality of hybrid polypeptides with a pharmaceutically acceptable adjuvant and optionally and preferably at least one pharmaceutically acceptable excipient.

The vaccine compositions may be in the form of a solid, liquid, or gas (aerosol). Alternatively, vaccine compositions described herein may be formulated as a lyophilizate (i.e., a lyophilized composition), or may be encapsulated within liposomes using technology known in the art.

The compositions and preparations described herein may be formulated for any appropriate manner of administration, including, for example, topical, buccal, lingual, oral, intranasal, intrathecal, rectal, vaginal, intraocular, subconjunctival, transdermal, sublingual or parenteral administration, including subcutaneous, intravenous, intramuscular, intrastemal, intracavemous, intrameatal or intraurethral injection or infusion.

For parenteral administration, such as subcutaneous injection or intramuscular injection, the carrier or excipient preferably comprises water, saline, alcohol, a fat, a wax or a buffer, and the vaccine composition is sterile. For oral administration, any of the above excipients or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactic galactide) as well as nanoparticles may also be used as carriers for the compositions described herein. A vaccine composition described herein may be lyophilized or otherwise formulated as a lyophilized product using one or more appropriate excipient solutions (e.g. , sucrose, physiological saline) as diluents upon administration. Nanoparticles may be used to deliver the lyphophilized product and appropriate excipient(s).

The vaccine compositions disclosed herein may be intended for topical administration, such as directly to mucosal tissue, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in a vaccine composition for topical administration (e.g., oral or vaginal). The vaccine compositions described herein may be administered topically by using any one of several delivery vehicles described herein and used in the art, including but not limited to a sponge, gel cap, suppository, gauze (or other suitable fabric for application to the tissue to be treated), nanoparticles, and a lozenge. With respect to certain delivery vehicles, such as a sponge, fabric, or gauze, the composition or preparation is attached to, absorbed by, adsorbed to, or in some manner applied to the vehicle that permits release of the composition upon contact with the tissue to be treated. A vaccine composition disclosed herein may be intended for rectal, oral, or vaginal administration, in the form, e.g., of a suppository or lozenge, which will melt in the rectum, oral, or vaginal space, respectively, and release the components of the composition. A composition described herein that is administered orally may also be in the form of a liquid. The composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.

A method of manufacture of the vaccine compositions described herein is also disclosed herein. Methods of manufacture comprise combining or mixing together an immunogenic peptide, polypeptide, peptide, and/or hybrid polypeptide or the desired plurality of immunogenic peptides, polypeptides and/or hybrid polypeptides, with a pharmaceutically suitable adjuvant and optionally with one or more physiologically suitable (or pharmaceutically acceptable) excipients as described herein.

Vaccine compositions described herein may be used for immunizing a subject (particularly, a human or non-human animal) to induce an immune response against GAS. Hence, a further aspect relates to the aforementioned hybrid polypeptide, nucleic acid, or vaccine composition, for use in the prevention of a GAS infection. A related aspect provides a method for treating a GAS infection in a subject in need thereof, comprising administering to the subject an effective amount of the aforementioned hybrid polypeptide, nucleic acid, or vaccine composition. Also provided is use of the aforementioned hybrid polypeptide, nucleic acid, or vaccine composition, for the manufacture of a medicament for the prevention of a GAS infection.

Hence, the actives taught herein (including immunogenic peptides or polypeptides, optionally hybrid ones, as well as various conjugated or fusion polypeptides, nucleic acids, and any combinations thereof) and vaccine compositions containing such are useful for prophylactic and/or therapeutic treatment of a subject in need thereof who has inadequate immunity to GAS and is susceptible to infection. In addition, induction of secretory or mucosal anti-GAS antibodies in the subject may prevent (i.e., reduce or decrease likelihood of occurrence) initial colonization by GAS.

Subjects or hosts who may be immunized with the vaccine compositions described herein include human and non-human hosts and subjects and hosts. Human subjects/hosts include infants, children, and/or adults. Vaccine compositions suitable for administration to an adult may further be prepared appropriately depending on whether the adult is a young adult, middle-aged, or a senior adult.

Administration may be once, twice, three times, four times, or more times at appropriate time intervals to evoke and maintain the desired anti-GAS immune response. For example, an immunization regimen (i.e., protocol) may include an initial administration (i.e., administration of the primary dose) of a vaccine composition followed by one or more booster immunizations. Booster immunizations may be administered multiple times (e.g., two times or three times or four times or more) at desired time intervals ranging from about 2 weeks to about 26 weeks, such as 2, 4, 8, 12, 16, 20, 24, 26, or 28 week intervals. The time intervals between different doses (e.g., between the primary dose and second dose, or between the second dose and a third dose) may not be the same, and the time interval between each two doses may be determined independently. Additional booster immunizations for prophylaxis of microbial infections, such as GAS infections, may be administered one year or more years after the initial immunization regimen, such as any time between one and ten years after the initial regimen.

The dose of the vaccine composition, the number of doses administered to the subject, and the time intervals between two doses of the composition may be determined by a person skilled in the medical art. An appropriate dose and a suitable duration and frequency of administration will be determined by factors such as the condition of the patient, age of the patient, the type and severity of the patient's disease to be treated or prevented, the particular form of the active ingredient, and the method of administration. In general, an appropriate dose and treatment regimen provides the vaccine composition in an amount sufficient to provide therapeutic and/or prophylactic benefit (e.g., an improved clinical outcome, overall survival, or a lessening of symptom severity). For prophylactic use, a dose should be sufficient to prevent or delay the onset of, and/or to diminish the severity of a GAS infection in a statistically, biologically, or clinically significant manner. Optimal doses may generally be determined using experimental in vitro assays, in vivo animal models, and/or human clinical trials. The optimal dose may depend upon the body mass, weight, or blood volume of the subject. The appropriate amount of each active in the vaccine composition administered to the subject may depend upon the subject’s or patient's (e.g., human's) condition, that is, stage of the disease, general health status, as well as age and weight, and other factors familiar to a person skilled in the medical art. Without limitation, the amount of each active present in a dose, may range from about 10 pg to about 10 mg; such as from about 10 pg to about 100 pg, such as about 10 pg, about 20 pg, about 30 pg, about 40 pg, about 50 pg, about 60 pg, about 70 pg, about 80 pg, about 90 pg, or about 100 pg; or from about 100 pg to 1 mg, such as about 100 pg, about 200 pg, about 300 pg, about 400 pg, about 500 pg, about 600 pg, about 700 pg, about 800 pg, about 900 pg, or about 1 mg; or from about 1 mg to about 10 mg, such as about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, or about 10 mg. When administered in a liquid form, suitable dose sizes may vary with the size (i.e., weight or body mass) of the patient, but will typically range from about lOOpl to about 1 ml, such as about lOOpl, about 200pl, about 300pl, about 400pl, about 500pl, about 600pl, about 700pl, about 800pl, about 900pl, or about 1 ml (comprising an appropriate dose) for a 10-100 kg subject. The use of the minimum dosage that is sufficient to provide effective therapy and/or prophylaxis is usually preferred. Patients may generally be monitored for therapeutic or prophylactic effectiveness using assays suitable for the condition being treated or prevented, which assays will be familiar to those having ordinary skill in the art and are described herein. For example, to monitor the immune response of an immunized subject during pre- clinical studies in animals, sera is obtained from the animals prior to the first dose (i.e., pre-immune sera) and obtained after the final boosting dose. Sera may also be obtained after any one or more of the boosting doses between the primary dose and final boosting dose. To monitor the immune response of an immunized subject during clinical studies or during post-marketing studies, sera may also be obtained from humans before the first immunization and after one or more administrations of the vaccine compositions. The vaccine compositions may be administered in a manner appropriate to the disease to be treated (or prevented) as determined by persons skilled in the medical art. For example, the vaccine compositions described herein may be administered via a route including oral, enteral, parenteral, transdermal/transmucosal, and inhalation. The term enteral, as used herein, is a route of administration in which the agent is absorbed through the gastrointestinal tract or oral mucosa, including oral, rectal, and sublingual. The term parenteral, as used herein, describes administration routes that bypass the gastrointestinal tract, and are typically administered by injection or infusion, including intraarterial, intradermal, subdermal, intramuscular, intranasal, intraocular, intraperitoneal, intravenous, subcutaneous, submucosal, intravaginal, intrastemal, intracavemous, intrathecal, intrameatal, and intraurethral injection. The term transdermal/transmucosal, as used herein, is a route of administration in which the agent is administered through or by way of the skin, including topical. The term inhalation encompasses techniques of administration in which an agent is introduced into the pulmonary tree, including intrapulmonary or transpulmonary and includes intranasal administration. In particular embodiments, the vaccine compositions described herein may be administered orally, intramuscularly, or intranasally. All doses of the vaccine compositions may not necessarily be administered by the same route. In certain embodiments, different doses of the vaccine compositions may be delivered by different routes, such as by two or more of oral, intramuscular, and intransal routes.

GAS serotypes targeted by the actives in vaccine compositions described herein include those that cause non-invasive infections (e.g., pharyngitis, impetigo, erysipelas, and cellulitis) and GAS serotypes that cause invasive infections (e.g., GAS infections of the blood (bacteremia), muscle, and lung (pneumonia), necrotizing fasciitis, and streptococcal toxic shock syndrome), and nonsuppurative sequelae such as acute rheumatic fever, reactive arthritis, and glomerulonephritis. In embodiments, the vaccine compositions described herein may be used for the prevention and/or treatment of a GAS infection that causes any one of the following: pharyngitis, scarlet fever, necrotizing fasciitis, cellulitis, meningitis, pneumonia, streptococcal toxic shock syndrome, bacteremia, septicemia, septic arthritis, pyoderma, skin infections (invasive and non-invasive), impetigo, erysipelas, soft-tissue infection, nephritis, and GAS pyrogenic reaction. The vaccine compositions described herein may also be used for the prevention and/or treatment of a GAS infection that causes nonsuppurative sequelae such as acute rheumatic fever, rheumatic heart disease, reactive arthritis, and glomerulonephritis.

Immunized subjects may be monitored for therapeutic or prophylactic effectiveness using assays suitable for the infection or condition being treated or prevented, which assays will be familiar to those having ordinary skill in the art and which are described herein. The immune response evoked by administering the vaccine compositions described herein e.g. according to the methods described above comprises an adaptive immune response that includes a humoral response and may also include a cellular response (which comprises a CD4 immune response and a CD 8 immune response) specific for each immunogenic peptide or polypeptide represented in the vaccine composition. The humoral immune response (i.e., antibody response) can be monitored throughout an immunization protocol using, for example, immunoassays (e.g., ELISA, immunoblotting), in vitro functional assays (e.g., opsonic, phagocytic and killing assays, indirect bactericidal assays) and the like. Such methods are useful for monitoring and determining the level of binding (i.e., titer) of specific antibodies present in a biological sample (e.g., a blood sample (from which serum or plasma may be prepared), a biopsy specimen, body fluids (e.g., lung lavage, ascites, mucosal washings, synovial fluid), bone marrow, lymph nodes, tissue explant, organ culture, or any other tissue or cell preparation from the subject) from an immunized subject. Based on the results from one or more of these assays, the dose or timing of the next dose or the necessity for an additional dose may be determined. A cell- mediated immune response involves various types of T cells (i.e., T lymphocytes). In a cell mediated response, T cells act to eliminate an antigen by a number of mechanisms. For example, helper T cells that are capable of recognizing specific antigens may respond by releasing soluble mediators such as cytokines to recruit additional cells of the immune system to participate in an immune response. Also, cytotoxic T cells are capable of specifically recognizing an antigen and may respond by binding to and destroying or damaging an antigen-bearing cell, such as a GAS bacterial cell. Assays routinely practiced in the art to examine a cellular immune response include determining the presence and level of soluble mediators such as cytokines, lymphokines, chemokines, hormones, growth factors, as well as other mediators. Immunoassays also include determining cellular activation state changes of immune cells by analyzing altered functional or structural properties of the immune cells, for example, cell proliferation, altered motility, induction of specialized activities such as specific gene expression or cytolytic behavior; cell maturation, and alteration in relationship between a Thl response and a Th2 response. Procedures for performing these and similar assays are may be found, for example, in Lefkovits (Immunology Methods Manual: The Comprehensive Sourcebook of Techniques, 1998). See also Current Protocols in Immunology; Weir, Handbook of Experimental Immunology, Blackwell Scientific, Boston, MA (1986); Mishell and Shigii (eds.) Selected Methods in Cellular Immunology, Freeman Publishing, San Francisco, CA (1979); Green and Reed, Science 281 : 1309 (1998) and references cited therein). Determining the effectiveness of immunization with the vaccine compositions described herein may also include clinical evaluation. By way of example, the presence of a GAS infection may be determined by performing routine assays (bacteria cell culture; immunofluorescence assays) available to the clinician to determine quickly if streptococci are present in a body fluid or at a site on the body, such as the throat, mucosal tissue, or skin. Symptomatology, such as fever, inflammation, pain, and various other and numerous symptoms of GAS infections can be monitored by persons skilled in the clinical art. The present application also provides aspects and embodiments as set forth in the following Statements. In these statements, the wording “The [subject] according to Statement [number], wherein... ” or “The [subject] according to any one of Statements [numbers], wherein... ” also discloses and may be replaced by the simple wording “In certain embodiments. . . ”.

Statement 1. An immunogenic peptide or polypeptide from a group A Streptococcus (GAS) Enn protein, for use in the prevention of a GAS infection.

Statement 2. The immunogenic peptide or polypeptide for use according to Statement 1, wherein the immunogenic peptide or polypeptide comprises at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of the hypervariable region (HVR) of the Enn protein.

Statement 3. The immunogenic peptide or polypeptide for use according to Statements 1 or 2, wherein the immunogenic peptide or polypeptide comprises the hypervariable region (HVR) of the Enn protein.

Statement 4. An immunogenic peptide or polypeptide for use in the prevention of a GAS infection, wherein the immunogenic peptide or polypeptide: is from, or preferably comprises at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95% identical to the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96; or comprises the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96.

Statement 5. The immunogenic peptide or polypeptide for use according to Statement 4, wherein the immunogenic peptide or polypeptide: is from, or preferably comprises at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96; or the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96.

Statement 6. The immunogenic peptide or polypeptide for use according to Statement 4 or 5, wherein the immunogenic peptide or polypeptide is selected from the group consisting of the peptides as set forth in SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96. Statement 7. A hybrid peptide or polypeptide, or an immunogenic composition, comprising at least two immunogenic peptides or polypeptides selected from the group consisting of: an immunogenic peptide or polypeptide from a GAS Enn protein; an immunogenic peptide or polypeptide which is from, or preferably comprises at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95% identical to the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96, or comprising the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96; and mixtures thereof; wherein the hybrid peptide or polypeptide, or the immunogenic composition, is capable of conferring host immunity to an infection by a group A Streptococcus (GAS).

Statement 8. A hybrid peptide or polypeptide, or an immunogenic composition, comprising at least one immunogenic peptide or polypeptide selected from an immunogenic peptide or polypeptide from a GAS Enn protein; an immunogenic peptide or polypeptide which is from, or preferably comprises at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95% identical to the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96, or comprising the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96; and mixtures thereof; and further comprising at least one other GAS immunogen, optionally selected from the group consisting of an immunogenic peptide from a GAS M protein, an immunogenic peptide from a GAS Mrp protein, and mixtures thereof; wherein the hybrid peptide or polypeptide, or the immunogenic composition, is capable of conferring host immunity to an infection by a group A Streptococcus (GAS).

Statement 9. The hybrid peptide or polypeptide, or the immunogenic composition, according to Statements 7 or 8, wherein: the immunogenic peptide or polypeptide, or the at least two immunogenic peptides or polypeptides each independently, is from, or preferably comprises at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of the hypervariable region (HVR) of the Enn protein; the immunogenic peptide or polypeptide, or the at least two immunogenic peptides or polypeptides each independently, comprises the hypervariable region (HVR) of the Enn protein; the immunogenic peptide or polypeptide, or the at least two immunogenic peptides or polypeptides each independently, is from, or preferably comprises at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96; or the immunogenic peptide or polypeptide, or the at least two immunogenic peptides or polypeptides each independently, comprises the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96.

Statement 10. The hybrid peptide or polypeptide, or the immunogenic composition, according to any one of Statements 7 to 9, wherein:

A) the at least two immunogenic peptides or polypeptides are from different GAS Enn proteins;

B) the at least two immunogenic peptides or polypeptides are from GAS Enn proteins belonging to different GAS Enn vaccine clusters selected from vaccine clusters VE1, VE2, VE3, VE4, VE5, VE6, VE7, VE8, VE9, and VE10;

C) the at least two immunogenic peptides or polypeptides are from different subgroups a) to j), wherein subgroup a) consists of immunogenic peptides and polypeptides which are from, or preferably comprise at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95%, identical to the amino acid sequence of any one of SEQ ID NO: 1 to 15, preferably SEQ ID NO: 4 or 9, subgroup b) consists of immunogenic peptides and polypeptides, which are from, or preferably comprise at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95%, identical to the amino acid sequence of any one of SEQ ID NO: 16 to 20, preferably SEQ ID NO: 17, subgroup c) consists of immunogenic peptides and polypeptides, which are from, or preferably comprise at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95%, identical to the amino acid sequence of any one of SEQ ID NO: 21 to 37, preferably SEQ ID NO: 35, subgroup d) consists of immunogenic peptides and polypeptides, which are from, or preferably comprise at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95%, identical to the amino acid sequence of any one of SEQ ID NO: 38 to 39, preferably SEQ ID NO: 38, subgroup e) consists of immunogenic peptides and polypeptides, which are from, or preferably comprise at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95%, identical to the amino acid sequence of any one of SEQ ID NO: 40 to 43, preferably SEQ ID NO: 40, subgroup f) consists of immunogenic peptides and polypeptides, which are from, or preferably comprise at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95%, identical to the amino acid sequence of any one of SEQ ID NO: 44 to 63, preferably SEQ ID NO: 53 or 60, subgroup g) consists of immunogenic peptides and polypeptides, which are from, or preferably comprise at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95%, identical to the amino acid sequence of any one of SEQ ID NO: 64 to 65, preferably SEQ ID NO: 64, subgroup h) consists of immunogenic peptides and polypeptides, which are from, or preferably comprise at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95%, identical to the amino acid sequence of any one of SEQ ID NO: 66 to 78, preferably SEQ ID NO: 76, subgroup i) consists of immunogenic peptides and polypeptides, which are from, or preferably comprise at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95%, identical to the amino acid sequence of any one of SEQ ID NO: 79 to 89, preferably SEQ ID NO: 86, and subgroup j) consists of immunogenic peptides and polypeptides, which are from, or preferably comprise at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95%, identical to the amino acid sequence of any one of SEQ ID NO: 90 to 101, preferably SEQ ID NO: 96; or

D) the at least two immunogenic peptides or polypeptides are from different subgroups a*) to j*), wherein subgroup a*) consists of immunogenic peptides and polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 1 to 15, preferably SEQ ID NO: 4 or 9, subgroup b*) consists of immunogenic peptides and polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 16 to 20, preferably SEQ ID NO: 17, subgroup c*) consists of immunogenic peptides and polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 21 to 37, preferably SEQ ID NO: 35, subgroup d*) consists of immunogenic peptides and polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 38 to 39, preferably SEQ ID NO: 38, subgroup e*) consists of immunogenic peptides and polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 40 to 43, preferably SEQ ID NO: 40, subgroup f*) consists of immunogenic peptides and polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 44 to 63, preferably SEQ ID NO: 53 or 60, subgroup g*) consists of immunogenic peptides and polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 64 to 65, preferably SEQ ID NO: 64, subgroup h*) consists of immunogenic peptides and polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 66 to 78, subgroup i*) consists of immunogenic peptides and polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 79 to 89, preferably SEQ ID NO: 76, and subgroup j*) consists of immunogenic peptides and polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 90 to 101, preferably SEQ ID NO: 96.

Statement 11. The hybrid peptide or polypeptide, or the immunogenic composition, according to any one of Statements 7 to 10, comprising at least two peptides selected from the group consisting of the peptides as set forth in SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96. Statement 12. A nucleic acid molecule encoding the hybrid peptide or polypeptide according to any one of Statements 7 to 11, wherein the hybrid polypeptide is a fusion polypeptide.

Statement 13. A vaccine composition comprising a pharmaceutically acceptable adjuvant and or more of:

(i) an immunogenic peptide or polypeptide from a GAS Enn protein;

(ii) an immunogenic peptide or polypeptide which is from, or preferably comprises at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95% identical to the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96, or comprising the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96;

(iii) the hybrid peptide or polypeptide, or the immunogenic composition, according to any one of Statements 7 to 11 ; or

(iv) the nucleic acid molecule according to Statement 12, wherein the vaccine composition is capable of conferring host immunity to an infection by a group A Streptococcus (GAS).

Statement 14. The vaccine composition according to Statement 13, wherein: the immunogenic peptide or polypeptide is from, or preferably comprises at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of the hypervariable region (HVR) of the Enn protein; the immunogenic peptide or polypeptide comprises the hypervariable region (HVR) of the Enn protein; the immunogenic peptide or polypeptide is from, or preferably comprises at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96; or the immunogenic peptide or polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96.

Statement 15. The hybrid polypeptide according to any one of Statements 7 to 11, the nucleic acid molecule according to Statement 12, or the vaccine composition according to Statement 13 or 14, for use in the prevention of a GAS infection. While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing description. Accordingly, it is intended to embrace all such alternatives, modifications, and variations as follows in the spirit and scope of the appended claims.

The herein disclosed aspects and embodiments of the invention are further supported by the following non-limiting examples.

EXAMPLES

Example 1: Typing systems for GAS based on Enn protein

The present inventors teach typing of group A Streptococcus (GAS) based on the hypervariable region (HVR) of the Enn protein. Moreover, the inventors demonstrate in later examples that Enn proteins, in particular HVR thereof, can function as useful target antigens in anti -GAS vaccines. Consequently, Enn protein-based GAS typing and grouping the bacteria into clusters can inform vaccine design, since a representative antigen selected from a given Enn GAS cluster can provide protection more broadly against bacteria belonging to that cluster (and, as demonstrated by the inventors, even inter-cluster protection, especially within the same clade). Multivalent antigen designs, including one or more antigens per cluster selected from two or more distinct Enn GAS clusters, are thus also informed by the inventors’ findings. As explained below, the inventors identified a way of Enn protein-based typing of GAS into vaccine clusters VE1-3 (belonging to group 1) and VE4-10 (belonging to group 2). This typing scheme can be used in Enn antigen-based anti- GAS vaccine design, especially when designing multivalent vaccines including antigens from two or more of such clusters or subgroups.

The enn genes from a genetically diverse collection of 1668 contiguous Mga regulons were extracted based on genetic probes, open reading frame predictions and sequence similarity to published gene sequences. Mature sequences were generated by in silico removal of the signal peptide up to the cleavage site and from the glycine residue of the 99 LPXTG-sortase motif, used to attach the protein to the bacterial surface. The final database contained 275 Enn protein sequences.

Multiple sequence alignments (MSA) of unique protein sequences were generated using MAFFT version 7.311 using the G-INS-i method which performs global alignments using the Needleman- Wunsch algorithm (Katoh et al. Nucleic Acids Res. 2005, vol. 33(2), 511-8). The Enn protein MSA had 487 sites (ungapped length mean = 287.9; Std Dev = 17.0) including 92 complete sites, 48 variable sites and 40 informative sites. Outliers (13 Enn proteins) were detected based on divergence in MSA and removed. The sequences of the hypervariable regions of the 262 Enn proteins, from N- to C-terminus, are shown in Table 1.

Table 1. Sequences of Enn HVR. Sequence analyses of 262 Enn mature proteins indicated two groups of structurally related Enn. A systematic Enn nomenclature was established, in which each unique protein sequence has a numerical identifier (e.g., Ennl), and any allele that produces the same protein sequence is named as a subtype (e.g., Enn 1.1).

Classification into group 1 and 2 and clusters VE1-3 and VE4-10

The alignment of the 262 Enn protein sequences was analysed to infer maximum likelihood (ML) phylogenetic trees, whereby clusters and individual proteins that were strongly phylogenetically related (approximate likelihood-ratio test, aLRT, value >80%) were combined or separated into clusters with an average pairwise identity of >85% and a minimum pairwise identity of >75%. Clusters were numbered based on phylogenetic distance. The phylogeny revealed 2 distinct phylogenetic groups (group 1 and 2) with 10 distinct vaccine clusters, 3 in group 1 (VE1-3) and 7 vaccine clusters in group 2 (VE4-10) - see Table 1. The sequence relationships within these clusters are captures in Table 2.

Table 2. Sequence relationship within Enn protein Vaccine clusters VE1-3 and VE4-10

8 representative Enn proteins were chosen based on the Vaccine cluster (VE) they belong to. The 8 enn genes were expressed as recombinant proteins and by co-purification in human serum it was shown that these 8 proteins have different profiles of C4BP, albumin, vitronectin and human IgG binding, indicating that the Enn Vaccine clusters reflect a functional classification (Fig. 4). Example 2 - Materials and methods used in Examples 3-5

Peptide antigens

50-amino acid peptide test antigens (denoted Enn antigens GAS001, GAS002, and EA1-10) were custom synthesized by Proteogenix (France) using solid-phase peptide synthesis, desalted and supplied at 85% purity. The test antigens contained the hypervariable regions (HVR) of Enn proteins selected - based inter alia on a ‘heptad-based’ score reflecting the conservation of some key residues in the coil-coiled structure (Aranha et al. The Journal of Biological Chemistry 2020, vol. 295 (12), 3826-36) - from each of the VE1-3 and VE4-10 Enn vaccine clusters. The amino acid sequences of the peptide test antigens, from N- to C-terminus, are shown in Table 3. Table 3: Enn HVR peptides used for immunisation

Immunization

One New Zealand white rabbit per peptide (Proteogenix, France) was immunized intramuscularly with 200 pg of the Enn HVR GAS001, GAS002, or EA1-10 (the latter series coupled to Keyhole limpet hemocyanin (KLH)), mixed to Alun hydroxide 2% (wt/wt) at 0, 2, 4 and 6 weeks. Serum was obtained 2 weeks after the final immunization. ELISA was performed with synthetic Enn peptides.

Immunization of 10 New Zealand white rabbit (1/per cluster) (Proteogenix, France) with 200 pg of the EnnHVR peptide vaccine, coupled to KLH, and mixed to Alun hydroxide 2% (wt/wt) at 0, 2, 4 and 6 weeks. Serum was obtained 2 weeks after the final immunization. ELISA was performed with synthetic Enn peptides (HVR).

Enzyme linked immunosorbent assays (ELISA)

1) Rabbit ELISA

An enzyme-linked immunosorbent assay was used to measure specific antibody in pre-immune and immune rabbit sera. Peptides (5 pg/ml) were bound to flat-bottomed microtiter wells (Nunc-Immuno modules; Nalge Nunc International, Roskilde, Denmark) in 0.1 M sodium carbonate, pH 9.8 (100 pl/well), overnight at 4°C. Excess peptide was removed, and wells were washed three times with 0.15 M NaCl containing 0.05% Tween 20 (PBS/TW). Blockage was performed by adding 200 pl of Phosphate Buffered Saline (PBS)-Tween (PBS/TW) with 3% pig and 2% goat serum for 1 hour. Rabbit sera were serially diluted from 1:200 in PBS/TW. Diluted sera (2 -fold dilution) were added to wells (100 pl/well) and incubated at 37°C for 1 h. Unbound primary antibody was removed, and the wash step was repeated. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (Tebu-bio BA1054-1) was diluted 1:2,500 in PBS/TW, added to the wells (100 pl/well), and incubated at 37°C for 1 h. After removal of unbound secondary antibody and washing, chromogenic substrate (3,3 ’,5,5 ’-tetramethylbenzidine (TMB)) was added (100 pl/well). Substrate was allowed to develop for 20 min. Serum antibody titre was defined as the reciprocal of the highest dilution of serum which yielded an absorbance of 0.1 at 450 nm.

2) Human ELISA

An enzyme-linked immunosorbent assay was used to measure specific antibody in human sera. Peptides (5 pg/ml) were bound to flat-bottomed microtiter wells (Nunc-Immuno modules; Nalge Nunc International, Roskilde, Denmark) in 0.1 M sodium carbonate, pH 9.8 (100 pl/well), overnight at 4°C. Excess peptide was removed, and wells were washed three times with 0.15 M NaCl containing 0.05% Tween 20 (PBS/TW). Blockage was performed by adding 200 pl of PBS-Tween with 3% pig and 2% goat serum for 1 hour. Human sera were serially diluted from 1 : 100 in PBS/TW. Diluted sera (2-fold dilution) were added to wells (100 pl/well) and incubated at 37°C for 1 h. Unbound primary antibody was removed, and the wash step was repeated. HRP -conjugated goat antihuman immunoglobulin G (Abeam Ab6858) was diluted 1:2,500 in PBS/TW, added to wells (100 pl/well), and incubated at 37°C for 1 h. After removal of unbound secondary antibody and washing, TMB was added (100 pl/well). Substrate was allowed to develop for 20 min. Serum antibody titre was defined as the reciprocal of the highest dilution of serum which yielded an absorbance of 0. 1 at 450 nm.

Measurement of antibodies-dependent cellular phagocytosis

One milligram of each peptides was biotinylated and incubated with FITC beads during 2hours at 37°C. After two washes, beads were resuspended in PBS. Antibodies from rabbit sera were purified using MelonGel kit. Purified antibodies were mixed with peptides-coated beads in a 1/10 ratio and incubated 2 hours at 37°C. Immune complexes were then incubated with HL-60 cells (final concentration 2500.000 cells/ml) for 1 hour at 37°C. Anti-CD35 AF647 and anti-CD71 BV421 in PBS-BSA were added to each well. After Ih at 4°C, cells were washed and fixed in 2% PFA before flow cytometry analysis. The assays were measured with BD X20 Fortessa flow cytometer. Neutrophil bead internalization was quantified using FlowJo (FlowJo, LLC) software. A minimum of 2000 cells were acquired and a phagocytic-score (phagoscore) was calculated for each sample comprising of the percent neutrophils that had taken up beads multiplied by the fluorescent signal of beads taken up by the neutrophils (geometric mean fluorescence intensity (gMFI) of (bead+ neutrophils).

Example 3: Immunogenicity of Enn HVR peptides

Proof of concept study

2 Enn peptides (GAS001 and GAS002) derived from the HVR region of Enn proteins belonging to VE1 and VE6 cluster, respectively, were injected in rabbits using Alun as adjuvant. Table 4 shows antibody titres against the peptides EA2, EA8, and against the control peptides Enn 336 (VE10, SEQ ID NO: 91) and Enn 62 (VE3, SEQ ID NO: 34) in the respective rabbit sera, as assessed by ELISA with synthetic Enn peptides. No cross-recognition inter-cluster was observed in this experiment.

Table 4: Anti-Enn peptide antibody titres in rabbits injected with GAS001 (rabbit 1) or GAS002 (rabbit 2) (ELISA)

Systematic immunisation study

Additional 10 Enn peptides (EA1-10) derived from the HVR region of Enn proteins belonging to each one of the VE1-3 and VE4-10 vaccine clusters, and coupled to KLH, were injected in rabbits using Alun as adjuvant. All injected Enn HVR peptides were immunogenic in rabbits, providing for antibody titres between 25,600 and 51,200. Fig. 1 shows anti-Enn antibody titres in the rabbits injected with peptides EA1-10 (identified by reference to their respective Enn clusters, see Table 1), as assessed by ELISA with synthetic Enn peptides. Cross-recognition inter-cluster was observed.

Detection of full-length Enn proteins

Table 5 shows antibody titres of rabbit serum immunised with a VE1 vaccine cluster Enn peptide HVR antigen (EA1) against full-length proteins from different Enn vaccine clusters (N=3). The data shows that antibodies against Enn HVR can recognise full-length Enn proteins, and cross-recognition inter-vaccine cluster is observed.

Table 5. Anti -full-length Enn antibody titres in rabbit VE1 serum (ELISA)

Example 4: Detection of anti-Enn antibodies in human plasma

ELISA analysis showed that human sera harbour high levels of natural antibodies against Enn74 (cluster VE1, peptide GAS001), Ennl99 (cluster VE6, peptide GAS002), Enn336 (cluster VE10), Enn62 (cluster VE3), and Enn232 (cluster VE5) (Table 6). Hence, anti-Enn antibodies are raised during natural infection with GAS in human hosts.

Table 6. Anti-Enn antibody titres in human donors (ELISA)

Example 5: Phagocytosis of anti-Enn HVR antibody-coated beads

To show the ability of the antibodies raised against the HVR of Enn to induce antibodies-dependent cellular phagocytosis, a fluorescent bead-based assay was conducted. Enn HVR antigens from across the various Enn vaccine clusters were coupled to fluorescent beads and incubated with anti-Enn HVR antibodies raised against the VE1 Enn HVR peptide (EA1). Insofar the antibodies bound to the various antigens, this produced beads decorated with the anti-VEl Enn HVR antibodies. Subsequently, the beads were incubated in vitro with phagocytes, and flow cytometry was used to detect the uptake of the fluorescent beads into the phagocytes. The results are shown in Fig. 2. These results demonstrate that anti-Enn HVR antibodies bound to HVR antigen-coated beads can allow phagocytosis of the beads, and that anti-VEl Enn HVR antibodies are able to cause crossphagocytosis of beads coupled to Enn HVR antigens from non-VEl clusters.

Example 6: Multivalent Enn HVR antigens

Genetic constructs encoding certain embodiments of multivalent Enn HVR antigens, as shown in Fig. 3 (identified by reference to their respective Enn vaccine clusters), are prepared by arranging the coding sequences for the mentioned HVR antigens in-frame in a single nucleotide sequence, operably linked to a promoter suitable for expression of the construct in a host cell, such as a bacterial cell, such as E. coli. A C-terminal polyhistidine tag (6xHis-tag) is genetically fused to the recombinant protein, to allow metal ion affinity-based purification of the expressed protein. Endo- or exopeptidases can be used to remove the 6xHis-tag as generally known in the art.

Example 7: Distinct functional roles of emm and enn proteins

Bacteria were grown in THY medium, washed and either incubated in presence of pharyngeal Detroit 562 cells or in blood. Bacterial RNA was extracted and DNase-digested RNAs were reverse- transcribed in complementary DNA (cDNA). The cDNAs were quantified in a CFX96 with SYBR Green and gene-specific primers for emm, enn, mrp, mga and recA (control). RT-qPCR data for each gene are presented as a fold change of expression in the different conditions compared to expression in THY medium and normalized with the expression of the housekeeping recA gene. The experiment demonstrates that M-like genes were all expressed and this expression was differentially regulated (Fig. 5). This evidences that emm, enn, and mrp genes have different functions and that enn and mrp genes do not serve as merely a genetic reservoir for emm diversity.

For whole cell binding experiments, bacteria were grown in THY medium, washed and incubated in presence of 100% human serum for 2h at room temperature (RT). Bacteria were centrifuged and washed with PBS + Tween20 0.05%. Serum proteins were then eluted in SDS-PAGE loading buffer with rotation for 15min at RT. Whole cell binding samples were then resolved on SDS-PAGE and either stained with Coomassie blue or western blotting were performed with antibodies specific to complement component 4 binding protein (C4BP), Albumin, IgG and Fibrinogen. Knock out mutants of enn, mrp and emm genes in a M25 strain did not present the same phenotype in term of binding capacities (Fig. 6A). Mrp was mainly binding fibrinogen and IgG while Enn bound C4BP and Emm albumin. Additionally, bactericidal assays were performed using whole, non-immune human blood. After 3h of incubation, bacteria were plated on THY agar plates and incubated O/N at 37°C + 5% CO2. Percentage of survival was calculated using the formula: [(total CFU of the tested strain (WT or Aenn mutant)/total CFU of the WT strain) x 100], Data were reported as the average percent survival +/- the standard deviation calculated after averaging the CFU in the four control samples. Fig. 7 shows that knock-out mutant of enn has very reduced survival in whole blood even in presence of M and Mrp proteins at its surface.

Together these data underscore that Emm, Enn, and Mrp proteins have distinct functional roles.

Example 8: C4BP-binding motif in Enn

We have also identified the motif in Enn responsible for C4BP binding using copurification experiments between C4BP and an N-terminal deletion mutant or a number of point mutants of Enn314. For these copurification experiments, tandem GST-Enn-His-tagged proteins were expressed in E. coli and purified on Cobalt resin (His-tag). GST was removed on-resin using thrombin. Serum or recombinant C4BP (rC4BP) were then incubated with the resin overnight (O/N) at 4°C with rotation. After several washing steps, proteins were eluted in SDS-PAGE loading buffer with rotation for lOmin at RT. Copurification samples were then resolved on SDS-PAGE and either stained with Coomassie blue or a western blotting was performed with antibodies specific to C4BP. As shown in Fig. 6B the C4BP-binding motif was part of the HVR region that embodiments of the invention employ as an antigen for raising anti -Enn antibodies. Antibodies directed to HVR can thus be particularly advantageous as they may interfere with the Enn-C4BP binding.

Claims

1. An immunogenic peptide or polypeptide from a group A Streptococcus (GAS) Enn protein, for use in the prevention of a GAS infection.

2. The immunogenic peptide or polypeptide for use according to claim 1, wherein the immunogenic peptide or polypeptide comprises at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of the hypervariable region (HVR) of the Enn protein.

3. The immunogenic peptide or polypeptide for use according to claim 1 or 2, wherein the immunogenic peptide or polypeptide comprises the hypervariable region (HVR) of the Enn protein.

4. An immunogenic peptide or polypeptide for use in the prevention of a GAS infection, wherein the immunogenic peptide or polypeptide: is from, or preferably comprises at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95% identical to the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96; or comprises the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96.

5. The immunogenic peptide or polypeptide for use according to claim 4, wherein the immunogenic peptide or polypeptide: is from, or preferably comprises at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96; or the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96.

6. The immunogenic peptide or polypeptide for use according to claim 4 or 5, wherein the immunogenic peptide or polypeptide is selected from the group consisting of the peptides as set forth in SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96.

7. A hybrid peptide or polypeptide, or an immunogenic composition, comprising at least two immunogenic peptides or polypeptides selected from the group consisting of: an immunogenic peptide or polypeptide from a GAS Enn protein; an immunogenic peptide or polypeptide which is from, or preferably comprises at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95% identical to the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96, or comprising the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96; and mixtures thereof; wherein the hybrid peptide or polypeptide, or the immunogenic composition, is capable of conferring host immunity to an infection by a group A Streptococcus (GAS).

8. A hybrid peptide or polypeptide, or an immunogenic composition, comprising at least one immunogenic peptide or polypeptide selected from an immunogenic peptide or polypeptide from a GAS Enn protein; an immunogenic peptide or polypeptide which is from, or preferably comprises at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of an amino acid sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95% identical to the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96, or comprising the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96; and mixtures thereof; and further comprising at least one other GAS immunogen, optionally selected from the group consisting of an immunogenic peptide from a GAS M protein, an immunogenic peptide from a GAS Mrp protein, and mixtures thereof; wherein the hybrid peptide or polypeptide, or the immunogenic composition, is capable of conferring host immunity to an infection by a group A Streptococcus (GAS).

9. The hybrid peptide or polypeptide, or the immunogenic composition, according to claim 7 or 8, wherein: the immunogenic peptide or polypeptide, or the at least two immunogenic peptides or polypeptides each independently, is from, or preferably comprises at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of the hypervariable region (HVR) of the Enn protein; the immunogenic peptide or polypeptide, or the at least two immunogenic peptides or polypeptides each independently, comprises the hypervariable region (HVR) of the Enn protein; the immunogenic peptide or polypeptide, or the at least two immunogenic peptides or polypeptides each independently, is from, or preferably comprises at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96; or the immunogenic peptide or polypeptide, or the at least two immunogenic peptides or polypeptides each independently, comprises the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96.

10. The hybrid peptide or polypeptide, or the immunogenic composition, according to any one of claims 7 to 9, wherein:

11. The hybrid peptide or polypeptide, or the immunogenic composition, according to any one of claims 7 to 10, comprising at least two peptides selected from the group consisting of the peptides as set forth in SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96.

12. A nucleic acid molecule encoding the hybrid peptide or polypeptide according to any one of claims 7 to 11, wherein the hybrid polypeptide is a fusion polypeptide.

13. A vaccine composition comprising a pharmaceutically acceptable adjuvant and one or more of:

(i) an immunogenic peptide or polypeptide from a GAS Enn protein;

(iii) the hybrid peptide or polypeptide, or the immunogenic composition, according to any one of claims 7 to 11 ; or

(iv) the nucleic acid molecule according to claim 12, wherein the vaccine composition is capable of conferring host immunity to an infection by a group A Streptococcus (GAS).

14. The vaccine composition according to claim 13, wherein: the immunogenic peptide or polypeptide is from, or preferably comprises at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of the hypervariable region (HVR) of the Enn protein; the immunogenic peptide or polypeptide comprises the hypervariable region (HVR) of the Enn protein; the immunogenic peptide or polypeptide is from, or preferably comprises at least 25 contiguous amino acids, preferably at least 40 contiguous amino acids, of the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96; or the immunogenic peptide or polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 1 to 101, preferably any one of SEQ ID NO: 4, 9, 17, 35, 38, 40, 53, 60, 64, 76, 86, and 96.

15. The hybrid polypeptide according to any one of claims 7 to 11, the nucleic acid molecule according to claim 12, or the vaccine composition according to claim 13 or 14, for use in the prevention of a GAS infection.

16. The immunogenic peptide or polypeptide for use according to any one of claims 1 to 6, or the hybrid polypeptide, the nucleic acid molecule, or the vaccine composition for use according to claim 15, wherein the GAS belongs to emm cluster D4.