Protein-based vaccine composition for protection against streptococcus suis
Patent Information
- Application Number
- PCT/CA2026/050551
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2025-04-14
- Filing Date
- 2026-04-13
- Publication Date
- 2026-10-22
Smart Images
Figure CA2026050551_22102026_PF_FP_ABST
Abstract
Description
PROTEIN-BASED VACCINE COMPOSITION FOR PROTECTION AGAINST STREPTOCOCCUS SUIS CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of Great Britain patent application serial number 2505606.0 filed on April 14, 2025. The entire contents of the abovereferenced documents are incorporated herein by reference in their entirety.TECHNICAL FIELD
[0002] The present invention relates generally to the field of immunology and veterinary medicine, and more particularly to protein-based immunogenic or vaccine compositions for protection against Streptococcus suis.SEQUENCE LISTING STATEMENT
[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format in accordance with WIPO Standard ST.26 and is hereby incorporated by reference in its entirety. Said XML file, created on March 11, 2026, is named " SaoVaccineSeqListing.xml" and is 125 kilobytes in size. The information in the electronic format of the Sequence Listing is part of the present application and is incorporated herein by reference in its entirety.BACKGROUND
[0004] Streptococcus suis (S. suis) is a Gram-positive bacterial pathogen of global significance that poses substantial food production and public health challenges. In swine populations, S. suis infection causes severe systemic diseases including meningitis, septicemia, endocarditis, polyarthritis, and septic shock, with high morbidity and mortality rates. The diversity of capsular polysaccharide (CPS) antigens has led to the identification of more than 30 serotypes of S. suis, of which serotype 2 is the most virulent and most frequently isolated from clinical cases. Beyond its veterinary impact, S. suis serotype 2 is recognized as an emerging zoonotic pathogen, causing severe invasive infections (such as, sepsis and meningitis) in humans with occupational exposure to swine or uncooked pork products.
[0005] Vaccination is widely recognized as a critical strategy for controlling bacterial infectious diseases in livestock. Despite decades of research, no broadly efficacious and commercially viable vaccine against S. suis has been successfully deployed.Currently available commercial bacterin vaccines and herd-specific autogenous vaccines have demonstrated only limited efficacy in the field. Alternative approaches involving live attenuated strains have been explored, but these have yielded inconsistent protection and raise substantial safety concerns, including the potential for reversion to virulence and zoonotic transmission. These limitations have prevented widespread adoption of these S. suis vaccines to date.
[0006] Capsular polysaccharide (CPS) antigens have also been investigated as vaccine candidates. Although CPS antigens can induce IgM antibody responses, they act as T-cell independent antigens and therefore fail to elicit robust, durable immunological memory. Consequently, CPS-based vaccines typically confer only short-lived, transient protection and are unsuitable for long-term disease control. Additionally, S. suis possesses immune evasion mechanisms that reduce the effectiveness of IgM-mediated bacterial clearance (such as enzymes which degrade IgM antibodies), further limiting the protective value of CPS-directed immune responses. These inherent limitations of CPS-based vaccine approaches underscore the need for alternative antigens capable of eliciting T-cell dependent, memoryinducing immune responses.
[0007] The S. suis surface antigen one (Sao) protein is a conserved, surface-anchored protein expressed by a substantial proportion of clinically important S. suis strains. Prior art (WO 2007 / 025390) has demonstrated that immunization of piglets with recombinant full-length Sao polypeptide can provide protection against experimental S. suis challenge. However, the full-length Sao protein presents significant challenges that impede commercial vaccine development, complicate manufacturing, formulation, and regulatory development of a commercial vaccine product.
[0008] Accordingly, there remains a significant unmet need in the art for an efficacious S. suis vaccine that demonstrates both protective efficacy and industrial-quality attributes suitable for commercialization.SUMMARY
[0009] The following Summary provides a general overview of aspects of the invention. This Summary is not intended to be a comprehensive description of all features, elements, or aspects of the invention, nor is it intended to limit the scope of the claims appended hereto.
[0010] The present disclosure is based on the identification and experimental validation of a minimal protective epitope within the Streptococcus suis surface antigen one (Sao) polypeptide. Through systematic testing of Sao polypeptide fragments using in vivo challenge studies, the inventors have demonstrated that a complete 30-amino-acid repeat sequence of the SEK type (SEQ ID NO: 79) constitutes a minimal protective epitope sufficient to confer protective immunity against S. suis disease when appropriately presented to the immune system. In contrast, Sao polypeptide fragments comprising 1) N-terminal structured domains D1 and D2 alone, 2) D1 and D2 with a complete SKE repeat domain, or 3) D1 and D2 with a complete SKI repeat domain plus a partial SEK repeat domain failed to confer protection despite inducing robust antigen-specific IgG responses.
[0011] The inventors further demonstrate that immunogenic compositions incorporating this protective epitope can be implemented in various engineered Sao polypeptide fragments. These include C-terminally truncated forms of the Sao protein that retain at least one complete SEK repeat domain, as well as peptide-based constructs wherein a single SEK repeat domain is conjugated to an immunogenic carrier. These engineered fragments provide significant advantages in recombinant production yield, protein stability, and shelf life compared to full-length Sao, thereby enabling commercial-scale vaccine manufacture.
[0012] All features of exemplary embodiments which are described in this disclosure and are not mutually exclusive can be combined with one another. Elements of one embodiment can be utilized in the other embodiments without further mention. Other aspects and features of the present disclosure will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments in conjunction with the accompanying Figures.BRIEF DESCRIPTION OF THE DRAWINGS
[0013] A detailed description of specific exemplary embodiments is provided herein below with reference to the accompanying drawings in which:
[0014] FIG. 1 is a non-limiting illustration of an in silico predicted model of Sao parent polypeptide AAX57440.1, highlighting amino acid positions from the beginning of the polypeptide sequence (N-terminal, or “N”), [3-barrel domain 1 (D1), [3-barrel domain 2 (D2), and the end of the polypeptide sequence (C-terminal, or “C”);
[0015] FIG. 2 is a non-limiting graph showing an analysis of the reliability of the polypeptide structure of the Sao parent polypeptide of FIG. 1, assessed by the QMEAN scoring protocol. A single column represents an amino acid position. Any score below the threshold of 0.6 is considered unreliable. The QMEAN estimate shows the structural model is more reliable for the N-terminal and C-terminal sequences than for amino acids between ~240 and 480;
[0016] FIG. 3 is a non-limiting illustration showing predicted B-cell epitopes of different amino acid lengths in the Sao protein as determined by SVMtrip tool. Potential epitopes, with a score of 0.8 (after rounding), were selected, as recommended by the tool, and are scattered throughout the Sao parent polypeptide sequence. This confirms that D1 and D2 have predicted B cell epitopes. It also predicts antibody epitopes in repeats 6-10.. As shown herein, predicted immunogenic epitopes do not necessarily correspond to protective epitopes, underscoring the necessity of in vivo challenge models;
[0017] FIG.4A to FIG. 4J each illustrates the key polypeptide elements of the full-length Sao polypeptide and fragments tested as vaccine antigens. The 10 boxes downstream of D2 represent the 10 repeat domains. The first box is the lone SKE repeat domain and the following 9 boxes are SKE repeat domains with varying sequences. The V5 fragment is the 30 amino acid sequence of repeat domain 4 (aa 385-414), which has 100% identity with repeat 5 (aa 415-444);.
[0018] FIG. 5A is a non-limiting histogram that shows the production yield (milligrams per liter of culture) of the Vm, V1, V3 and V4 polypeptides produced by recombinant E. coli:
[0019] FIG. 5B is a non-limiting picture of an SDS-PAGE of purified Vm, V1, V3 and V4 polypeptides, showing the difference in polypeptide sizes for the recombinant constructs expressed in E. coli. Note: The gel was loaded with the same quantity of protein for each polypeptide to ensure ease of visualization;
[0020] FIG. 6A and FIG. 6B are non-limiting graphs showing the thermostability by differential scanning fluorimetry (DSF) of purified V1, V3, and V4 polypeptides (5 µg each). FIG. 6A shows DSF results at recovery (day 0) and FIG. 6B shows DSF results after 1 week of storage in a refrigerator (2-8°C);
[0021] FIG. 7A is a non-limiting picture of an SDS-PAGE gel showing the long-term stability of V1 and 3 polypeptides, when kept at 2-8°C, over 18 months, in accordance with embodiments of the present disclosure;
[0022] FIG. 7B is a non-limiting graph representing the long-term stability by differential scanning fluorimetry (DSF) of V1 and V3 polypeptides, when kept in storage at 2-8°C, over 18 months. The assay demonstrates thermal stability of 57°C to 59°C, in accordance with embodiments of the present disclosure;
[0023] FIG. 8A and FIG. 8B are non-limiting graphs representing the specific IgG response induced following immunization with (A) V1 polypeptide vs. V1 in combination with an oil-in-water adjuvant or (B) V3 polypeptide vs. V3 in combination with an oil-in-water adjuvant. The antigens with or without an oil-in-water adjuvant, were administered to piglets at 3 weeks of age, followed by a booster dose at 5 weeks of age. Significant differences are shown on the graph (* p<0.05; **p<0.01), in accordance with embodiments of the present disclosure;
[0024] FIG. 9 is a non-limiting illustration of the animal study timeline in which piglets were vaccinated with either V1, V3 or unvaccinated (control). The antigens combined with an oil-in-water adjuvant were administered intramuscularly twice, namely a first time at 4 weeks of age and a second time at 6 weeks of age. Fourteen days after the second vaccination, animals were intramuscularly challenged with 2 x 107CFU of a S. suis serotype 2 strain;
[0025] FIG. 10 is a non-limiting graph representing the IgG titers against V1 following ELISA analysis of serum samples obtained at Day 28 in pigs vaccinated with V1 or unvaccinated (control) as per the protocol set forth in FIG. 9. A significant difference is shown on the graph (**** p<0.0001). The low-level anti-S. suis antibodies in the controls are likely due to residual maternal antibodies and / or natural immunity development at D28;
[0026] FIG. 11 is a non-limiting graph representing the IgG titers against V3 following ELISA analysis of serum samples obtained at Day 28 in pigs vaccinated with V3 or unvaccinated (control) as per the protocol set forth in FIG. 9. A significant difference is shown on the graph (**** p<0.0001). The low-level anti-S. suis antibodies in the controls are likely due to residual maternal antibodies and / or natural immunity development at D28;
[0027] FIG. 12 is a non-limiting workflow that illustrates an assessment of the Sao domains conservation and the repeats diversity, in accordance with embodiments of the present disclosure;
[0028] FIG. 13A is a non-limiting illustration of the Sao polypeptide showing D1-D2, the junction segment, SKE repeat domain (cluster position: Pos 2) and SEK repeat domains 2-4 (cluster position: Pos 3 to Pos 5). This nomenclature with respect to cluster position is used in FIG. 13B.
[0029] FIG. 13B is a non-limiting illustration of the prevalence of repeat sequences within each cluster position within 232 Sao parent polypeptides across a 295 S. suis genome dataset;
[0030] FIG. 14 is a non-limiting graph that shows the distribution of Sao repeats per genome. Histogram depicting the number of repeats identified from 232 Sao proteins across the 295-genome dataset. The x-axis represents the number of repeats per genome, while the y-axis indicates the frequency of genomes containing each repeat count, in accordance with embodiments of the present disclosure;
[0031] FIG. 15A is a non-limiting schematic representation of the Sao polypeptide organization. A database of 295 S. suis genomes was analyzed to identify Sao protein in 232 genomes. FIG. 15B is a non-limiting schematic representation of the amino acid variability in 12 SKE repeat domains that were analyzed. Full-sized letters indicate 100% homology and smaller letters show relative frequency of amino acid variations in the 12 repeats analyzed. FIG. 15C is a non-limiting schematic representation of the 58 SEK repeat domains that were analyzed. Amino acid variability is depicted as described for SKE repeats;
[0032] FIG. 16 is a non-limiting alignment of the amino acid sequences of the 10 domain repeats present in the Sao polypeptide of SEQ ID NO: 23;
[0033] FIG. 17A and FIG.17B are non-limiting graphs showing the specific IgG response induced following immunization with V1 or 3 at Day 28 against (A) Domain 1 (D1) and Domain 2 (D2) or (B) each of the 10 repeats from the Sao polypeptide of SEQ ID NO: 23. Significant differences are shown on the graph (**p<0.01, ****p<0.0001).
[0034] In the drawings, exemplary embodiments are illustrated by way of example only. The description and drawings are provided for purposes of illustration andunderstanding, and are not intended to limit the scope of the invention as defined by the appended claims.DETAILED DESCRIPTION
[0035] The present invention is described in detail below with reference to specific embodiments. The following description is not intended to constitute an exhaustive account of all possible implementations or of all features that may be incorporated into the invention. Features described with respect to one embodiment may be combined with features described with respect to other embodiments. Accordingly, the following description illustrates certain embodiments of the invention by way of example only and is not to be construed as limiting the scope of the claims.Identification of a Protective Epitope within the Sao Polypeptide
[0036] The present invention arises from the experimental identification and validation of a specific protective epitope within the Sao polypeptide. While Sao was previously known to be immunogenic, the inventors have now demonstrated through controlled in vivo challenge experiments that protective immunity depends on the presence of at least one complete 30-amino-acid SEK repeat domain (SEQ ID NO: 79 or a sequence variant retaining the conserved motifs ISEKQ4, I4TPI6, and22NKE24). This discovery is significant because it establishes a structure-function relationship between Sao domain architecture and protective efficacy that was previously unknown. Sao polypeptide constructs lacking a complete SEK repeat domain, including constructs containing only the N-terminal structured domains D1 and D2, e.g., the V1 polypeptide (FIG. 4D), failed to confer protection in challenge models despite inducing robust antigen-specific IgG responses. In contrast, constructs including at least one complete SEK repeat domain, e.g., the V3 polypeptide (FIG. 4F), the V5 polypeptide (FIG. 4H), and the V6 polypeptide (FIG. 4I) provided statistically significant protection, as evidenced by reduced mortality and clinical scores. These findings demonstrate that immunogenicity, as measured by antigen-specific IgG titers, does not correlate with protective efficacy, which is a critical insight for rational vaccine design.
[0037] The present invention thus relates to a modular repeat-based Sao antigenic system, in which protective efficacy is associated with complete SEK repeat units. This modular architecture enables rational design of vaccine antigens that retain the minimal protective epitope while eliminating polypeptide regions that impair manufacturability. The modular nature of the system permits flexibility in constructdesign, including minimal peptide-based constructs (e.g., V5), truncated polypeptide fragments (e.g., V3, V4, V6), and conjugate or fusion approaches, all sharing the common feature of at least one complete SEK repeat domain.Discovery of Manufacturing and Stability Limitations of Sao Polypeptides
[0038] The present inventors unexpectedly discovered that attempts to produce a commercial vaccine composition comprising the full-length S. suis Sao surface antigen polypeptide of SEQ ID NO: 23 revealed several critical drawbacks that impede commercial development. As demonstrated in the working examples of the present specification, these drawbacks include: (a) low production yield in recombinant expression systems, rendering commercial-scale manufacture economically unfeasible; (b) protein instability and degradation during storage at 2-8°C, failing to meet regulatory requirements for minimum product stability and shelf life; and (c) lack of characterization of the minimal protective epitope, which limits rational protein engineering for improved manufacturability. These drawbacks became apparent only upon systematic evaluation under conditions relevant to industrial-scale production and regulatory requirements for veterinary vaccines.
[0039] Accordingly, the present invention provides immunogenic compositions containing recombinant polypeptide fragments of the Sao polypeptide which exhibit improved manufacturing yield and stability for industrial manufacture, while maintaining protective immunogenicity.
[0040] Through systematic investigation involving structure-based protein engineering and in vivo challenge studies, the inventors made the surprising discovery that a single complete 30-amino-acid SEK repeat domain is sufficient, in the experimental models described herein, to confer protective immunity against S. suis infection. This finding directly addresses the technical problem of identifying a minimal protective epitope suitable for vaccine development. The discovery is significant because prior work in the field focused on immunoreactivity of various Sao epitopes without demonstrating whether such immunogenicity correlates with protective efficacy in animal challenge models. The inventors have now established that immunogenicity, as measured by antigen-specific IgG titers, does not necessarily predict protective efficacy, which is a finding with important implications for vaccine antigen selection. The inventors further discovered that deliberate removal of polypeptide segments at the N-terminal and / or C-terminal ends of the full-length Sao polypeptide significantly improves production yield and protein stability, thereby enabling commercial-scale manufacture whileretaining the minimal protective epitope required for vaccine efficacy. Specifically, removal of the signal sequence and C-terminal anchor domain (amino acids 445-670) resulted in polypeptides with improved expression yields and stability profiles (see Example 2, FIGS. 5-7).Structural Organization of the Sao Polypeptide
[0041] In a broad aspect, the recombinant polypeptide described herein is a polypeptide fragment of a S. suis surface antigen one (“Sao”) parent polypeptide (also referred to herein, as “Sao parent polypeptide”). In the present text, the term “recombinant polypeptide” will thus also be referred to as “Sao polypeptide fragment”, interchangeably. The Sao parent polypeptide is described in further details later in this text.
[0042] In some embodiments, the Sao polypeptide fragment can be obtained with recombinant technology, for expression through recombinant expression in bacteria (E. coli or another bacteria), in yeast or in mammalian cells (e.g. CHO). In some embodiments, a recombinant DNA coding for the Sao polypeptide fragment can be inserted in a virus (replication competent or incompetent) that can induce expression of the polypeptide after injection into an animal.
[0043] In some embodiments, the Sao polypeptide fragment is comprised in a bacterial, yeast, or mammalian extract. For example, a bacterial extract can be obtained with any known process in the art, such as the process described in US20240424077, which is incorporated by reference herein in its entirety. The use of bacterial, yeast, or mammalian extracts containing the Sao polypeptide fragment is within the scope of the present invention and represents an alternative formulation approach to isolated recombinant polypeptides.
[0044] In some embodiments, the Sao polypeptide fragment is an isolated recombinant polypeptide. In other words, the Sao polypeptide fragment in such cases has been separated from its natural environment, either through chemical synthesis or purification methods, and is distinct from its naturally occurring state.
[0045] The term “fragment” with respect to a polypeptide, as used herein, refers to a polypeptide constructed artificially, which is a portion of a corresponding parent polypeptide. For example, through expression of a nucleic acid molecule that contains a portion of the nucleic acid sequence of the corresponding parent polypeptide, where the nucleic acid molecule has been constructed artificially (e.g., by chemical synthesis)or by cleaving a natural product into multiple pieces, using restriction endonucleases or mechanical shearing, or a portion of a nucleic acid synthesized by PCR, DNA polymerase or any other polymerizing technique well known in the art, or expressed in a host cell by recombinant nucleic acid technology well known to one of skill in the art. Accordingly, the Sao polypeptide fragments described herein do not exist in nature and are obtained by deliberate truncation or engineering of the Sao full-length polypeptide.
[0046] As such, a fragment of the Sao parent polypeptide does not contain the complete amino acid sequence of the Sao parent polypeptide. The Sao polypeptide fragment is free from an N-terminal amino acid segment and / or a C-terminal amino acid segment of the Sao parent polypeptide. The Sao polypeptide fragment can be characterized as comprising less than the full-length Sao parent polypeptide amino acid residue sequence. Although selected N-terminal and / or C-terminal segments of the Sao parent polypeptide are removed in certain embodiments to improve manufacturability, the Sao polypeptide fragments described herein retain protective vaccine properties, as evidenced by their recognition by Sao-specific antibodies and their ability to elicit a measurable protective immune response in challenge models. Accordingly, the truncations described herein do not abolish the herein described protective epitope, as demonstrated by the challenge and immunologic assays reported in the present application (see, e.g., Examples 4, 5, and 8), and are compatible with use of the Sao polypeptide fragments in immunogenic or vaccine compositions.
[0047] In some embodiments, the Sao polypeptide fragment is free from an N-terminal amino acid segment of the Sao parent polypeptide. For example, the N-terminal amino acid segment can correspond to at least amino acid residues 1 to 30 of the Sao parent polypeptide, more preferably to at least amino acid residues 1 to 42 of the Sao parent polypeptide. In further examples, the N-terminal amino acid segment can correspond to at least amino acid residues 1 to 164 of the Sao parent polypeptide, preferably to amino acid residues 1 to 276 of the Sao parent polypeptide. It has been demonstrated that such polypeptide amino acid sequence truncations improve manufacturing yield while retaining the polypeptide’s protective immune response capability.
[0048] In some embodiments, the Sao polypeptide fragment is free from a C-terminal amino acid segment of the Sao parent polypeptide. For example, the C-terminal segment can correspond to at least amino acid residues 600 to 670 of the Sao parent polypeptide. In further examples, the C-terminal amino acid segment can correspond to amino acid residues 445 to 670 of the Sao parent polypeptide. Expression of theSao polypeptide fragment which is free from the C-terminal amino acid segment of the Sao parent polypeptide results in a polypeptide that does not exist in nature, as the Sao polypeptide fragment is no longer associated with the cell membrane, in contrast to the Sao parent polypeptide, which includes an LPXTG-motif cell wall anchor domain and is anchored to the cell wall on the surface of Streptococcus suis. It has been demonstrated that such polypeptide amino acid sequence truncations improve manufacturing yield while retaining the polypeptide’s protective immune response capability.
[0049] As used herein, the expression “free from” encompasses complete deletion, replacement, or interruption of a given amino acid sequence.
[0050] Throughout the disclosure, the numbering of the amino acid residues is based on the amino acid sequence from GenBank deposit accession number AAX57440.1 (SEQ ID NO: 23) for ease of reference. While the Sao polypeptide fragment can have an amino acid sequence which corresponds to a fragment of a Sao parent polypeptide from any other strain, i.e., having an amino acid sequence from a parent polypeptide from any strain listed in Table 1 other than that one depicted in SEQ ID NO: 23, nevertheless, the numbering will refer to that one depicted in SEQ ID NO: 23 for ease of reference unless otherwise stated.
[0051] In some embodiments, the Sao polypeptide fragment includes at least one SEK repeat domain having the consensus amino acid sequence set forth in SEQ ID NO: 73 (SEKQMPSWNDNAVTPEKQMTNKENDNVET) or a variant thereof as defined herein, referred to herein as the “SEK” domain. For example, a variant SEK domain may have any one of the naturally occurring amino acid substitutions disclosed in FIG.15C with respect to the amino acid sequence set forth in SEQ ID NO: 73. For example, the variant SEK domain may have one or more substitutions selected from: M5K, M5I, P6T, S7V, V8I, V8N, V8S, V9M, V9E, N10K, D11E, N12M, D12T, A13V, V14I, E17Q, K18E, K18R, M20T, M20I, T21 A, T21D, E24V, N25K, N25R, D26E, N27K, N27E, V28I, E29V, E29L, T30I, and T30A, where the 1stletter is the consensus sequence amino acid, # is the amino acid position in the repeat, and the last letter is the amino acid substitution. These substitutions represent naturally occurring variants identified across 58 S. suis isolates as described in Example 6 and are expected to retain protective efficacy based on the conservation of the key functional motifs.
[0052] It will be understood by the reader that in the present specification, %-identity is calculated over the full-length of the polypeptide fragment unless otherwise noted.
[0053] In some embodiments, the SEK domain sequence includes conserved segments amongst several Sao parent polypeptides, namely the first amino acid residues 1-4 SEKQ (“1SEKQ4”).
[0054] In some embodiments, the SEK domain sequence further includes amino acid residues at positions 14-16 VTP (“14VTP16”).
[0055] In some embodiments, the SEK domain sequence further includes amino acid residues at positions 22-24 NKE (“22NKE24”).
[0056] In some embodiments, the SEK domain sequence includes conserved segments amongst several Sao parent polypeptides, namely amino acid residues 1-4 SEKQ (“1SEKQ4”), amino acid residues at positions 14-16 VTP (“14VTP16”), and amino acid residues at positions 22-24 NKE (“22NKE24”). For example, the SEK domain sequence variant may have at least 50% identity over its full length to the amino acid sequence set forth in SEQ ID NO: 73 and retains the conserved motifs 1SEKQ4, 1 VTP16, and 22NKE2. These conserved motifs are consistently present across SEK repeat sequences identified from 232 Sao proteins in the 295-genome dataset (see Example 6) and are believed to be functionally important for the protective immune response elicited by SEK repeat domain-containing antigens. Preferably, the SEK domain sequence variant retains the expanded conserved motifs 1SEKQMP6, 1 VTP16, 19QM20, and 22NKE2, which are present within the 10 repeat domains of the Vm polypeptide tested experimentally in the present disclosure. These extended conserved motifs are characteristic of the SEK repeat domains tested in the challenge studies described herein and are expected to retain protective efficacy.
[0057] In some embodiments, the SEK domain variant has any one of the following 30-mer amino acid residues sequence, where the repeat # corresponds to the repeat illustrated in FIG. 4E:Table 0 (the expanded conserved motifs is shown in bold)
[0058] In some embodiments, the Sao polypeptide fragment includes at least one SEK repeat domain of SEQ ID NO: 79. As demonstrated in Example 8, the presence of a single complete SEK repeat domain is sufficient to provide protection against S. suis infection. Accordingly, in a preferred embodiment, the Sao polypeptide fragment includes or consists essentially of a single SEK repeat domain of SEQ ID NO: 79. Due to the limited size of this 30-amino acid peptide, the SEK repeat domain, when used alone, is preferably conjugated to or presented in association with an immunogenic carrier protein, such as keyhole limpet hemocyanin (KLH), to enhance immunogenicity and ensure adequate presentation to the immune system. This carrier-conjugation approach is employed to demonstrate that the SEK repeat sequence itself constitutes a sufficient protective epitope, rather than to suggest that the peptide alone would elicit an effective immune response. Alternatively, the person skilled in the art could fuse the sequence of the single SEK repeat domain of SEQ ID NO: 79 into a recombinant expressed immunogenic protein, thus avoiding the need for peptide synthesis and conjugation to a carrier.
[0059] In some embodiments, the Sao polypeptide fragment includes a plurality of SEK repeat domains selected from SEQ ID NO: 77 to 82. These SEK repeat domains each retain the conserved motifs 1SEKQ4, 1 VTP16, and 22NKE24, and preferably further retain the extended conserved motifs iSEKQMPe, 14 TP16, 19QM20, and 22NKE24. Based on the conservation of these functional motifs across multiple SEK repeat domain variants identified in the 295-genome dataset analysis (see Example 6) or in Table 0, SEK repeat domains having the amino acid sequence set forth in any one of SEQ ID NOs: 77, 78, 79, 80, 81, or 82 are expected to retain protective efficacy. Preferably, the plurality of SEK repeat domains includes two repeat domains each having the amino acid sequence set forth in SEQ ID NO: 79.
[0060] In some embodiments, the Sao polypeptide fragment further includes an SKE repeat domain having the consensus amino acid sequence set forth in SEQ ID NO: 72 (SKEAMTDKEKGNIEPPKEQIANEEKDNIEA) or a variant thereof. For example, a variant SKE domain may have any one of the naturally occurring amino acid substitutions disclosed in FIG. 15B with respect to the amino acid sequence set forth in SEQ ID NO: 72. For example, the variant SKE domain can have an amino acid sequence having at least 90% identity over its full length to the amino acid sequenceset forth in SEQ ID NO: 72. Alternatively, the variant SKE domain may have one or more substitutions selected from D7G, P16S, A21T, N22T, E29K, A30S, A30T, and A30V. These substitutions represent naturally occurring variants identified across 58 S. suis isolates as described in Example 6.
[0061] In some embodiments, the SKE domain variant has the following 30-mer amino acid residues sequence: SKEAMTGKEKGNIEPPKEQIANEEKDNIEA (SEQ ID NO: 76).
[0062] In some embodiments, the Sao polypeptide fragment includes the 30-amino-acid SKE repeat domain positioned N-terminal to at least one SEK repeat domain. For example, the Sao polypeptide fragment can include a 30-amino-acid SKE repeat domain positioned N-terminal to from 2 to 10 SEK repeat domains, preferably 4 to 7 SEK repeat domains, more preferably 4 SEK repeat domains.
[0063] In some embodiments, the Sao polypeptide fragment includes the amino acid residues having the sequence set forth in SEQ ID NO: 8 (V3 polypeptide, FIG 4F). For example, the Sao polypeptide fragment can consist essentially of the amino acid residues having the sequence set forth in SEQ ID NO: 8 (V3 polypeptide).
[0064] In some embodiments, the Sao polypeptide fragment includes the amino acid residues having the sequence set forth in SEQ ID NO: 9 (V4 polypeptide, FIG 4G). For example, the Sao polypeptide fragment can consist essentially of the amino acid residues having the sequence set forth in SEQ ID NO: 9 (V4 polypeptide).
[0065] In some embodiments, the Sao polypeptide fragment includes the amino acid residues having the sequence set forth in SEQ ID NO: 79 (V5 polypeptide, FIG 4H). For example, the Sao polypeptide fragment can consist essentially of the amino acid residues having the sequence set forth in SEQ ID NO: 79 (V5 polypeptide).
[0066] In some embodiments, the Sao polypeptide fragment includes the amino acid residues having the sequence set forth in SEQ ID NO: 91 (V6 polypeptide, FIG 4I). For example, the Sao polypeptide fragment can consist essentially of the amino acid residues having the sequence set forth in SEQ ID NO: 91 (V6 polypeptide). The V6 polypeptide corresponds to amino acids 164 to 444 of the full-length Sao polypeptide (SEQ ID NO: 23), comprising the D2 domain, the junction segment, the SKE repeat domain, and four complete SEK repeat domains, but lacking the D1 domain. As demonstrated in Example 8, the V6 polypeptide provides 54% reduction in mortality against S. suis challenge, confirming that the D1 domain is not required for protectiveimmunity and that constructs comprising at least the D2 domain together with complete SEK repeat domain(s) retain protective efficacy.
[0067] Without being bound by any theory, the present disclosure demonstrates that while the full-length Sao polypeptide includes several predicted B-cell immunogenic epitopes distributed throughout the amino acid sequence (see FIG. 3), protective immunity in vivo is conferred by a complete SEK repeat domain (SEQ ID NO: 79), whereas a Sao polypeptide fragment containing predicted B-cell immunogenic epitopes but lacking a complete SEK repeat domain was unable to confer protective immunity in vivo, as demonstrated in Examples 4, 5, and 8. Interestingly, B cell epitopes in this sequence SEK repeat domain (SEQ ID NO: 79) were not predicted using in silico tools. This finding reveals that immunogenicity, as measured by antigenspecific IgG titers, does not necessarily correlate with protective efficacy-a key insight that guides the rational design of the claimed vaccine compositions. Through systematic structure-based engineering, the inventors have developed Sao polypeptide fragments that: retain at least one complete SEK repeat domain comprising the conserved motifs 1SEKQ4, 1 VTP16, and 22NKE24; and exhibit improved production yield and stability in recombinant expression systems. These engineered Sao polypeptide fragments, when formulated as immunogenic compositions, elicit robust protective immunity against Streptococcus suis in swine and mice, as demonstrated by statistically significant reductions in mortality and clinical signs in challenge studies (see Examples 4 and 8). This approach overcomes the limitations of the prior art, which neither addresses manufacturability nor demonstrates protective efficacy of minimal or incomplete Sao fragments.
[0068] In some embodiments the Sao polypeptide fragment includes in its N-terminal segment, a structured domain featuring a [3-barrel display, as detected with X-ray crystallography.
[0069] In some embodiments, the structured domain featuring a [3-barrel display, as detected with X-ray crystallography, may have the amino acid residue sequence DGGQATSKAVNVKIPAWRLFGRELLENEFKFELREANGEELPVLDTAQNTKEGQV RFKNLSFDKPGKYWYTISEVKDELGGIEYDSKYIVAKITVEDRNGQLQAMIEFIDNDN VFNNFYTP (SEQ ID NO: 74) or a sequence variant thereof. This structured domain will be referred to as the “D1 domain” and spans from amino acid 43 to 164 in SEQ ID NO: 23. For example, the D1 domain sequence variant can have an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% identity over its full length to the amino acid sequenceset forth in SEQ ID NO: 74. For example, the D1 domain variant may have an amino acid sequence that varies from the amino acid sequence set forth in SEQ ID NO: 74 due to natural mutations or artificial mutations (i.e., human-made mutations), wherein a mutation may include a substitution, a deletion, or an insertion of one or more amino acid residues. For example, the D1 domain sequence variant may have at least one mutation selected from G45R, A51T, V54I, E66D, E75A, A79V, N80D, S81G, K83E, L84-l87insPV, I87L, T90A, E95D, R99S, D106N, E110K, S116L, V118I, N120D, S123G, V125I, Y127F, 1132V, A134S, R141-N143insN, A154D, and D155N, wherein the amino acid numbering is based on the amino acid sequence set forth in SEQ ID NO: 23.
[0070] In some embodiments, the structured domain featuring a [3-barrel display, as detected with X-ray crystallography, may have the amino acid residue sequence APAAASLSIKKVLEGRTLNTGEFEFVLKNEKGDEIEKVSNQADGSVNFSALTFTKEG TYTYTVSEVDGGLGDIIYDKSDIKATVTVKDNNHGQLVSTVTYENSDQIFENILN(SEQ ID NO: 75) or a sequence variant thereof. This structured domain will be referred to as the “D2 domain” and spans from amino acid 164 to 276 in SEQ ID NO: 23. For example, the D2 domain sequence variant can have an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% identity over its full length to the amino acid sequence set forth in SEQ ID NO: 75. For example, the D2 domain sequence variant may have an amino acid sequence that varies from the amino acid sequence set forth in SEQ ID NO: 75 due to natural mutations or artificial mutations (i.e., human-made mutations), wherein a mutation may include a substitution, a deletion, or an insertion of one or more amino acid residues. For example, the D2 domain sequence variant may have at least one mutation selected from V177G, E179K, G186S, A215T, G234R, D237N, V249I, D269N, I275N, and N277K, wherein the amino acid numbering is based on the amino acid sequence set forth in SEQ ID NO: 23.Sao Wild Type Polypeptide Structure and Sequence
[0071] As used herein, the expression “Sao parent polypeptide” (or “full-length polypeptide”) refers to a wild-type bacterial surface protein with conserved protein domains used for cell surface attachment and colonization that is expressed among several strains of S. suis. By “wild type” is meant here a polypeptide that includes an amino acid sequence found in nature, i.e., which is of natural origin, including allelic variations, and which has not been intentionally modified by molecular biology techniques, such as mutagenesis of the corresponding coding nucleic acid molecule.
[0072] As shown in FIG. 4A, the full-length polypeptide comprises the following structural regions: (i) an N-terminal segment; (ii) a structured domain D1; (iii) a structured domain D2; (iv) a junction sequence; (v) a plurality of 30-amino-acid domains; and (vi) a C-terminal region containing the LPXTG cell wall anchor motif and hydrophobic tail.
[0073] Each of the plurality of 30-amino-acid domains consists of a 30-amino-acid residues with a strong consensus pattern. For example, a non-limiting example of a plurality of such repeat domains can be found at amino acid residues 295 to 595 of the amino acid sequence set forth in SEQ ID NO: 23. For example, the strong consensus pattern consists of the amino acid sequence set forth in SEQ ID NO: 29. The Sao parent polypeptide may include a plurality of repeat domains.
[0074] Non-limiting examples of Sao wild type polypeptides that can be used as “parent polypeptide” are summarized in Table 1, below.Table 1: Sao Wild Type polypeptides
[0075] The Sao protein from 232 field strains was analyzed (see Example 6). FIG. 15B and FIG. 15C illustrate sequence logos depicting the amino acid conservation among the 12 SKE repeats and the 58 SEK repeats, respectively. These sequence logos were generated using WebLogo tool at the weblogo.berkeley.edu internet website. Prior to logo generation, unique repeat sequences were identified from 232 Sao proteins using CD-Hit clustering and aligned with MUSCLE. Low-frequency amino acid positions in the alignment (<=10%) were removed. Repeat lengths ranged from 26 to 33 amino acids, with low representation (~5.1%) in the dataset of repeats differing from thepredominant length of 30 amino acids. Percentage of similarity and number of substitutions were assessed using BLASTp.Immunogenic Composition
[0076] In a broad aspect, the Sao polypeptide fragment described herein can be administered in the form of an immunogenic composition to elicit a protective immune response in an animal against subsequent natural exposure to, or experimental challenge with, S. suis. Such immunogenic composition that elicits a protective immune response in an animal is also referred to herein as a vaccine composition.
[0077] In particular, the immunogenic composition can be administered to any animal that can be infected by S. suis. For example, swine, mice, or human. For example, swine (pigs) includes gilts, sows, piglets, or any combinations thereof. The immunogenic composition can be administered for protecting the animal against an S. suis disease. For example, the immunogenic composition can be administered for prophylactic, metaphylactic or therapeutic purposes.
[0078] In some embodiments, administration of the immunogenic composition containing the Sao polypeptide fragment described herein to an animal induces protective antibodies against Streptococcus suis in the animal. As demonstrated in Examples 3 and 4, administration of Sao polypeptide fragments V1 and V3 to piglets elicited antigen-specific IgG responses, and administration of V3 (which contains at least one complete SEK repeat domain) conferred protective immunity against S. suis challenge.
[0079] In some embodiments, administration of the immunogenic composition comprising the Sao polypeptide fragment described herein or causing expression of the Sao polypeptide fragment (e.g., using recombinant technology, such as virus coding for the Sao polypeptide fragment) to an animal reduces the incidence of or severity of one or more clinical signs or death associated with or caused by infection with S. suis. Such clinical signs can include, for example, behavioral changes, lameness, meningitis, bacteremia, endocarditis, arthritis, and septic shock. As demonstrated in Examples 4 and 8, vaccination with Sao polypeptide fragments containing at least one complete SEK repeat domain resulted in statistically significant reductions in mortality and clinical signs compared to unvaccinated control groups.
[0080] In some embodiments, the immunogenic composition may further comprise a pharmaceutical or veterinary-acceptable excipient, diluent, or carrier, and optionally anadjuvant. For example, any pharmaceutical or veterinary-acceptable adjuvant may be used. The inclusion of an adjuvant in the immunogenic composition can enhance the protective immune response, as demonstrated in the working examples herein.
[0081] Pharmaceutical or veterinary-acceptable excipients, diluents, or carriers are well known to the one skilled in the art. Examples include water, a 0.9% NaCI solution (saline), Tris-buffered saline, Hepes buffered saline, or a phosphate buffer. Details on doses and volumes are provided in the general description and can be determined by skilled artisans through careful consideration of this disclosure alongside existing knowledge in the field, without undue experimentation.
[0082] In some embodiments, the immunogenic composition comprises the immunogenic polypeptide fragment described herein in association with an immunogenic carrier. Linking the polypeptide fragment to an immunogenic carrier can enhance the immunogenicity of the fragment, particularly when the fragment corresponds to a short peptide epitope that may otherwise elicit a limited immune response when administered alone. For example, immunogenic polypeptide carriers or other synthetic compounds with immunogenic properties.
[0083] In some embodiments, the polypeptide fragment is linked to the immunogenic carrier by covalent or non-covalent association. For example, the polypeptide fragment may be chemically conjugated to the carrier, genetically fused to the carrier, or associated with the carrier through linker molecules, cross-linking reagents, or affinity interactions.
[0084] In some embodiments, the polypeptide fragment is conjugated to a carrier protein. Conjugation may be achieved using conventional peptide-protein conjugation techniques known in the art, including cross-linking chemistries that target amino, sulfhydryl, or carboxyl functional groups on the peptide and the carrier protein.
[0085] Non-limiting examples of suitable conjugation chemistries include carbodiimide-mediated coupling, maleimide-based sulfhydryl coupling, glutaraldehyde cross-linking, succinimidyl ester coupling, and heterobifunctional linker systems. Such approaches allow the peptide antigen to be covalently attached to the carrier protein while preserving the antigenic determinants of the polypeptide fragment.
[0086] In some embodiments, the carrier protein is selected from proteins commonly used as immunogenic carriers in peptide-based vaccines. Suitable carrier proteins include, without limitation, keyhole limpet hemocyanin (KLH), tetanus toxoid, diphtheriatoxoid, cross-reactive material 197 (CRM 197), bovine serum albumin (BSA), ovalbumin, or combinations thereof. Preferably, the carrier protein is KLH.
[0087] In some embodiments, particularly when the Sao polypeptide fragment consists of a single SEK repeat domain (such as the V5 construct), conjugation to a carrier protein such as KLH is preferred to enhance immunogenicity. The V5 construct, comprising a single SEK repeat domain of SEQ ID NO: 79 conjugated to KLH, demonstrated 86% efficacy in a murine challenge model (see Example 8). These data demonstrate that a single complete SEK repeat domain, when conjugated to an immunogenic carrier, is sufficient to elicit protective immunity against S. suis infection.
[0088] In some embodiments, the Sao polypeptide fragment is expressed as a fusion protein with another antigen or carrier moiety. Such fusion proteins may be produced by genetic fusion of the nucleic acid sequence encoding the Sao polypeptide fragment with a nucleic acid sequence encoding the carrier moiety or additional antigen. Fusion proteins may be advantageous for producing bivalent or multivalent vaccine compositions. For example, such fusion protein may include a single SEK repeat domain of SEQ ID NO: 79 fused to another antigen or carrier moiety.
[0089] In some embodiments, the present invention relates to a nucleic acid molecule encoding a recombinant polypeptide as described herein. The nucleic acid molecule may comprise a coding sequence for a Sao polypeptide fragment that includes at least one complete SEK repeat domain of SEQ ID NO: 79 or a sequence variant thereof retaining the conserved motifs 1SEKQ4, 1 VTP16, and 22NKE24. The nucleic acid molecule may be codon-optimized for expression in a suitable host cell, such as Escherichia coli. Non-limiting examples of nucleic acid constructs encoding Sao polypeptide fragments are set forth in SEQ ID NOs: 17 and 20, which encode the V1 and V3 polypeptides, respectively. For example, the nucleic acid molecule may further include appropriate upstream (e.g. promoter, transcription start codon) and downstream (e.g. polyadenylation signals) to facilitate protein production in a host cell.
[0090] In some embodiments, the present invention relates to a host cell comprising a nucleic acid molecule encoding a recombinant polypeptide as described herein. The host cell may be a prokaryotic cell, such as Escherichia coli, or a eukaryotic cell suitable for recombinant protein expression. In preferred embodiments, the host cell is E. coli BL21 (DE3), which has been demonstrated in the present disclosure to efficiently express the Sao polypeptide fragments V1, V3, V4, V5, V6, and Vm. The host cell may be transformed with an expression plasmid comprising the nucleic acid molecule underthe control of a suitable promoter for inducible expression, such as an IPTG-inducible promoter.
[0091] Methods of administering the immunogenic composition described herein encompass well-known techniques within the expertise of those with ordinary skills in the field.
[0092] For example, when administering to a host the immunogenic composition described herein, the immunogenic composition may contain the Sao polypeptide fragment in an amount sufficient to elicit a protective immune response in the host against S. suis. For example, the amount may include any amount in the range of from about 4 pg to about 500 pg per dose, including any ranges or any value therein. For example, any amount such as about 4 pg, about 5 pg, 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, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, and the like. As demonstrated in the working examples herein, protective immune responses were observed at doses of 50 pg (Examples 5 and 8) and 300 pg (Examples 3 and 4) of Sao polypeptide fragment per dose. Alternatively, the amount can be any amount in the range of for example 0.01-10 pg per kg body weight. Details on doses and volumes can be determined by skilled artisans through careful consideration of this disclosure alongside existing knowledge in the field, without undue experimentation. Routine experimentation will easily establish the required amount.
[0093] For example, the immunogenic composition described herein can be formulated for parenteral or enteral administration.
[0094] For example, the immunogenic composition described herein can be for a suitable administration such as oral, transdermal, transmucosal, intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, intracapsular, intraspinal, intrasternal, intrapulmonary, intranasal, vaginal, rectal, intraocular, intrathecal, buccal (e.g., sublingual), respiratory, topical, ingestion, local delivery (e.g., by aerosol or transdermal), and similar approaches. Intramuscular administration is preferred for swine vaccination. As demonstrated in Examples 3 and 4, intramuscular administration of the V3 polypeptide fragment formulated with an oil-in-water adjuvant resulted in robust antigen-specific antibody responses and protective efficacy against S. suis challenge in piglets.
[0095] In some embodiments, the immunogenic composition is administered in a prime-boost regimen. The prime-boost regimen comprises a first dose (prime dose) followed by a second dose (boost dose) administered 2 to 4 weeks after the first dose. As demonstrated in Examples 3, 4, and 8, a prime-boost regimen with a 2-week interval between doses was employed in the vaccination studies, resulting in enhanced antigen-specific antibody responses and protective efficacy. For example, in Example 3, piglets were administered a primer dose at 3 weeks of age followed by a booster dose at 5 weeks of age (2-week interval). In Example 4, piglets were administered a primer dose at 4 weeks of age followed by a booster dose at 6 weeks of age (2-week interval). In Example 8, mice were administered two doses at a 2-week interval.
[0096] In some embodiments, the immunogenic composition described herein can be formulated for injectable administration. An injectable formulation can take the form of a ready-to-use solution for parenteral injection or the form of a lyophilized composition, intended for reconstitution in a physiologically acceptable solution. Lyophilisation processes are known in the art and typically involve a freezing step, a primary drying step and a secondary drying step. For conciseness’ sake, lyophilisation processes will not be further described here.
[0097] In some embodiments, a commercial package may include in separate containers the lyophilized composition and the physiologically acceptable solution. Such commercial packages may also further include instructions for using, mixing, or handling the lyophilized composition and the physiologically acceptable solution. Such a commercial package may also include a dispenser adapted for administration of the reconstituted composition to an animal.
[0098] In some embodiments, a physiologically acceptable solution may include one or more elements suitable for reconstitution of the lyophilized composition for injectable administration. For example, sterile water for injection, which is water that has been purified and sterilized to meet the standards for injectable use. For example, normal saline (0.9% sodium chloride), which is a solution that contains a concentration of sodium chloride similar to that of the body’s extracellular fluid. For example, Ringer’s solution, which is an isotonic electrolyte solution containing salts that closely resemble the electrolyte composition of the body’s fluids. For example, a dextrose solution, which is a solution containing glucose (sugar). The choice of a specific physiologically acceptable solution for reconstitution may depend on the requirements of the formulation. For example, the specific physiologically acceptable solution should not adversely affect the stability and efficacy of the composition. Additionally, the solutionshould be free from contaminants and suitable for injection into the bloodstream or other tissues.
[0099] In some embodiments, an injectable formulation may also include one or more additional constituents, such as one or more of: preservatives, stabilizers, emulsifiers, other veterinary-acceptable components, carrier polypeptides, buffering agents, or diluents. For example, stabilizers may contribute to maintaining the composition’s effectiveness over time. For example, emulsifiers can enhance the stability and distribution of the composition components. For example, inclusion of other veterinary-acceptable components may contribute to improving the solubility, shelf life, and overall stability of the composition. For example, a carrier polypeptide can be employed to boost the immune response to the one or more polypeptide(s) described herein. For example, buffering agents may contribute to maintaining the pH of the composition, ensuring stability. For example, diluents may be provided in separate containers for mixing with the composition before administration.
[0100] In some embodiments, one or more adjuvants may enhance the immune response to promote a stronger and more enduring immunity. As demonstrated in Example 3, the V1 and V3 polypeptide fragments elicited significantly higher antigenspecific IgG responses when administered in combination with an oil-in-water adjuvant compared to administration without adjuvant. Furthermore, as demonstrated in Example 4, protective efficacy against S. suis challenge was observed in piglets vaccinated with V3 polypeptide formulated with an oil-in-water adjuvant. In Example 8, protective efficacy was demonstrated in mice vaccinated with V3, V4, V5, or V6 polypeptides formulated with TiterMax™ Gold adjuvant. Numerous considerations come into play when choosing an adjuvant. In some cases, the selected adjuvant may facilitate a gradual release and absorption of the antigen, ensuring efficiency while minimizing toxic, allergenic, irritating, and other adverse effects on the host. Some desirable adjuvant characteristics may include non-viricidal properties, biodegradability, consistent high-level immunity, compatibility with multiple antigens, efficacy across various species, non-toxicity, and safety for the host (e.g., absence of injection site reactions). Additional favorable attributes may encompass micro-dosing capability, dose-sparing qualities, excellent shelf stability, suitability for drying, potential for oil-free formulation, adaptability as either a solid or liquid, isotonic nature, ease of manufacturing, and cost-effectiveness. Ideally, an adjuvant should be configurable to induce either a humoral or cellular immune response, or both, depending on the vaccination scenario requirements. The choice of adjuvant mayhinge on vaccine needs, whether to enhance antibody response magnitude or function, elevate cell-mediated immune response, induce mucosal immunity, or reduce antigen dose.
[0101] Various adjuvants are suitable for use in swine vaccines to enhance immune responses. Oil-based adjuvants, such as Montanide™ ISA series (e.g., ISA 201, ISA 206, ISA 50), Emulsigen®, MF59, and TiterMax® Gold, are commonly used to induce strong and long-lasting immunity. Aluminum-based adjuvants, including aluminum hydroxide and aluminum phosphate, are widely employed to stimulate robust humoral responses. Saponin-based adjuvants, such asQuil-A® and Matrix-M™, enhance both humoral and cell-mediated immunity. Bacterial-derived adjuvants, including CpG oligonucleotides (CpG-ODN), lipopolysaccharide (LPS), and monophosphoryl lipid A (MPLA), activate innate immune pathways via Toll-like receptors. Polymer-based adjuvants, such as Carbopol® and chitosan, are known to enhance both mucosal and systemic immune responses. Cytokine-based adjuvants, including granulocytemacrophage colony-stimulating factor (GM-CSF) and interleukins such as IL-2 or IL-12, can further enhance antigen-presenting cell activity and cellular immunity. Additionally, liposome and nanoparticle-based adjuvants, such as immune-stimulating complexes (ISCOMs) and poly(lactic-co-glycolic acid) (PLGA) nanoparticles, facilitate antigen delivery and immune activation. The selection of an appropriate adjuvant depends on the desired immune response, the nature of the antigen, and the intended route of administration, such as intramuscular, intradermal, or mucosal delivery.DEFINITIONS
[0102] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art to which the present disclosure pertains. As used herein, and unless stated otherwise or required otherwise by context, each of the following terms shall have the definitions set forth below.
[0103] As used herein, the terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues.
[0104] As used herein, the term “Sao parent polypeptide” refers to a wild-type or naturally occurring Streptococcus suis surface antigen one (Sao) polypeptide that is expressed by Streptococcus suis and is anchored to the bacterial cell surface. The Sao parent polypeptide comprises an N-terminal signal sequence, one or morestructured N-terminal domains (including domains commonly referred to as D1 and D2), a junction region, a plurality of tandem repeat domains comprising SKE and / or SEK repeat motifs, and a C-terminal cell wall sorting signal (CWSS) including an LPXTG motif and hydrophobic membrane-spanning region. The Sao parent polypeptide includes polypeptides encoded by Sao genes from diverse S. suis strains, including allelic variants, serotype-specific variants, and naturally occurring sequence variants, provided that such polypeptides retain the overall domain architecture characteristic of Sao proteins. Non-limiting examples of Sao parent polypeptides include those having the amino acid sequences set forth in SEQ ID NOs: 23-28 and 30-70. A Sao parent polypeptide is of natural origin and has not been intentionally modified by recombinant truncation, deletion, or engineering to remove N-terminal or C-terminal segments.
[0105] As used herein, the term “Sao polypeptide fragment” refers to a non-naturally occurring polypeptide derived from a Sao parent polypeptide, wherein the fragment comprises less than the full-length amino acid sequence of the Sao parent polypeptide and is obtained by deliberate truncation, deletion, or engineering of the Sao parent polypeptide. A Sao polypeptide fragment is free from at least one N-terminal segment and / or at least one C-terminal segment of the Sao parent polypeptide and does not include the complete amino acid sequence of the Sao parent polypeptide as it exists in nature.
[0106] Expressions such as “protection against disease,” “protective immunity,” and “functional immunity” within this disclosure signify a response to a disease or condition induced by the administration of one or more therapeutic compositions, or a combination thereof. This response results in fewer deleterious effects than expected in a non-immunized subject exposed to disease or infection. Specifically, the severity of deleterious effects from infection is diminished in a vaccinated subject, encompassing the reduction, slowing, or potential prevention of infection.
[0107] The term “reduction of the incidence and / or severity of clinical signs” or “reduction of clinical symptoms” encompasses diminishing the number of infected subjects, eliminating subjects displaying clinical signs, or lessening the severity of any clinical signs compared to wild-type infection. It includes the reduction of pathogen load, pathogen shedding, transmission, or any clinical sign symptomatic of S. suis (S. suis) infection. Preferably, these clinical signs are reduced by at least 10% in subjects that have received the immunogenic composition described herein compared to non-receiving infected subjects. For example, these clinical signs can be reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, or more.
[0108] “Increased protection” denotes a statistically significant reduction in one or more clinical symptoms associated with infection by an infectious agent, preferably S. suis, in a vaccinated group compared to a non-vaccinated control group. A “statistically significant reduction of clinical symptoms” signifies a frequency in the incidence of at least one clinical symptom in the vaccinated group that is at least 10%, preferably 20%, 30%, 40%, 50%, 60%, 70%, or more than in the non-vaccinated control group after challenge with the infectious agent.
[0109] “Long-lasting protection” refers to “improved efficacy” persisting for at least 3 weeks, more preferably at least 3 months, and most preferably at least 6 months. In the case of livestock, the utmost preference is for long-lasting protection to endure until the average age at which animals are marketed for meat.
[0110] An “immunogenic or immunological composition” pertains to a composition comprising at least one polypeptide eliciting an immunological response, preferably conferring protective immunity against one or more clinical signs of S. suis infection.
[0111] “Immunogenic” polypeptide or “antigen” refers to a polypeptide that elicits an immunological response.
[0112] An “immune response” includes the development of a cellular and / or antibody-mediated immune response to the composition or vaccine. The host’s display of a therapeutic or protective immunological response enhances resistance to new infection and / or reduces clinical severity of the disease.
[0113] “Pharmaceutically or veterinary acceptable carrier or carrier” encompasses solvents, dispersion media, coatings, adjuvants, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, etc. Stabilizers for lyophilisation are preferred in certain embodiments.
[0114] “Isolated” signifies alteration “by the hand of man” from its natural state.
[0115] “Vaccination” refers to the administration of an immunogenic composition that elicits a protective immune response against S. suis.
[0116] “Mortality” refers to death caused by S. suis infection, including situations where euthanasia is necessary to prevent suffering.
[0117] “Therapeutically effective amount” means an amount of the recombinant polypeptide which when administered to an animal elicits an immune response against S. su / sthat reduces clinical signs or mortality.
[0118] “Cellular immune response” or “cell mediated immune response” is one mediated by T-lymphocytes or other white blood cells or both, and includes the production of cytokines, chemokines and similar molecules produced by activated T-cells, white blood cells, or both; or a T lymphocyte or other immune cell response that kills an infected cell.
[0119] “Humoral immune response” refers to one that is mediated by antibodies. “Immune response” in a subject refers to the development of a humoral immune response, a cellular immune response, or a humoral and a cellular immune response to an antigen. Immune responses can usually be determined using standard immunoassays and neutralization assays, which are known in the art.
[0120] “Therapeutic treatment” is the application of healthcare measures when ill, in the present disclosure this may include administration of the herein described immunogenic composition when a piglet is ill.
[0121] “Metaphylaxis treatment” the application of healthcare measures to a group of animals when at least one is diagnosed with a clinical infection, in the present disclosure this may include administration of the herein described immunogenic composition to the group of animals when at least one is diagnosed with a S. suis infection.
[0122] “Preventive healthcare” or “prophylaxis treatment” is the application of healthcare measures to prevent diseases, in the present disclosure this may include administration of the herein described immunogenic composition to a piglet to elicit an immune response against S. su / sthat reduces clinical signs or mortality.
[0123] As used herein, the transitional phrase “consisting essentially of’ is intended to limit the scope of a composition, method, or article to the specified elements or steps, together with such additional elements or steps that do not materially affect the basic and novel characteristics of the invention. Accordingly, components or steps that do not alter the functional or technical contribution responsible for the advantageous effects described herein are encompassed within the scope of claims reciting “consisting essentially of,” whereas components or steps that materially affect such characteristics are excluded.
[0124] The basic and novel characteristics of the present invention include, without limitation, the ability of the claimed compositions to elicit a protective immune response against Streptococcus suis mediated by one or more complete SEK repeat domains. Additional components, excipients, carriers, linkers, fusion partners, conjugates, adjuvants, tags, or formulation aids that do not materially affect this protective immune function are not excluded by use of the phrase “consisting essentially of’.EXAMPLES
[0125] The following examples describe exemplary modes of making and practicing certain compositions and methods described herein. These examples are provided for illustrative purposes only and are not intended to limit the scope of the invention as defined by the appended claims. Unless otherwise indicated, all parts and percentages are by weight.Example 1: Structure-Based Design and Truncation of Sao Polypeptides
[0126] Example 1 describes the structure-based rationale used to design truncated Sao polypeptide fragments for experimental evaluation. Because the location of the protective epitope within Sao was unknown at the outset, constructs were designed to systematically include or exclude structurally defined regions, including the D1 and D2 domains and the C-terminal repeat region, in order to enable subsequent correlation of structure, immunogenicity, and protective efficacy.Structure prediction
[0127] Reference is made to FIG. 1 and FIG. 2.
[0128] The prediction of the Sao polypeptide structure was approached through computational methods which involve the utilization of homology modeling, ab initio and machine learning approaches, considering several Sao wild type sequences.
[0129] The initial step involved the identification of the optimal experimental polypeptide templates, and multiple sequence alignments were generated using bioinformatics tools to establish conserved regions and evolutionary relationships among the chosen templates and target sequences. Computational models were generated using the above-described computational methods. The generated models were then subjected to an independent classification process that assessed their structural characteristics. This step allowed for systematic and unbiased evaluation ofthe models based on their intrinsic features. Post model generation and classification, a selection process was implemented to choose at least one model from each generation protocol. The selection was based on the scoring functions inherent to each modeling system, considering quality scores specific to the employed methodology to ensure a diverse representation of the models each chosen for its optimal structure attributes.
[0130] Following the model selection, a comprehensive evaluation phase was undertaken using independent bioinformatics tools. These tools assessed the quality of the top-ranked models chosen in the previous step. The evaluation process included a thorough examination of structural parameters, such as Ramachandran plots, RMSD (Root Mean Square Deviation), and MolProbity scores, providing a holistic understanding of the overall model quality. The best overall model was then identified based on the results of this rigorous evaluation, contributing to a robust and reliable prediction of the Sao parent polypeptide’s three-dimensional structure.B-cell epitope prediction
[0131] The SVMTriP tool (available on the internet website of System Biology Laboratory Of Chi Zhang Center for Plant Innovation) was chosen for B-cell epitope prediction, using the full-length Sao sequence (accession number: AAX57440.1; SEQ ID NO: 23), as it combines a Support Vector Machine (SVM) with tripeptide similarity and propensity scores (Yao et al., SVMTriP: A Method to Predict Antigenic Epitopes Using Support Vector Machine to Integrate Tri-Peptide Similarity and Propensity. PLoS ONE. 2012; 7:e45152). SVMTrip prediction method combines both linear and conformational analyses for B-cell epitope.
[0132] To achieve optimal performance, the predicted epitope length was set to 20 amino acids (a. a.), corresponding to sensitivity and specificity values of 80% and 55%, respectively. However, most experimentally validated epitopes in the Immune Epitope Database (IEDB) database (a freely available resource funded by the National Institute of Allergy and Infectious Diseases of the United States National Institute of Health) are shorter than 20 residues (Vita et al., (2010)). The immune epitope database 2.0. Nucleic acids research 38: D854-862). Therefore, additional predictions were performed using epitope lengths of 10 and 16 amino acids. A minimum score of 0.8,once rounded up, as recommended by the tool, was used as the cut-off for epitope selection.Results
[0133] The improvement of protein production yield and stability through sequence truncation requires an understanding of protein structure and the role played by specific amino acids in folding, stability, and solubility. Accordingly, this investigation was initiated by predicting the three-dimensional structure of the Sao polypeptide. A combination of computational methods was employed to generate 58 models, each evaluated to identify the most reliable structural candidate. One model was identified as the best, achieving a high-confidence QMEAN score (FIG. 1). Evaluation of the structural reliability of the Sao polypeptide using the QMEAN scoring protocol identified high prediction accuracy within amino acids 51 to 270, corresponding to the structured domains D1 and D2 (FIG. 2). These domains feature well-structured cores with a / 3-barrel fold, while the remaining regions of the protein are largely disordered, comprising ten tandem amino acid sequences of thirty amino acids each. Sequence analysis of the Sao polypeptide further revealed a signal sequence at the N-terminus and an LPXTG motif forming part of the cell wall sorting signal (CWSS) at the C-terminal region, which is characteristic of cell wall-anchored proteins of Gram-positive bacteria. This structural information indicates that the Sao polypeptide is anchored to the cell wall via its C-terminal region.
[0134] Epitope prediction tools were then employed to identify specific regions within the protein that could serve as potential epitopes for rational fragment design. The SVMTriP epitope prediction software, which can predict both linear and structural 13-cell epitopes, was utilized. However, rather than having a small, well-defined epitope region, the analysis revealed multiple putative immunogenic epitopes distributed throughout the Sao parent polypeptide, spanning both structured and disordered regions (FIG. 3). This analysis confirms that D1, D2, and repeats 6-10 have predicted B cell epitopes. This widespread distribution complicates the rational design of epitopebased fragments. Indeed, it was not possible, using prediction tools, to predict with confidence whether a single region of the Sao parent polypeptide provides protection against S. suis.Conclusion
[0135] Structural analysis of the Sao parent polypeptide indicates that the C-terminal region comprises the membrane insertion and cell wall anchoring sequences, including the LPXTG motif and associated hydrophobic segment. As a result, regions of the polypeptide located distal to the C-terminus (particularly the N-terminal structured domains and adjacent repeat regions) are predicted to be positioned farther from the bacterial cell wall and therefore more likely to be exposed on the surface of Streptococcus suis. Such surface exposure is consistent with accessibility to host antibodies and immune effector mechanisms.
[0136] As shown in FIG. 2, reliability analysis of the predicted structural model using the QMEAN scoring protocol further indicates that the model is most reliable for the N-terminal region encompassing domains D1 and D2, while confidence is reduced for amino acid positions approximately 240 to 480, corresponding largely to the disordered repeat domain region. This reduced structural confidence is consistent with the repetitive and flexible nature of the Sao repeat domains and does not preclude their surface exposure or immunological relevance, as demonstrated by subsequent experimental immunogenicity and protection studies described herein.Initial fragment design based on structural prediction
[0137] Based on structural and sequence analysis and given the absence of data indicating a well-defined antigenic region within Sao, a rational fragment design was developed using insights from the generated three-dimensional structure (see FIG. 1). Initially, four truncated versions of the Sao protein were designed, as illustrated in FIG.4D to FIG. 4G:a) Version 1 (V1, amino acids 43-276), which includes N-terminal domains 1-2, which are well-structured. See FIG. 4D.b) Version 2 (V2, amino acids 43-599), which includes N-terminal domains 1-2, the junction segment, and ten repeat domains. See FIG. 4E.c) Version 3 (V3, amino acids 43-444), which includes N-terminal domains 1-2, the junction segment, and five repeat domains. See FIG. 4Fd) Version 4 (V4, amino acids 277-444), which consists of the junction segment, and first five repeats. See FIG. 4G.
[0138] This example establishes the conceptual framework for the invention by demonstrating that rational fragment design based solely on predicted structure or in silico B-cell epitope mapping was insufficient to identify a protective epitope within the Sao polypeptide. Accordingly, a series of Sao polypeptide fragments were deliberately engineered to include regions predicted to be surface-exposed and to contain at least some of the predicted B-cell epitopes, while enabling systematic experimental evaluation of immunogenicity and protection.
[0139] The C-terminal region of the Sao parent polypeptide was deleted in all constructs to remove the LPXTG-containing cell wall sorting signal and associated hydrophobic membrane-spanning domain, thereby preventing anchoring of recombinant proteins to the E. coli cell envelope and avoiding toxicity, mislocalization, or degradation during heterologous expression. In addition, selected N-terminal sequences, including the signal peptide and portions of the N-terminal region upstream of the structured D1 domain, were deleted to eliminate sequences that are not required for immunogenicity, are cleaved or processed during native secretion in Streptococcus suis, or that may impair recombinant expression, solubility, or stability in E. coli. Removal of these N-terminal sequences further enabled production of non-naturally occurring, cytosolic recombinant fragments that are more amenable to industrial manufacturing.
[0140] Having established these structurally and biologically rational truncations, the next step was to determine which designed Sao polypeptide fragments could be produced efficiently in E. coli at commercially viable levels and which exhibited suitable physicochemical stability in liquid formulations. Accordingly, the truncated fragments were subjected to comparative expression, purification, and stability analyses prior to evaluation in animal immunization and challenge studies.Example 2: Manufacturing Yield and Stability of Sao Fragments
[0141] The manufacturability and physicochemical stability of the Sao polypeptide fragments designed in Example 1 was next evaluated. Because commercial vaccine development imposes constraints on protein yield and stability, these experiments were conducted to determine whether whether the Sao polypeptide fragments could be made cost effectively using E. coli and whether the expressed proteins would be stable in liquid.Materials and MethodCloning of V1, V2, V3, V4, Vm and SP1 polypeptides
[0142] The method used for the cloning of the SP1 polypeptide is described in WO 2007 / 025390. For the V1, V2, V3, V4 and Vm polypeptides, Escherichia coli (E. coli) strains TOP10 and BL21(DE3) were used for subcloning and expressing the different Sao polypeptide versions, respectively. The following plasmids containing a coding nucleic acid molecule were used for expressing the various Sao polypeptides:a) For V1: amino acid sequence set forth in SEQ ID NO: 5 and the plasmid pD454-SR (ATUM) - the resulting construct corresponds to pD454_V1 (SEQ ID NO: 17);b) For V2: amino acid sequence set forth in SEQ ID NO: 6 and the plasmid pET21b(+) (GenScript Biotech);c) For V3: amino acid sequence set forth in SEQ ID NO: 8 and the plasmid pD454-SR (ATUM) - the resulting construct corresponds to pD454_V3 (SEQ ID NO: 20);d) For V4: amino acid sequence set forth in SEQ ID NO: 9 and the plasmid pD454-SR (ATUM); ande) For Vm: amino acid sequence set forth in SEQ ID NO: 4 and the plasmid pET21b(+) (GenScript Biotech).
[0143] Gene synthesis, codon optimization and cloning in pET21b(+) and pD454-SR were performed by GenScript (V2 and Vm) and ATUM (V1, V3 and V4), respectively. All these sequences contain a histidine tag (his6) and a TEV cleavage site, or an MBP tag and Factor Xa cleavage site (for SP1 polypeptide), which are underlined in the amino acid sequences in Table 2 and are not part of the SEQ ID amino acid sequence. The constructions were confirmed by sequencing using the primers listed in Table 2. All the plasmids were transformed into competent BL21(DE3) cells by heat-shock, for the expression of the different Sao polypeptides.Table 2: Amino acid sequence for each Sao versionsTable 3: List of primersProtein expression and purification of V1, V3, V4, Vm, SP1
[0144] The method used for the expression of the SP1 polypeptide, and its purification are described in WO 2007 / 025390. E. coli BL21(DE3) strains carrying the expression plasmid for V1, V3, V4 and Vm were cultivated to produce a cell paste that could be used for protein purification. Briefly, an overnight culture in LB broth supplemented with ampicillin was used to inoculate TB broth supplemented with ampicillin. The cultures were grown at 37°C at 180RPM until an optical density (600nm) of 0.5-0.8 was reached. The cultures were then induced with IPTG at a final concentration of 0.5mM to 1mM. The cultures were incubated at 21 °C for 18 to 22 hours. Pellets were recovered from 2L cultures following centrifugation and the cell pellets (i.e., cell paste) were frozen in an ultra-low temperature freezer (-60 to -90°C) until purification.
[0145] Following the cell paste production, the proteins were purified using a protein purification system. Briefly, cell pastes were resuspended in 10mL of 20mM HEPES, 200mM NacL, pH 7.5 per gram of pellet. The suspensions were lysed using chemicals (0.1 mg / mL Lysobac, 2.4 U / mL benzonase nuclease, 2 mM MgCI2and 0.5mM phenylmethylsulfonyl fluoride (PMSF)) and ultrasounds (3 times - 3 minutes). The lysates were then centrifuged (1 h, 27200g, 4°C), and the soluble fractions were loaded to an Ni Sepharose affinity chromatography column. Proteins were eluted by increasing the concentration of imidazole. Fractions were then analyzed on SDS-PAGE and the fractions containing the protein of interest were pooled and concentrated using ultrafiltration devices prior to being purified on a size exclusion chromatography column, using 20mM HEPES, 200mM NaCL, pH 7.5. Fractions were analyzed on SDS-PAGE and the fractions containing the protein of interest were pooled and concentrated, if needed. Concentration was then evaluated using a BCA assay kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. All polypeptides were then aliquoted and conserved for long-term storage at -20°C in 20mM HEPES, 200mM NaCI at pH 7.5. Aliquots of V1 and V3 proteins were also kept in a refrigerator (2-8°C) to assess their long-term stability.Assessment of purity levels
[0146] To evaluate the purity of the purified Vm, V1, V3 and V4 polypeptides, a sample of each polypeptide was loaded into an SDS-PAGE, the gel was stained and analyzed using the Coomassie parameters of a ChemiDoc™ Imaging System (Bio-Rad).Long-term stability
[0147] To evaluate the long-term stability of the V1 and V3 polypeptides, a respective sample of these proteins was stored in a refrigerator (2-8°C), up to 18 months postpurification. Protein concentration was first evaluated using A280 and BOA assay, according to manufacturer’s instruction. Two methods were then used to assess their stability: observation of a degradation pattern via SDS-PAGE and evaluation of their thermostability by differential scanning fluorimetry (DSF).
[0148] To evaluate the degradation profile of the V1 and V3 proteins over 18 months storage in a refrigerator, a total of 10 pg of each protein was loaded on an SDS-PAGE gel, which was revealed using the stain-free parameters of a ChemiDoc Imaging System (Bio-Rad).
[0149] To evaluate the thermostability of the purified proteins, differential scanning fluorimetry (DSF) was performed. Briefly, 5 pg of the purified V1, V3 or V4 protein plus 5X Sypro™ Orange Protein Gel Stain were processed in a 96-well PCR plate. The plate was heated from 10 to 95°C with a heating rate of 0.05°C for 10 seconds in a qPCR. The fluorescence was measured using the FRET channel of the equipment. The melting curves were then analyzed, and the melting temperature was calculated using the derivative curve determination method.Results
[0150] Expression plasmids encoding for codon optimized nucleic acid sequences of the truncated Sao polypeptides (V1-V4) and the mature protein (Vm) were transformed into E. coli BL21(DE3) for protein production. However, we observed that the transformation of the plasmid encoding V2 was toxic to the cells, suggesting that this truncation results in a protein version whose expression induces a toxic effect. As a result, V2 could not be further characterized. The remaining polypeptide versions were expressed in E. coli BL21(DE3) and subjected to a meticulous two-step purification process involving affinity and size exclusion chromatography, as in Example 2. The resulting production yields were compared with those obtained from the purification of the SP1 and Vm polypeptides, as illustrated in FIG. 5A. Notably, V1, V3 and V4 polypeptides exhibited significantly higher production yields, demonstrating an impressive improvement ranging from from 90 to 430-fold compared to the SP1 polypeptide.
[0151] SP1 was difficult to purify and quickly degraded into a smear of protein sizes when stored in liquid (data not shown). The Sao fragments Vm, V1, V3, and V4 were easily purified, as shown in FIG. 5B.
[0152] This comparative analysis provides valuable insights into the purification efficiency and stability of the truncated Sao variants, supporting informed decisions regarding their commercial potential.
[0153] Long-term stability is a key requirement for a commercial vaccine to meet regulatory shelf-life standards. Given the poor protein yields obtained for SP1 and Vm, these versions were excluded from long-term stability assessment. A preliminary evaluation of the most promising candidates, V1, V3, and V4 polypeptides, was conducted to assess their stability over a period ranging from one week to 18 months post-purification with storage in a refrigerator (2-8°C). Degradation profiles were analyzed using SDS-PAGE, alongside protein thermostability measurements, which indicate structural integrity. The results showed no detectable degradation for V1, V3, or V4 polypeptides after one-week post-purification (data not shown). Using the DSF assay, only only V1 and V3 were confirmed to be stable with storage in a refrigerator (2-8°C) as shown on FIG 6A. and FIG. 6B.
[0154] After 18 months, the thermostability data for V1 and V3 polypeptides confirmed the maintenance of their thermal stability (Tm) by DSF, given that there is no shift in melting temperature; DSF (FIG 7B) shows no shift in melting temperature when compared to time 0 (Fig 6A). Moreover, there is no detectable degradation by SDS-PAGE given that the polypeptides are still present at the expected size. These results, reinforce the potential of V1 and V3 for commercialization (FIG. 7A and FIG. 7B).
[0155] These results demonstrate that removal of N-terminal (amino acids 1 to 42) and C-terminal (amino acids 445 to 670 for V3; or amino acids 277 to 670 for V1) regions of Sao substantially improves recombinant expression yield and protein stability. Importantly, this example establishes that truncated Sao fragments are industrially feasible vaccine antigens, enabling downstream evaluation of their immunogenic and protective propertiesExample 3: Immunogenicity of Sao Fragments
[0156] Example 3 was conducted to assess whether the Sao polypeptide fragments induce antigen-specific immune responses in vivo. As Streptococcus suis is an extracellular bacterium, antibody responses were evaluated as an initial indicator ofbiological activity, with the understanding that immunogenicity alone may not correlate with protection.Production of immunogenic composition candidates
[0157] V1 and V3 polypeptides were produced and purified as described in Example 2. Level of purity of the purified polypeptides was assessed by SDS-PAGE and LC / MS-MS and was confirmed to be at least of 98% for each candidate. Protein concentration for administration was determined by BOA method, using BSA as a standard.Study Design
[0158] Three-week-old healthy piglets from a farm with low S. suis antibody levels, determined using a commercial S. suis Antibody ELISA test kit (Green Spring™ (S. suis) Antibody ELISA test kit; Gentaur), were selected and were randomly assigned to each group. Each parity and sex were represented in each group. Animals were immunized intramuscularly with a primer dose at three-week old and a booster two-weeks later with 1mL of either 300pg V1 or 300pg V3 alone (V1 or V3 control group; n=5) or in combination with 20% of the commercially available Emulsigen-D adjuvant (Phibro Animal Health; V1+ or V3+ adjuvant; n=8) in phosphate buffer saline. Blood sampling was carried out throughout the experiment to obtain sera before the first dose (DO), then each week (D7, D14 and D21) following immunization until two-weeks postbooster (D28).
[0159] Blood samples were collected from all pigs prior to the first and second vaccinations, as well as before the challenge, to assess the IgG antibody response specific to the V1 or V3 candidate vaccine using the ELISA test. Briefly, 10OpL V1 (0.3 pg / mL) or 3 (0.0125 pg / mL) in carbonate buffer were coated in Flat-Bottom Immuno 96-Well Polysorp plates (ThermoFisher) overnight at 4°C. Between each step, plates were washed 3 times with phosphate buffer saline with 0.5% Tween-20 (PBST). The wells were blocked with 1% bovine serum albumin fraction (BSA; Sigma Aldrich) at room temperature for 1h. All sera were diluted 1 / 100 in PBST and 100pL of sera were added to each well and incubated for 1h at room temperature. For determination of swine total IgG, 100pL of a 1 / 4000 dilution of HRP-Goat anti-Swine IgG(Fc) (BioRad) in PBSTwas added to the well and incubated for 1h at room temperature. Finally, 100pL of TMB single solution (ThermoFisher) was added and incubated for 20 minutes at room temperature. After stopping the reaction with 50 pL of 1N H2SO4, theabsorbance was measured with a microplate reader (BioTek) at 450nm. The optical density at 450nm, after subtracting the blank, gave the intensity of the IgG response.
[0160] Statistical differences in antigen-specific IgG responses between control animals (receiving the corresponding Sao polypeptide without adjuvant) and vaccinated animals (receiving the same polypeptide formulated with adjuvant) were evaluated using the non-parametric Mann-Whitney U test.Results
[0161] To evaluate the ability of the Sao-based antigens V1 and V3 to elicit an immune response in piglets, we conducted an animal study using weaned piglets. The antigens were administered either with or without an oil-in-water adjuvant, at 3 weeks of age, followed by a booster dose at 5 weeks of age. The immunogenicity of the different vaccine formulations was assessed by measuring the generation of specific antibodies against the administered antigens. The results demonstrated that both V1 and V3 antigens successfully induced a specific immune response in piglets when administered with an adjuvant and following two doses (FIG. 8A and FIG. 8B). Based on strong antibody response, pigs vaccinated with V1 or V3 were challenged with virulent S. suis to assess protection from disease
[0162] This example demonstrates that multiple Sao fragments, including constructs lacking C-terminal repeat regions, are strongly immunogenic. However, these data do not establish protective efficacy and instead serve to distinguish immunogenicity from protection in subsequent challenge studies.Example 4: Evaluation of Sao Polypeptide Fragment Vaccines to Protect against S. suis Experimental Challenge
[0163] Example 4 was designed to determine whether Sao polypeptide fragments that are immunogenic are also capable of conferring protection against Streptococcus suis challenge.Study Design
[0164] The study design used in this example is similar as the one described in Example 3, with the following additional details. The timeline of the study is presented in FIG. 9. Controls animals were unvaccinated.
[0165] Animals were monitored for 14 days post-challenge (2 times per day) for clinical signs, including depression / lethargy (behavioral changes), locomotion issues (lameness), and neurological signs. A scoring system was used to assess clinical signs, ranging from 0 (normal) to 4 (severe) for behavior, 0 (normal) to 3 (severe) for locomotion, and 0 (normal) to 3 (severe) for clinical neurological signs (CNS), such as tremors, head tilt, ataxia, nystagmus, seizure and pedaling.
[0166] Pigs reaching humane endpoints (considered as severe above for at least one clinical sign) were euthanized immediately, while all surviving animals were euthanized at the end of the study (14 days post-challenge). The timeline of the study is presented in FIG. 9. Blood samples were collected from all pigs prior to the first and second vaccinations, as well as before the challenge, to assess the IgG antibody response specific to the V1 or V3 candidate as vaccines using the ELISA test. Briefly, 10OpL V1 or V3 (0.3 pg / mL) in carbonate buffer were coated in Flat-Bottom Immuno 96-Well Polysorp plates (ThermoFisher) at 37°C for 1h. Between each step, plates were washed 3 times with phosphate buffer saline with 0.5% Tween-20 (PBST). The wells were blocked with 1% bovine serum albumin fraction (BSA; Sigma Aldrich) at room temperature for 1h. Serial dilution (1:4) of sera were prepared and 100pL of the serially diluted sera were added to each well and incubated for 1h at room temperature. For determination of swine total IgG, 100pL of a 1 / 4000 dilution of HRP-Goat anti-Swine IgG(Fc) (BioRad) in PBST was added to the well and incubated for 1h at room temperature. Finally, 100pL of TMB single solution (ThermoFisher) was added and incubated for 15-20 minutes at room temperature. After stopping the reaction with 50 pL of 1N HCI, the absorbance was measured with a microplate reader (BioTek) at 450nm. Antibody titers were determined as reciprocal of the last dilution that resulted in an optical density at 450 nm of 0.2, after subtracting the blank. If the sample did not reach the 0.2 threshold, a value of 10 was assigned. The statistical difference in immunological results between control group and vaccinated groups was compared using the Mann- Whitney test.Results
[0167] To assess the efficacy of the V1 and V3 antigen candidate as vaccines, an intramuscular challenge model in pigs was used. In susceptible animals, the intramuscular administration of a high concentration of S. suis serotype 2 strain induced behavioral and locomotor symptoms, as well as neurological signs. In most cases, clinical symptoms progressed to a humane endpoint requiring euthanasia. Bythe end of the study, the mortality rate in the unvaccinated control group reached 76% (see, Table 4).
[0168] Although this model results in more severe outcomes compared to natural farm infections, where mortality rarely exceeds 20%, it effectively replicates S. su / s-related clinical signs, making it a robust tool for vaccine evaluation.
[0169] In the V3-vaccinated group, mortality was reduced by 30% (Table 4) accompanied by a statistically significant reduction in clinical signs (Table 5). Vaccine efficacy is reported using Prevented Fraction: PF = 1- (ARV / ARC), where ARV = attack rate vaccinates, and ARC - attack rate controls.
[0170] Additionally, the number of piglets in which the S. suis challenge strain was detected in the blood at least once post-vaccination was 49% lower than in the control group (Table 4), indicating improved infection control in pigs vaccinated with V3.
[0171] For the V1-vaccinated group, the immunogenic or vaccine candidate was not able to protect pigs from the challenge administration. Indeed, the mortality rate was 84% (higher than the control group) with no reduction of clinical signs when compared to controls and the reduction in bacteremia was similar to controls.Table 4: Post-challenge data
[0172] +Number of animals in which the S. suis challenge strain was detected in the blood at least once for 7 days post-challenge.Table 5: Sum of the daily average clinical scores upon 14 days post-challenge
[0173] ++statistical diminution of clinical scores between V3-vaccinated group and Control group (p<0.0001), based on Wilcoxon Ranked Sum analysis.
[0174] Since serological data demonstrated that both V1 and V3 candidate vaccine antigens induced a strong and significant IgG response (FIG. 10 and FIG. 11, respectively), the lack of protection in pigs vaccinated with V1 does not indicate an absence of immune response but rather the absence of protective antibodies. The only difference between V1 and V3 was the inclusion of five repeated sequences downstream of D1 and D2 domains.
[0175] Despite inducing strong antigen-specific IgG responses, only the vaccine candidate containing repeat domains (V3) provided protection. Since 5 repeat domains were present, the next step was to determine how many repeat domains were required. Example 5: Evaluation of Vaccine Antigen SP1 containing all 10 Repeat Domains vs. SPIAwith truncated repeats
[0176] The SP1 vaccine antigen was described in WO 2007 / 025390 and contains D1 / D2 structural domains plus all 10 downstream repeats (FIG. 4B). In this study, the efficacy of the SP1 vaccine antigen was compared to a truncated protein containing D1 / D1, all of the first repeat, and part of the second repeat domain (SP1A, FIG. 4J) using a mouse vaccination and challenge model.Cloning of SPIA and SP1 polypeptides
[0177] The method used for the cloning of SP1 version is described in WO 2007 / 025390. Forthe truncated version SP1 A, an amplification of the region containing amino acid 28-342 was performed. Restriction sites for Ndel or BamHI were included in the forward and reverse primers, respectively:a) SP1A-Reverse (BamHI): GTT GGATCC TTA CTTTTCAGGTGTTACGACCATG (SEQ ID NO: 1)b) SP1A-Forward (Ndel): GTT CATATG TCGGCACAAGAAGTAAAAAATACC (SEQ ID NO: 2)
[0178] These restriction sites were used to clone the resulting PCR fragment in the Ndel and BamHI sites of the pET16b expression vector (Novagen) - the his-tag and cleavage site are underlined and are not part of the SEQ ID NO amino acid sequence.Table 6: Amino acid sequence for the SP1 A truncated versionProtein expression and purification of SP1 A and SP1
[0179] The method used for the expression of SP1 polypeptide, and its purification are described in WO 2007 / 025390. The vector for SP1 A polypeptide fragment expression was transformed into E. coli BL21(DE3) and expression of the proteins was performed at 37°C for 3h after induction with 1mM IPTG in LB broth. Extraction of the protein of interest was performed by sonication, then the extract was purified using affinity chromatography with an His-Pur Ni-NTA affinity (Thermo Fischer Scientific). Fractions obtained following elution at 250mM were pooled together and a dialysis in phosphate buffer saline was performed to remove imidazole. The proteins were then used in a mice immunization study.Study design immunization study with SP1 A polypeptide fragment
[0180] To assess the protective potential of his-tagged SP1A polypeptide fragment (amino acid 28-342), five-week-old CD1 mice were randomly assigned to two groups of 14 to 15 mice for immunization with one of the candidates or a placebo. Animals were immunized twice subcutaneously at approximately a 2-week interval with 50pg SP1 A polypeptide fragment combined with 20pg of Quil-A® as an adjuvant. The control group received 20pg of Quil-A adjuvant in combination with PBS. Approximately, two weeks after the second vaccination, animals were intraperitoneally challenged with 7x107CFU of S. suis serotype 2.
[0181] Mice were monitored post-infection for the development of clinical signs of sepsis, such as depression, swollen eyes, rough hair coat, prostration, and lethargy, as well as neurological signs. Mice were immediately euthanized upon reachingendpoint criteria using CO2, followed by cervical dislocation. All surviving mice were euthanized as described above at the end of the experiment (12 days post-infection). Study design immunization study with SP1 and SP1 A polypeptide fragments
[0182] To confirm the protective potential of the SP1 polypeptide, as previously described by WO 2007 / 025390, approximately four-week-old CD1 mice were randomly assigned to two groups of 15 mice for immunization with SP1 or a placebo. Animals were immunized twice subcutaneously at an approximatively 2-week interval with either 25pg (dose 1) or 40pg (dose 2) SP1 combined with 20pg of Quil-A® as an adjuvant. The placebo group received 20pg of Quil-A adjuvant in combination with PBS. Approximately two weeks after the second vaccination, animals were intraperitoneally challenged with 6.1x107CFU of S. suis serotype 2.
[0183] Mice were monitored post-infection for the development of clinical signs of sepsis, such as depression, swollen eyes, rough hair coat, prostration, and lethargy, as well as neurological signs. Mice were immediately euthanized upon reaching endpoint criteria using CO2, followed by cervical dislocation. All surviving mice were euthanized as described above at the end of the experiment (8 days post-infection). Results
[0184] The SP1A polypeptide fragment purified using his-tag did not show protection in a mouse model of infection (Table 7). Indeed, five days after a challenge infection with S. suis serotype 2, mice from all groups began to present severe clinical signs of disease including rough hair coat, depression, prostration, and general weakness, leading to the euthanasia of certain individuals. However, from day 6 post-challenge, several of the mice developed severe signs of central nervous system infection. A total of 9 / 14 mice immunized with SP1A polypeptide fragment, and 9 / 15 mice that received the placebo were euthanized for ethical reasons. No difference in survival between the control group (placebo) or any of the treated group could be observed (LogRank test; p > 0.05).Table 7: Post-challenge data
[0185] Given the absence of protection observed using the his-tagged SP1A, the SP1 protein was purified as described by WO 2007 / 025390 and was used to immunize CD1 mice. Results showed that this version of the Sao protein was able to induce significant protection (LogRank test; p=0.0002) of the mice following a challenge with S. suis serotype 2 (Table 8), when compared to a placebo group.Table 8: Post-challenge data
[0186] Conclusion: These results demonstrate that the SP1A polypeptide fragment, despite containing the N-terminal domains D1 and D2, a complete first repeat domain (Repeat 1), and an incomplete second repeat domain (Repeat 2) having the amino acid sequence SEKQMPSWNDNAVTPEK (FIG. 4J) is insufficient to provide protection against S. suis infection. In contrast, the full-length SP1 polypeptide, which contains all 10 repeat domains, confered significant protection.
[0187] Without being bound by theory, the lack of protection observed with the SP1A polypeptide fragment is both unexpected and instructive. Although the SP1 A fragment was previously shown by WO 2007 / 025390 to elicit a humoral (antibody) response in mice, the ability to induce an immune response alone does not translate into protective immunity in vivo, as demonstrated by the lack of protection from disease shown in Table 7.
[0188] The first repeat domain from the Sao protein of this S. suis strain has a starting amino acid sequence of SKE. The second domain and all subsequent repeat domains in this Sao full length protein have an N-terminal amino acid sequence starting with SEK. These data suggest that a full-length SEK repeat domain is required for protection.Example 6: Conservation of the SEK Repeat Among S. suis Isolates
[0189] Example 6 examines the prevalence and sequence conservation of Sao and its repeat domains among a diverse collection of Streptococcus suis isolates. This analysis was performed to assess whether the identified protective SEK repeat domain represents a conserved feature among clinically relevant strains.Genome Analyses
[0190] Most of the genomes analyzed were sourced from Roodsant et al. (2021; DOI: 10.5281 / zenodo.4686597). A subset of 235 S. suis genomes was selected from the 1,703 genomes, consisting of 124 strains isolated from pork disease and 111 S. suis specially belonging to serotype 9. Additionally, 18 S. suis strains sequenced by the inventors, two virulent strains confirmed experimentally by Nicholson et al. (2021, DOI: 10.3389 / fmicb.2020.620843), 21 strains isolated in North America by Estrada et al. (2022; DOI: 10.1186 / s13567-022-01039-8), and 19 strains isolated in Spain by Uruen et al. (2024, DOI: 10.1186 / s13567-024-01267-0) were included to the 235 S. suis datasets to enhance the diversity of Sequence Types (STs). Most bioinformatics analyses were conducted on this 295-genome subset. The 124 S. suis genomes isolated from swine disease were selected to reflect the proportions of the 19 most abundant STs observed in the 1,703 genomes dataset.
[0191] Using bioinformatics, the proportion of ST among the 1,703 genomes was calculated, and this information was used to randomly select the appropriate number of genomes per ST. Genomes were annotated using Prodigal v2.6.3 (Hyatt et al. 2010; DOI:10.1186 / 1471-2105-11-119) to predict the Open Reading Frames (ORFs) to identify the Sao proteins among the S. suis genomes. First the two conserved domains were searched. The domains of the Sao proteins were searched using BLASTp v 2.15.0+, with proteins showing >=85% coverage and >=70% similarity considered positive matches. BLASTp similarity was also reduced to >=60% to identify repeated sequences. The portion of the Sao protein after domain 2, corresponding to the repeat section was then analyzed with a Python script to define all the repeats. Identical domains and repeats were clustered using CD-Hit v4.8.1 (-aL 1 -c 1) (Fu et al. 2012; DOI: 10.1093 / bioinformatics / bts565) to remove redundancy and assess the diversity and frequency of each sequence. Each unique sequence was aligned using MUSCLE V3.8.1551 (Edgar, R. C., 2004; DOI: 10.1093 / nar / gkh340), and low-frequency amino acid positions (<=10%) were removed from the alignment (FIG. 12). Multiple Sequence Comparison by Log-Expectation (MUSCLE) is a widely used software program for creating multiple sequence alignments of DNA, RNA, and protein sequences (available at the Internet site of the European Bioinformatics Institute of the European Molecular Biology Laboratory).Sao Repeat Diversity
[0192] To investigate repeat diversity, the end position of domain D2 in the Sao protein was retrieved using the BLASTp alignment results. This information was then used to extract the downstream Sao section with samtools faidx, which contained the “SKE”and “SEK” repeats (FIG. 12). Subsequently, the repeats were annotated for each Sao protein using a Python script by searching the “SKE” and “SEK” motifs. Clustering of Sao repeats identified 12 distinct “SKE” and 58 “SEK” repeat sequences. The first repeat in each Sao protein always began with the “SKE” motif, while all subsequent repeats started with “SEK”. Conservation among the “SKE” repeats was assessed by calculating the number of substitutions using BLASTp. Results mainly revealed between one and three substitutions, corresponding to 90-96.7% of similarity, indicating a high conservation of “SKE” repeats across ST. T o further investigate where substitutions occurred, the 12 “SKE” repeats were aligned and six positions exhibited substitutions: D7G, P16S, A21T, N22T, E29Kand A30 (substituted by S, TorV) (Table 11).
[0193] The same strategy described above was applied to the 58 “SEK” repeat domains. BLASTp analysis revealed conservation levels among “SEK” repeats ranging from 96.7% to the threshold of 50%. Compared to the “SKE” motifs, the “SEK” repeats presented greater diversity, with at least 21 positions exhibiting one or more substitutions (Table 11).
[0194] At least three amino acid motifs of the “SEK” repeat domains, each consisting of 3 to 4 consecutive amino acids, were conserved across nearly all sequences (>97%). These conserved motif sequences include the first four amino acid residues (“1SEKQ4”), followed by amino acid residues at positions 14-16 (“14VTP16”), and finishing with amino acid residues at positions 22-24 (“22NKE24”) (Tables 11 and 12A-12E). Within the Vm, V3, V4, V5, and V6 polypeptides used in the herein described experimental assays, the SEK repeat domains retained the following expanded conserved motif sequences ISEKQMP6, 14 TP16, 19QM20, and 22NKE24.
[0195] A consensus SEK domain sequence was established as shown in Tables 12A-12E, and possible amino acid variations at each position were identified across 58 Strep suis isolates (Tables 12A-12E), thus representing variants having natural occurring mutations relative to the 30-mer consensus sequence.
[0196] The following table 10 illustrates a comparison of the amino acid sequence identity between the 10 repeats of Sao parent polypeptide AAX57440.1:Table 101 2 3 4 5 6 7 8 9 101 13% 23% 27% 27% 30% 30% 30% 33% 47% 2 73% 70% 70% 90% 90% 90% 83% 57% 3 97% 97% 63% 63% 63% 70% 73% 4 100% 60% 60% 60% 67% 77% 5 60% 60% 60% 67% 77% 6 100% 100% 93% 53% 7 100% 93% 53% 8 93% 53% 9 60%
[0197] The above table shows that repeat domains 4 and 5 are identical to each other, repeat domains 6 to 8 are identical to each other, repeat domain 3 is very similar to repeat domains 4 and 5 (1 amino acid difference), repeat domain 9 is very similar to repeat domains 6 to 8 (2 amino acid difference), repeat domain 2 is very similar to repeat domains 6 to 8 (3 amino acid sequence difference), and repeat domain 1 and repeat domain 10 are the most different from all repeat domains.Table 11: Main Sao repeats SKE and SEK sequence variations
[0198] Based on the amino acid variations set forth in Table 11, the SKE domain consensus sequence is SKEAMTDKEGNIEPPKEQIANEEKDNIEA (SEQ ID NO: 72), and a variant thereof has at least 90% identity over its full length. A variant of SEQ ID NO: 72 may have any one of the following amino acid mutations relative to SEQ ID NO: 72: D7G, P16S, A21T, N22T, E29K, A30S, A30T, and A30V (See FIG. 15B).Table 12A: Sao repeats SEK sequence variations in ISEKQMP6Table 12B: Sao repeats SEK sequence variations in 7SWNDNA13Table 12C: Sao repeats SEK sequence variations in 12VTPEKQM20Table 12D: Sao repeats SEK sequence variations in 21TNKEND26Table 12E: Sao repeats SEK sequence variations in 27K ET30
[0199] Based on the amino acid variations set forth in Tables 12A-12E, the SEK domain consensus sequence is SEKQMPSWNDNAVTPEKQMTNKENDNVET (SEQ ID NO: 73) and a variant thereof may have at least 50% identity over its full length and any one of the following amino acid mutations relative to SEQ ID NO: 73: M5K, M5I, P6T, S7V, V8I, V8N, V8S, V9M, V9E, N10K, D11E, N12M, D12T, A13V, V14I, E17Q, K18E, K18R, M20T, M20I, T21 A, T21D, E24V, N25K, N25R, D26E, N27K, N27E, V28I, E29V, E29L, T30I, and T30A (See Fig 15B). Preferably, the variant retains the conserved motifs 1SEKQ4, 14VTP16, and 22NKE24.
[0200] These results demonstrate that SEK repeat domains containing conserved motifs are widely distributed among Sao-positive S. suis isolates.Ordering and Distribution of the Repeats
[0201] The 232 identified Sao protein exhibited between 1 and 13 repeats - data not shown also demonstrate strains with 15 repeats. Sections of the Sao protein downstream of the D2 domain were separated based on repeat motifs “SKE / SEK”, where the sequence joining D2 to the first repeat (“SKE”) was designed as Pos=1, followed by the first repeat (“SKE”) named as Pos=2, and so on, until the last repeat (FIG. 13A). Each repeat was then assigned a position within the Sao protein. To assess whether the position of identical repeats is conserved among S. suis strains, repeat position data were included in the CD-Hit clustering analysis. Interestingly, repeat positions were generally conserved within the same cluster, highlighting the stability of the repeat order across strains (FIG. 13B).
[0202] Next, the distribution of repeat numbers across the 232 genomes was explored (FIG. 14). Notably, approximately 41% (96 / 232) of the Sao proteins contained seven repeats. Subsequently, the number of repeats per genome was also divided per ST (not shown). Distinct trends in repeat distribution were perceived, with nine STs, such as ST1, ST7, ST16, ST29, harboring an average of seven repeats. On the other hand, other STs, like ST25, ST27 and ST28, exhibited fewer repeats, averaging 2, 2 and 5 repeats, respectively.Example 7: Immunogenicity Mapping Demonstrates Lack of Correlation with Protection
[0203] Example 7 was conducted to determine whether antibody responses directed against specific regions of the Sao polypeptide correlate with protective efficacy. Antibody recognition of the D1 and D2 domains, the SKE repeat, and individual SEK repeats was therefore evaluated using sera from vaccinated animals.Materials and Method
[0204] Using sera obtained from the animal study described in Example 4, ELISA was performed to assess the recognition of the different regions ofthe m Sao polypeptide. To do so, the D1 domain (SEQ ID NO: 74) and D2 domain (SEQ ID NO: 75) were separately produced and purified as described in the previous example. The amino acid sequence of each domain is shown as per the following table 13. The his-tag and cleavage site are underlined and are not part of the SEQ ID NO amino acid sequence.Table 13
[0205] Peptides corresponding to each repeat of the Vm Sao polypeptide were synthesized (GenScript), as per the following table 14.Table 14: Amino acid sequence of synthesized peptides
[0206] The reader will understand that multiple repeat ID’s are listed in cases where repeated sequences are identical (e.g. Repeats 4-5 and 6-8).
[0207] ELISA were performed as described in Example 4 with a modification for the peptides for which 100pL of each peptide (1 pg / mL) in carbonate buffer were coated in Flat-Bottom Immuno 96-Well MaxiSorp plates (ThermoFisher) at 37°C for 1h. Antibody titers were determined as the reciprocal of the last dilution that resulted in an optical density at 450 nm of 0.2, after subtracting the blank. If the sample did not reach the 0.2 threshold, a value of 10 was assigned. The statistical difference in immunological results between control group and vaccinated groups was compared using the Mann- Whitney test.Results
[0208] Reference is made to FIG. 16, FIG. 17Aand FIG. 17B.
[0209] FIG. 16 presents a multiple sequence alignment of the ten 30-amino-acid repeat domains present in the Sao parent polypeptide of SEQ ID NO: 23. The alignment illustrates that, while the individual repeat domains exhibit varying degrees of amino acid divergence, they share a common repeat architecture characterized by conserved sequence motifs. In particular, several short consensus regions are retained across most repeats, including conserved residues for the SEK repeat domain at the N-terminal SEK motif, a central VTP motif, and a downstream NKE motif.
[0210] Despite this shared consensus framework, FIG. 16 also demonstrates that the repeat domains are not identical and differ substantially at multiple positions outside the conserved motifs. These sequence variations result in distinct repeat variants, even among repeats that occupy adjacent positions within the same Sao polypeptide. This intra-protein diversity highlights that the Sao repeat region is modular in nature rather than a uniform array of identical epitopes.
[0211] FIG. 17A illustrates that sera collected from pigs receiving two administrations of either the V1 polypeptide or the V3 polypeptide exhibited markedly elevated IgG titers against the D1 and D2 domains relative to control animals.
[0212] FIG. 17B demonstrates that sera obtained from animals immunized with the V3 polypeptide produced robust IgG antibodies that reacted with the SKE repeat domain (labelled “1”) and each of the SEK repeat domains (labelled “2”to “10”), which are present in the full length Sao parental polypeptide. The antibody titers against each of the 10 repeats were statistically significantly higher than found in sera from controls and V1. By contrast, animals immunized with the V1 polypeptide, which is devoid of repeat domains, failed to mount detectable IgG responses against any repeat, yielding titers indistinguishable from control values.
[0213] Within the V3-immunized group, there is a large and significant difference (similar across all groups), except for repeat 10. The observed reactivity against each of the isolated repeat domains 6 to 10 (each of which only share partial sequence identity with each of repeat domains 1 to 5 - see table 10) provides evidence that the repeat regions contain conserved epitopes.
[0214] Taken together, these findings confirm that immunization with the V3 polypeptide (a construct comprising a subset of Sao parent polypeptide repeat domains) elicits an antibody repertoire that recognizes repeat domains 6 to 10 that are beyond those explicitly encoded by the immunogen (which contains repeat domains 1 to 5). This is a surprising finding, since the number of repeat domains and their amino acid composition in Sao polypeptides from virulent S. suis strains can vary. Because only pigs vaccinated with V3 were protected from S. suis challenge, the inventors reasonably predict that the protective epitopes reside within the repeat regions of the Sao polypeptide.
[0215] Further experimentation revealed that immunization with a construct containing the D1 and D2 domains in combination with only the SKE repeat domain and a partial SEK repeat domain (the SP1A construct, Example 5) conferred no measurable protection against S. suis challenge. Likewise, a construct comprising solely the D1 and D2 domains (the V1 construct, Example 4) induced measurable anti-D1 and anti-02 IgG antibody titres but failed to provide in vivo protection.
[0216] Although strong antibody responses were observed against multiple Sao regions, including highly conserved D1 and D2 domains, these responses did not correlate with protection. In contrast, protective efficacy was consistently associated with constructs containing multiple SEK repeat domains.Example 8: Investigation of Smaller Peptides Derived from V3 as Candidate Vaccines
[0217] Example 8 was conducted to determine whether smaller peptides containing sequences found in V3 could be used as vaccine immunogens to protect mice from virulent S. suis experimental challenge..
[0218] The V3 (FIG 4F) and V4 (FIG 4G) Sao fragments were made a previously described in Example 2. The V5 polypeptide fragment (FIG 4H) consists of a single SEK repeat domain (Repeat 4, corresponding to amino acids 385-414 from SEQ ID NO 23). Note that Repeat 5 (corresponding to amino acids 415-444 of SEQ IS NO 23) has an identical amino acid sequence. The peptide having the sequence set forth in SEQ ID NO: 79 was chemically synthesized, and conjugated to keyhole limpet hemocyanin (KLH) carrier protein via the addition of a cysteine residue at the C-terminus (GenScript). The V6 polypeptide fragment (SEQ ID NO: 91; FIG 4I) corresponds to amino acids 164 to 444 of the full-length Sao polypeptide (SEQ ID NO: 23), comprising the D2 domain, the junction segment, the SKE repeat domain, andfour complete SEK repeat domains (Repeats 2-5). The V6 polypeptide is identical to the V3 polypeptide except that it lacks the D1 domain, thereby allowing evaluation of whether the D1 domain is necessary for protective immunity.Materials and Method
[0219] The V6 polypeptides was produced in E. coli and purified as described in Example 2. The amino acid sequence of V6 is set forth in Table 15.Table 15: Amino acid sequence of the V6 polypeptide fragment
[0220] The his-tag and cleavage site are underlined and are not part of the peptide used for vaccination.Study Design
[0221] To evaluate the protective potential of shorter versions of the Sao polypeptide, four-week-old CD1 mice were randomly assigned to 5 groups of 14 to 15 mice for immunization with V3, V4, V5, V6, or control phosphate buffer saline (PBS). Animals were immunized twice subcutaneously at a 2-week interval with either 50 pg (V3, V4, or V6) or 75 pg (V5-KLH conjugate) combined with TiterMaxGold (1:1) as an adjuvant. The placebo group received PBS only. Fourteen days after the second vaccination, animals were intraperitoneally challenged with 5 x 107CFU of a S. suis serotype 2 strain.
[0222] Mice were monitored post-infection for the development of clinical signs, such as activity / mobility, posture, fur condition, respiration, eyes, as well as neurological signs. Mice were immediately euthanized upon reaching endpoint criteria using CO2. All surviving mice were euthanized as described above at the end of the experiment. Results
[0223] The post-challenge data are summarized in Table 16. The mortality rate in the PBS control group was 93%. All vaccinated groups exhibited statistically significant reductions in mortality compared to the control group. The V3-vaccinated group showed a mortality rate of 53% (p<0.001), corresponding to 43% efficacy. The V4-vaccinated group showed a mortality rate of 60% (p<0.01), corresponding to 35% efficacy. The V6-vaccinated group (V3 without D1 domain) showed a mortality rate of 43% (p<0.01), corresponding to 54% efficacy.
[0224] Remarkably, the V5-vaccinated group, comprising only a single SEK repeat domain (SEQ ID NO: 79) conjugated to KLH, demonstrated protective immunity against S. suis challenge. The V5 group exhibited a mortality rate of only 13% (p<0.0001), corresponding to 86% efficacy.
[0225] Clinical scoring data further supported the protective efficacy of V5. The cumulative clinical scores were low in the V5-vaccinated group (18.6) compared to the control group (45.2). These data demonstrate that a single SEK repeat domain is sufficient to confer protective immunity against S. suis challenge.Table 16: Post-challenge mortality, efficacy, and cumulative clinical scores
[0226] 1Efficacy compared to the control group
[0227] 2Sum of the daily average cumulative clinical scores
[0228] ** statistically significant diminution of mortality between V4-vaccinated or 6-vaccinated group and Control group (p<0.01), using the LogRanktest.
[0229] *** statistically significant diminution of mortality between V3-vaccinated group and Control group (p<0.001), using the LogRanktest.
[0230] statistically significant diminution of mortality between V5-vaccinated group and Control group (p<0.001), using the LogRanktest.
[0231] Additionally, the V6 construct (V3 without D1 domain) further corroborated that presence of an N-terminal segment of the full-length Sao polypeptide (in this case, the N-terminal segment includes the signal segment and the D1 domain) is not required to confer protective immunity against S. suis challenge.Conclusion
[0232] The protective efficacy observed with a construct comprising only a single complete SEK repeat domain establishes that this domain alone contains epitope(s) recognized by the immune system that facilitate removal of the bacteria from the host. This most likely occurs by IgG antibody binding to epitopes within this peptide region (or similar repeat domains) in the full-length Sao parent protein expressed on the surface of the S. suis challenge strain. Surface bound IgG can directly kill bacteria by complement fixation or lead to killing by macrophages that recognize IgG Fc receptors (opsonization). Sao fragment polypeptides lacking any SEK repeat domain (see, e.g., V1) or containing only a partial SEK repeat (see, e.g., SP1A) failed to provide comparable protection. These data support the conclusion that a single SEK repeat domain is sufficient to induce an immune response against S. suis that protects against lethal infections.
[0233] Note that titles or subtitles may be used throughout the present disclosure for convenience of a reader, but in no way, these should limit the scope of the invention. Moreover, certain theories may be proposed and disclosed herein; however, in no way they, whether they are right or wrong, should limit the scope of the invention so long as the invention is practiced according to the present disclosure without regard for any theory or scheme of action.
[0234] As used herein, the wording “independently selected” in reference to a group of specified items refers to the fact that when more than one item is selected from the group of items, the decision of selecting a specific item is not influenced by the decision of selecting any of the previous or following item(s).
[0235] Reference throughout the specification to “some embodiments”, and so forth, means that a particular element (e.g., feature, structure, and / or characteristic) described in connection with the invention is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the inventive features described may be combined in any suitable manner in the various embodiments.
[0236] It will be understood by those of skill in the art that throughout the present specification, the term “a” used before a term encompasses embodiments containing one or more to what the term refers. It will also be understood by those of skill in the art that throughout the present specification, the term “comprising”, which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, un-recited elements or method steps.
[0237] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the case of conflict, the present document, including definitions will control.
[0238] As used in the present disclosure, when the terms “around”, “about” or “approximately” are before a quantitative value, the present disclosure also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the terms “around”, “about” or “approximately” refer to a ±10% variation from the nominal value unless otherwise indicated or inferred.
[0239] Unless otherwise noted, the expression “at least” or “at least one of’ as used herein includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression “and / or” in connection with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context.
[0240] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.
[0241] Unless otherwise noted, the order of steps or order for performing certain actions is immaterial so long as the present invention remain operable. Moreover, two or more steps or actions may be conducted simultaneously.
[0242] Unless otherwise noted, the use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present invention and does not pose a limitation on the scope of theinvention. No language in the specification should be construed as indicating any nonclaimed element as essential to the practice of the present invention.
[0243] Although various embodiments of the disclosure have been described and illustrated, it will be apparent to those skilled in the art considering the present description that numerous modifications and variations can be made. The scope of the invention is defined more particularly in the appended claims.INDUSTRIAL APPLICABILITY
[0244] The present invention has direct industrial applicability in the field of veterinary medicine and vaccine manufacturing. The engineered Sao polypeptide fragments described herein are produced using standard recombinant DNA technology and bacterial expression systems (e.g., E. coli), which are well-established in the pharmaceutical and biotechnology industries. Identification of a minimal protective epitope enables the design of vaccine antigens that are simpler, more stable, and more suitable for industrial manufacture than full-length Sao, without compromising protective efficacy. The immunogenic or vaccine compositions of the present invention are applicable to protecting swine populations against Streptococcus suis infection, addressing a significant economic burden in the global swine industry. Given the zoonotic potential of S. suis, particularly serotype 2, the disclosed vaccine compositions may also find application in human medicine for individuals at high risk of occupational exposure.
Claims
CLAIMS1. An immunogenic composition for inducing a protective immune response against Streptococcus suis (S. suis) infection in a mammal, the composition comprising a recombinant polypeptide and a pharmaceutically or veterinary acceptable excipient, diluent, or carrier,wherein the recombinant polypeptide consists essentially of one or more complete 30-amino acid SEK repeat domain(s), each SEK repeat domain having the amino acid sequence set forth in SEQ ID NO: 79 or a sequence variant thereof that retains the conserved motifs 1SEKQ, 1 VTP16, and 22NKE24, wherein the recombinant polypeptide optionally further comprises an SKE repeat domain positioned N-terminal to the one or more SEK repeat domain(s), wherein the SKE repeat domain comprises the amino acid sequence set forth in SEQ ID NO: 76 or a sequence variant thereof having at least 90% amino acid sequence identity thereto.
2. The immunogenic composition of claim 1, wherein the SEK repeat domain sequence variant comprises the conserved motifs ISEKQMP6, 1 VTP16, 19QM20, and 22NKE24.
3. The immunogenic composition of claim 1, wherein the SEK repeat domain sequence variant comprises the amino acid sequence selected from any one of SEQ ID NOs: 77, 78, 80, 81 and 82.
4. The immunogenic composition of claim 1, wherein the recombinant polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 9.
5. The immunogenic composition of claim 1, wherein the one or more complete 30- amino acid SEK repeat domain(s) is conjugated or fused to an immunogenic carrier, preferably keyhole limpet hemocyanin (KLH).
6. An immunogenic composition for inducing a protective immune response against Streptococcus suis (S. suis) infection in a mammal, comprising a recombinant polypeptide and a pharmaceutically or veterinary acceptable excipient, diluent, or carrier,wherein the recombinant polypeptide is a fragment of a Streptococcus suis surface antigen one (Sao) parent polypeptide,wherein the recombinant polypeptide comprises a protective epitope, wherein the protective epitope consists essentially of a complete 30-amino acid SEK repeat domain(s) having the amino acid sequence set forth in SEQ ID NO: 79 or a sequence variant thereof that retains the conserved motifs ISEKQ4, I4TPI6, and 22NKE24, preferably the sequence variant thereof retains the expanded conserved motifs iSEKQMPe, i4VTPi6, 19QM20, and 22NKE24,wherein the recombinant polypeptide is free from an N-terminal segment and a C-terminal segment from the parent polypeptide, wherein the C-terminal segment corresponds to at least amino acid residues 600 to 670 of SEQ ID NO: 23, and wherein the N-terminal segment corresponds to at least amino acid residues 1 to 30 of SEQ ID NO: 23.
7. The immunogenic composition of claim 6, wherein the Sao parent polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 23-28 or 30-70, preferably SEQ ID NO: 23.
8. The immunogenic composition of claim 6, wherein the N-terminal segment corresponds to at least amino acid residues 1 to 42 of SEQ ID NO: 23.
9. The immunogenic composition of claim 6, wherein the C-terminal segment corresponds to amino acid residues 600 to 670 of SEQ ID NO: 23, preferably corresponds to amino acid residues 445 to 670 of SEQ ID NO: 23.
10. The immunogenic composition of claim 6, wherein the N-terminal segment corresponds to at least amino acid residues 1 to 164 of SEQ ID NO: 23, preferably to amino acid residues 1 to 276 of SEQ ID NO: 23.
11. The immunogenic composition of claim 6, wherein the recombinant polypeptide consists essentially of the amino acid sequence set forth in SEQ ID NO:
8.
12. The immunogenic composition of claim 6, wherein the recombinant polypeptide consists essentially of the amino acid sequence set forth in SEQ ID NO: 9.
13. The immunogenic composition of claim 6, wherein the recombinant polypeptide consists essentially of the amino acid sequence set forth in SEQ ID NO: 91.
14. The immunogenic composition of any one of claims 1 to 13, further comprising an adjuvant.
15. The immunogenic composition of any one of claims 1 to 14, wherein the recombinant polypeptide is comprised in a bacterial extract.
16. The immunogenic composition of any one of claims 1 to 14, wherein the recombinant polypeptide is an isolated recombinant polypeptide.
17. A method for protecting a mammal susceptible to Streptococcus suis infection, comprising administering to the mammal the immunogenic composition according to any one of claims 1 to 16 in an amount effective to elicit a protective immune response against Streptococcus suis.
18. The method of claim 17, wherein the mammal is a pig.
19. The method of claim 17 or 18, wherein the immunogenic composition is administered intramuscularly.
20. The method of any one of claims 17 to 19, wherein the immunogenic composition is administered in a prime-boost regimen comprising a first dose (prime dose) followed by a second dose (boost dose) administered 2 to 4 weeks after the first dose.
21. The method of any one of claims 17 to 20, wherein the immunogenic composition is administered in an amount of from about 4 pg to about 500 pg of the recombinant polypeptide per dose.
22. A recombinant polypeptide as defined in any one of claims 1 to 13 for use in the manufacture of an immunogenic composition for protecting a mammal against Streptococcus suis infection.
23. A recombinant polypeptide as defined in any one of claims 1 to 13 for use in protecting a mammal against Streptococcus suis infection.
24. A nucleic acid molecule coding for the recombinant polypeptide as defined in any one of claims 1 to 13.
25. A host cell comprising a nucleic acid molecule coding for the recombinant polypeptide as defined in any one of claims 1 to 13.