Compositions and methods for enhancing immune responses against streptococci

Abstract

A vaccine composition against Streptococcus for use in mammals / fish, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and a pharmaceutically acceptable carrier.

Description

Invention Field

[0001] This invention relates to the fields of microbiology and vaccinology, and more specifically to the development of vaccines that can confer immunity against Streptococcus infection. Background of the Invention

[0002] Streptococci are a large genus of Gram-positive cocci that form long chains of cells. This genus is vast and diverse, harboring many important human and agricultural pathogens that cause a wide range of diseases. Streptococcal infections have significant global mortality, morbidity, and economic impacts. Although treatments have been developed for Streptococcus pneumoniae (Streptococcus pneumoniae...),... S. pneumoniae While effective vaccines against pneumococcus are available, there remains an urgent need for affordable and effective vaccines against other streptococcal species that infect humans and animals, such as Streptococcus suis. S. suis (Pathogens causing zoonotic diseases in pigs and humans), Streptococcus agalactiae ( S. agalactiae (Animal, human, and fish pathogens).

[0003] Like many genera, the classification of *Streptococcus* has undergone numerous revisions. Several classification methods exist for streptococci (Lancefield, hemolytic, metabolic), but the mainstream and most comprehensive method for measuring genetic affinity uses 16S rDNA sequencing, which has led taxonomists to divide the genus into six major groups containing at least 75 distinct species. Genomic plasticity within the genus further complicates classification and ultimately successful immunization; this plasticity allows individual strains to develop new traits that enable them to evade immune responses or become more pathogenic. Strains within a species are typically referred to as sequence types (STs) or serotypes to indicate their shared genetic or serological traits, respectively.

[0004] Capsular polysaccharides (CPS) are the extracellular matrix that encapsulates bacterial cells. They consist of repeating linear or branched oligosaccharide units linked by amino acids linked to hydroxyl groups on sugar rings. CPS limit the host immune system's ability to engulf, destroy, and eradicate bacterial cells. In most streptococci, cps The locus consists of eight genes encoding the biosynthetic pathways used for CPS production. For decades, CPS from Streptococcus and other pathogenic bacteria have been a target of vaccine research due to their high immunogenicity, but success has been limited to Streptococcus pneumoniae. Unfortunately, CPS are also highly variable, due to the highly plastic genome of Streptococcus, which exhibits high levels of genomic recombination and gene conversion. cps Recombination within loci produces numerous serotypes within each species of Streptococcus, making it extremely difficult to develop broadly effective vaccines against specific species.

[0005] Besides CPS (Cellular Pathogens), pathogenic streptococci encode an extremely diverse array of surface-associated components (SABs), secretory components, regulatory genes, and metabolic pathways, all of which contribute to their ability to colonize the host and evade the immune system. For example, at least 100 known or presumed pathogenic factors exist in the pangenome of *Streptococcus suis*, but due to genomic plasticity and recombination, their distribution within the species is uneven, rendering vaccination strategies ineffective. Targeting individual traits, serogroups, or sequence types are also ineffective vaccination strategies.

[0006] The advent of high-throughput genome sequencing has revolutionized scientists' ability to classify and organize species. More importantly, it allows scientists to define the core genome and pangenome of bacteria separately; essential genes found in all genera and species, and transferable genes found in certain lineages within a species. By analyzing and comparing the core genome of Streptococcus, highly conserved essential genes containing potential antigenic and immunoprotective epitopes across species can be identified. These antigens have the potential to provide far greater efficacy because they train the host's immune system to target proteins essential for bacterial cell growth and replication, and are conserved throughout Streptococcus, providing broad protection across species.

[0007] Therefore, there is a clear need for a broadly protective streptococcal vaccine that is antigenic and immunogenic in domesticated hybrid commercial pigs and provides protection against infection with *Streptococcus agalactiae* in Nile tilapia. Summary of the Invention

[0008] This invention includes broadly protective streptococcal vaccines that are antigenic and immunogenic. Attached Figure Description

[0009] Figure 1 illustrates the enzyme-linked immunosorbent assay (ELISA) for detecting anti-streptolysin epitope antibodies in saliva on day 34, intestinal mucosa on day 35, and serum IgG on day 34. Data were grouped by route of vaccination. All data are relative to untreated saline control animals. A ratio greater than 1 indicates higher antibody levels than in saline control animals.

[0010] Figure 2 shows the Kaplan-Meier survival analysis of Nile tilapia challenged with virulent Streptococcus agalactiae. The survival rates of the IP-AV (positive control) and SV1 10% immersion and 1% oral feeding groups were significantly higher than those of the unvaccinated control (p < 0.05).

[0011] Figure 3 illustrates the evaluation scheme.

[0012] Figure 4 shows the sIgA S / P ratio from saliva of pigs vaccinated with SV1.

[0013] Figure 5 shows the IgG S / P ratio from swine serum vaccinated with SV1. Detailed Implementation

[0014] The present invention will now be described more fully in the following detailed description, which describes some, but not all, embodiments of the invention. In fact, the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided to enable this disclosure to meet applicable legal requirements.

[0015] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. As used herein, the singular forms "a(a)," "an," and "described" are intended to include both the plural and singular forms unless the context clearly indicates otherwise. It should also be understood that when used in this specification, the terms "comprising" and / or "including" indicate the presence of the described features, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0016] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should also be understood that terms (e.g., those defined in common dictionaries) should be interpreted as having the same meaning as they have in the relevant field and in the context of this disclosure, and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0017] In describing this invention, it should be understood that numerous techniques and steps are disclosed. Each of these techniques and steps has its own advantages, and each can be used in combination with one or more other disclosed techniques, or in some cases with all other disclosed techniques. Therefore, for clarity, this description will avoid unnecessarily repeating every possible combination of the steps. However, the specification and claims should be interpreted as meaning that such combinations are fully within the scope of this invention and the claims.

[0018] Streptococcus is a genus of Gram-positive cocci or spherical bacteria belonging to the family Streptococcaceae within the order Lactobacillales (lactobacteria) of the phylum Firmicutes. Besides streptococcal pharyngitis (streptococcal laryngitis), certain species of Streptococcus are also the cause of many cases of conjunctivitis, meningitis, bacterial pneumonia, endocarditis, erysipelas, and necrotizing fasciitis ("flesh-eating" bacterial infections). However, many Streptococcus species are not pathogenic and form part of the symbiotic human microbiota found in the oral cavity, skin, intestines, and upper respiratory tract.

[0019] This invention identifies epitopes in Streptococcus species that are part of a protective antigen. An epitope, also known as an antigenic determinant, is part of an antigen that is recognized by the immune system, particularly antibodies, B cells, or T cells. The portion of an antibody that binds to an epitope is called a paratope. Although epitopes are usually non-self proteins, recognizable host-derived sequences (such as in the case of autoimmune diseases) are also epitopes.

[0020] Epitopes of protein antigens are classified into two categories based on their structure and interaction with complementary sites: conformational epitopes and linear epitopes. Conformational and linear epitopes are based on the interaction between the epitope's three-dimensional conformation and its complementary site, which is determined by the surface features of the epitope residues involved and the morphology or tertiary structure of other segments of the antigen.

[0021] • Conformational epitopes are formed by the three-dimensional conformation adopted by the interaction of discontinuous amino acid residues.

[0022] • In contrast, linear epitopes are formed by a three-dimensional conformation employed by the interaction of consecutive amino acid residues.

[0023] Linear epitopes are not solely determined by the primary structure of the amino acid involved. Residues flanking these amino acid residues, as well as more distant amino acid residues on the antigen, influence the ability of primary structure residues to adopt the three-dimensional conformation of the epitope.

[0024] 90% of epitopes are conformational epitopes.

[0025] T cell epitopes are small fragments of antigens recognized by T cell receptors (TCRs). T cells are a type of white blood cell that play a crucial role in adaptive immune responses. They recognize and respond to foreign invaders, such as bacteria and viruses. T cell epitopes are typically 8 to 11 amino acids in length and are derived from proteins. They are presented to T cells on the surface of antigen-presenting cells (APCs). APCs are cells that absorb foreign antigens and process them into peptides. These peptides then bind to MHC molecules displayed on the surface of APCs.

[0026] When a TCR binds to a T cell epitope, it triggers a series of events leading to T cell activation. Activated T cells can then release cytokines, which helps recruit other immune cells to the site of infection. They can also directly kill infected cells. The ability of a T cell to recognize a specific T cell epitope is determined by the structure of the TCR. Each TCR has a unique binding site that is specific to a particular set of T cell epitopes. The binding site is formed by a combination of amino acids in the TCR molecule.

[0027] B-cell epitopes are small portions of antigens recognized by B-cell receptors (BCRs). B cells are a type of white blood cell that produces antibodies. Antibodies are proteins that bind to antigens and help neutralize them. B-cell epitopes are typically 7 to 11 amino acids long and are derived from proteins. They can be linear epitopes or conformational epitopes. Linear epitopes are short amino acid sequences recognized by BCRs that are independent of the three-dimensional structure of the antigen. Conformational epitopes are three-dimensional structures recognized by BCRs. The ability of a B cell to recognize a specific B-cell epitope is determined by the structure of the BCR. Each BCR has a unique binding site that is specific to a particular set of B-cell epitopes. The binding site is formed by a combination of amino acids in the BCR molecule.

[0028] T-cell epitopes and B-cell epitopes are each small fragments of an antigen, recognized by T cells and B cells, respectively. Each is important for understanding immune responses and developing new vaccines and therapies. Epitope-based vaccines use isolated B-cell or T-cell epitopes to stimulate humoral and cellular immune responses. These vaccines can use multiple epitopes to enhance their potency. To identify epitopes for vaccines, computer simulations are typically used. After candidate epitopes are identified, the construct is engineered and vaccine efficacy is tested.

[0029] Using Bacillus subtilis ( Bacillus subtilis Nucleotide codon optimization is performed to obtain the desired nucleotide sequence for the DNA sequence, and recombinant synthetic DNA sequences are created (Genscript Biotech Corporation - Piscataway NJ, USA). Bacillus subtilis is a safe, non-pathogenic bacterium that can be easily genetically modified and mass-produced. Bacillus subtilis has been used to develop vaccines against a variety of diseases, including anthrax, cholera, and tuberculosis. One advantage of using Bacillus subtilis as a vaccine vector is that it can be administered orally, intranasally, and parenterally. This is a major advantage over traditional vaccines, which are typically administered only by injection. Another advantage of using Bacillus subtilis as a vaccine vector is that it can be easily mass-produced. This is important for developing vaccines against emerging diseases that may require the production of large quantities of vaccines in a short period of time.

[0030] Recombinant synthetic DNA sequences containing homology restriction sites were amplified using conventional PCR (using sequence-specific primers) to produce amplification products. The amplification products were purified and concentrated using gel extraction, digested overnight with BamHI and XbaI, and purified again. BamHI is a precursor for isolating Bacillus amyloliquefaciens (Bacillus subtilis). Bacillus amyloliquefaciens BamHI is a restriction enzyme found in bacteria. It is a type II restriction endonuclease that recognizes a specific DNA sequence and cuts DNA within that sequence. BamHI recognizes the sequence GGATCC and cuts DNA between G and A. This produces two DNA fragments, each with a sticky end. The sticky ends are complementary to each other, so they can be ligated (joined) together. BamHI is a very versatile restriction enzyme because it can be used to cut DNA from a wide variety of sources, including bacteria, viruses, and plants. It is also easy to use and inexpensive, making it a cost-effective option. XbaI is isolated from *Xanthomonas pasteurellis* (…). Xanthomonas badrii XbaI is a restriction enzyme found in bacteria. It is a type II restriction endonuclease that recognizes a specific DNA sequence and cuts DNA within that sequence. XbaI recognizes the sequence T^CTAGA and cuts DNA between T and A. XbaI is a very versatile restriction enzyme because it can be used to cut DNA from a wide variety of sources, including bacteria, viruses, and plants. Like BamHI, it is also easy to use and inexpensive, making it a cost-effective option.

[0031] The Bacillus expression plasmid pHT10 was digested, purified, concentrated, and treated with rSAP using BamHCl and Xbal. The digested synthetic insert and plasmid were then mixed into the T4 DNA ligase reaction mixture and incubated overnight at room temperature. T4 DNA ligase is an enzyme that catalyzes the formation of a phosphodiester bond between the 5'-phosphate group of one DNA strand and the 3'-hydroxyl group of another DNA strand, thus joining two DNA segments together. This reaction is essential for DNA replication, repair, and recombination.

[0032] The T4 DNA ligase reaction is performed according to the following steps: 1. T4 DNA ligase binds to the two DNA strands to be ligated.

[0033] 2. This enzyme breaks the phosphodiester bond between the 5'-phosphate group of one DNA strand and the 3'-hydroxyl group of another DNA strand.

[0034] 3. The enzyme then reforms the phosphodiester bonds between the two DNA strands, linking them together.

[0035] 4. T4 DNA ligase releases the two DNA strands that are now linked together.

[0036] The ligation reactant was converted into E. coli ( E. coli DH5α (New England Biolabs - Ipswich, Massachusetts, USA), and in the presence of ampicillin (100 µg / ml, LB) Amp Gene insertion fragments of transformants were screened on LB agar. The ligation reaction is the process of joining two DNA segments together. The resulting DNA molecules were then transformed into *E. coli* DH5α cells. These cells were then grown on plates containing antibiotics that killed any cells that did not contain the cloned gene. The surviving cells were then cultured and the cloned gene was harvested.

[0037] A novel plasmid, pStrep, was purified from *Escherichia coli* and transformed into *Bacillus subtilis*. The plasmid was then converted to a solution containing chloramphenicol (5 µg / ml, TSA). Cm Transformants were selected on tryptic soy agar to produce recombinant Bacillus subtilis strains, Strep Vaccine 1 (SV1).

[0038] Then, during SV1 fermentation at 28–37°C, protective subunits were generated by induction with isopropyl β-D-1-thiogalactoside (IPTG) at concentrations ranging from 0.1 to 0.5 mM. The culture was then inactivated using formalin. For the production of test vaccines and their iterative versions, the inactivated SV1 culture was mixed with naturally occurring polysaccharides, specifically methylcellulose (the encapsulation medium), which served as both an antigenic medium and a protectant.

[0039] Example Example 1: Immunogenicity study of streptococcal epitopes in weaned piglets: Title: A preliminary study to determine humoral immune responses in three-week-old piglets after vaccination with an experimental streptococcal vaccine via multiple routes. Basis for Argumentation: Streptococcus suis and streptococcal-related diseases are significant diseases and economic burdens in intensive indoor pig farming. Diseases associated with streptococcal infection include meningitis, septicemia, arthritis, polyserositis, valvular endocarditis, myocarditis, pericarditis, and abortion. Streptococcus suis is now the most common cause of swine meningitis submitted to veterinary diagnostic laboratories. Currently available vaccines do not induce sufficient cross-protection among the many serotypes of Streptococcus suis; therefore, vaccines that induce strong protective and cross-serotype-responsive immune responses are needed.

[0040] Materials and Methods: Thirty 3-week-old piglets from a highly healthy farm, free from previous streptococcal disease or other confounding factors (PRRS, Mycoplasma hyopneumoniae), were weaned. Mycoplasma hyopneumoniae (e.g., PEDv, APP, etc.) Pigs were tagged, weighed, and randomized to one of 10 groups (2 replicates × 5 groups (3 pigs / group)) and allowed to acclimatize for 2 to 3 days. On day 0 of the study, serum samples were collected from each pig, and the first dose of vaccine or saline was administered as shown in Table 1. As shown in Table 1, the second dose was administered after 14 days. Treatment groups were separate.

[0041] Vaccination: The vaccine is administered in two doses (2 mL each) via intramuscular injection (IM) behind the ear and in the neck, oral tube feeding, or nasal spray (1 mL / nostril). Saline is administered via oral tube feeding.

[0042] Table 2 outlines the research timeline and the samples to be collected.

[0043]

[0044]

[0045] The results are shown in Figure 1, where enzyme-linked immunosorbent assay (ELISA) detected anti-streptococcal epitope antibodies in A) secretory IgA in saliva on day 34, B) secretory IgA in intestinal mucosa on day 35, and C) serum IgG on day 34. Data were grouped by route of vaccination. All data are relative to untreated saline control animals. A ratio greater than 1 indicates higher antibody levels than in saline control animals.

[0046] Example 2: Survival of Nile tilapia after inoculation with SV1 vaccine and challenge with virulent Streptococcus agalactiae. Experimental design, sampling, and vaccination: A total of 180 fry were acclimatized for two days and distributed across four treatments, each in triplicate (3 tanks / treatment, 15 fish / tank). The fry were distributed in 12 80-liter experimental units, with a total of 15 fish per tank. The fish were treated as described below (Table 1) and allowed to grow for 54 days.

[0047] Fish were vaccinated with SV1 via immersion (imer) and oral administration (oral) by coating with food particles. Currently, only commercially available intraperitoneal injectable vaccines for the prevention of streptococcal disease are available; therefore, the commercially available agalactiae streptococcal vaccine (AquaVac) was used as a positive control (Table 3).

[0048]

[0049] intraperitoneally On day 41 of the experiment, 14 days prior to infection challenge, the vaccine was administered intraperitoneally. In the positive control group, the commercially available vaccine AQUAVAC® Strep SA, MSD was administered according to the label instructions. The commercially available vaccine (AV) was administered at a dose of 50 μL per fish.

[0050] bath In the immersion application, the immersion was conducted on two different days of the experiment. The first immersion was conducted at the start of the experiment (day 0), and the second immersion was conducted on day 45 of the experiment (day 45). The dosage was 500 mL of SV1 (10% v / v) in 4.5 L, in which the fish were kept immersed for 20 minutes.

[0051] oral In the oral route, SV1 was directly mixed into the feed. Fish received a diet containing SV1 for two consecutive days at two different times during the experiment. The first feeding containing SV1 was given on days 30 and 31 of the experiment, and the second feeding was given on days 45 and 46. SV1 was included in the diet 30 minutes before feeding administration. The dosage was defined as 100 μL of vaccine (10% v / w) per gram of feed.

[0052] Experimental attack: On day 55 of the experiment, fish in all groups were attacked with the bacterial pathogen Streptococcus agalactiae strain S13 (ST-552, 1b), which was isolated from a streptococcal disease outbreak in Nile tilapia in Paraná, Brazil (Facimoto et al., 2017).

[0053] Initially, a 50% lethal dose (LD50) test was conducted to determine the bacterial concentration (LD50) that could kill 50% of the unvaccinated and attacked population. 50 For this dosage test, 40 Nile tilapia were distributed across eight 30 L experimental units, with a total of five animals per unit. These units were aerated and at controlled temperatures. LD50 was performed in duplicate. 50 The bacterial concentration tested was 1 × 10⁻⁶. 5 ; 1 × 10 6 ; 1 × 10 7 ; 1 × 10 8 CFU mL -1 (Data not shown). LD50 of Streptococcus agalactiae S13 via intraperitoneal injection. 50 It was determined to be × 10 7 CFU mL -1 This concentration was used in the subsequent vaccination and attack phases described below.

[0054] The fish were fed for 54 days until they reached a weight of approximately 50 g, at which stage the animals were more susceptible to disease (Mian et al., 2009). After this fattening period, they were administered 1 × 10⁻⁶ mg of antibiotics via intraperitoneal injection. 7 CFU mL -1 Each fish was attacked with Streptococcus agalactiae S13. Prior to injection, the bacteria were precipitated and washed three times in cold, sterile PBS. The fish were anesthetized with eugenol before proceeding with the above procedure.

[0055] Clinical signs were observed daily for 7 days following the attack, and all dead fish were removed from the tank daily and recorded in the experimental documentation. Any fish unable to remain underwater or maintain normal orientation were humanely euthanized.

[0056] result:

[0057] Survival analysis of Nile tilapia challenged with virulent Streptococcus agalactiae: The results are shown in Figure 2, which illustrates the Kaplan-Meier survival analysis of Nile tilapia challenged with virulent Streptococcus agalactiae. The survival rates of the IP-AV (positive control) and SV1 10% immersion and 10% oral feeding groups were significantly higher than those of the unvaccinated control (p < 0.05).

[0058] Example 3: Determination of dosage, administration route, and humoral immune response kinetics in pigs vaccinated with candidate streptococcal vaccine 1 (SV1). Target Using different dose volumes and routes of administration, we evaluated humoral immune responses in pigs vaccinated with candidate streptococcal vaccine formulations to determine optimal vaccine delivery based on specific antibody levels identified by antigen-specific immunization-specific tests.

[0059] Materials and Methods Animals and their living environment "Las Chirus" is a fully enclosed pig farm with a total breeding stock of 450 head, producing 20 litters per week. It is located in French, 9 de Julio district, Buenos Aires province, Argentina.

[0060] During the farrowing and post-weaning phases, a separate all-in, all-out system was used, while the later fattening phase was conducted using a continuous flow system. A total of 105 animals were used (5 animals per group). These were females and males genetically selected by Agrosers Pig Improvement Company (PIC) from a cross between female Camborough 1050 and male Terminal 337.

[0061] They were kept in pigsties with slatted floors and water-filled manure pits. At the end of the fattening stage, the density was 0.33 animals per square meter.

[0062] Use curtain-style ventilation (i.e., natural ventilation).

[0063] Routine handling of food, water, and animals. food Birth to 15 days: Colostrum and milk 15 days to weaning (21 days): Milk and pre-starter food 21-30 days old: Food F1 (starter) 30-40 days old: Food F2 40-55 days old: Food F3 (containing 400 ppm amoxicillin) 55-70 days old: Food F4 (containing 100 ppm tiamulin + 300 ppm chlortetracycline) water Water is provided freely.

[0064] Number of nipples: 1 nipple for every 10 captive animals.

[0065] Other treatments during the experiment. After being transferred to the weaning box (around 21 days old), piglets receive vaccinations against PCV2 and Mycoplasma hyopneumoniae.

[0066] 42 days old: Pigs receive a second vaccination against mycoplasma.

[0067] For animals exhibiting neurological signs that may be caused by infection with Streptococcus suis or Haemophilus parasuis, administer ceftiofur for injection or amoxicillin for injection.

[0068] deal with Group Vaccine candidates under evaluation: • SV1: A candidate vaccine against Streptococcus species (Ss) Table 5

[0069] PO (oral administration); IM (intramuscular administration) Application date First dose: within the first 3 days after birth Second dose: 14-18 days after the first vaccination.

[0070] Vaccine administration Oral administration: Administer the volume shown above using a dispenser or auto-injector.

[0071] Intramuscular administration: The volume shown above is applied using an autoinjector to the neck area just below and behind the ear, in front of the shoulder.

[0072] Vaccine preparation in the laboratory Take 10 μL from a frozen tube containing SV1 working seed and spread it onto a TSA plate containing chloramphenicol (CP5: 5 mg / ml). Incubate the plate overnight at 37 °C. The next day, culture the colonies in TSB containing CP5. Incubate the pre-culture (PC) overnight at 37 °C. The next day, inoculate a volume of TSB + CP5 into a large volume of sterile TSB. Incubate the culture at 37 °C for 2 hours, then add a volume of 0.5 mg / ml TSB. M IPTG (1 ml / L) was used to induce antigen production, and incubation continued for another 6 hours.

[0073] After a total of 8 hours, samples were taken for colony-forming unit (CFU) calculation. Subsequently, the culture was inactivated by adding a certain volume of formaldehyde and incubated overnight at room temperature.

[0074] Excipients were prepared using conventional manufacturing processes.

[0075] Both the 1 ml and 2 ml dosage groups were prepared by mixing inactivated culture (SV1) and excipients in an equal ratio (1:1).

[0076] Package the formulation in 10 ml vials, label them, and store at 2–8 °C until use.

[0077] The samples and parameters being evaluated. Sampling date Samples were collected over the following days: • First sampling: On the day of initial vaccination (week 1) • Second sampling: Booster vaccination day (Week 3) • Third sampling: Week 4 • Fourth sampling: Week 6 • Fifth sampling: Week 8 Sampling and processing: serum samples For pigs, use an 18G x 1.5” needle and for piglets, use a 20G x 1” or 1.5” needle to collect blood samples from the jugular vein. Collect approximately 5 ml of blood and place it in a 15 ml conical tube without anticoagulant. To extract serum from the blood, allow the serum to coagulate in the tube at an angle of at least 60 degrees. Once the clot has formed, detach it from the glass with the aid of the needle. To obtain the maximum amount of serum, heat the tube to 37 °C for at least 30 minutes to at most 1 hour and then incubate at 4 °C overnight. The next morning, centrifuge the tube at 3000 rpm for 30 minutes and extract the serum (supernatant) directly using a pipette. The volume of serum collected should be 500 µl to 1 ml. Store the serum at -20 °C until analysis.

[0078] saliva sample The following steps were performed to determine IgA levels in saliva samples: Oral swabs: Collect saliva from the pig using a sterile cotton swab and place the swab in the pig's mouth until it is soaked with saliva. Then immerse the saliva-soaked swab in 1 ml of sterile PBS (pH 7.4) in a 15 ml conical tube and store at -20°C until analysis.

[0079] analyze ELISA for antibody assay The ELISA S / P ratio is used to determine the IgA and IgG antibody responses against SV1, as shown in the following formula: Sample - Negative Control / Positive Control - Negative Control ELISA scheme: 1) Coating: BioGreiner plates were coated with synthetic streptococcal peptide antigen covalently linked to BSA at a concentration of 5 µg / ml / well (100 µl / well) and then incubated overnight at room temperature.

[0080] 2) Wash the plates three times with TBS-T (Tris-buffered saline with 0.1% Tween 20) (200 µl / well). 3) Then block the plates with 200 µl / well of blocking buffer (Bethyl Lab, Cat. E104) and incubate at room temperature for 1 hour.

[0081] 4) Wash the plate three times with TBS-T (200 µl / well). 5) The test sample was diluted as follows: • Serum: in sample buffer (50 mL) MDiluted 1:100 in Tris-buffered saline, pH 8, 1% bovine serum albumin (BSA), and 0.05% Tween 20, 100 µl / well • Saliva: Undiluted, 100 µl / well Incubate the plates at room temperature for 1 hour.

[0082] 6) Wash the plates three times with TBS-T (200 µl / well). Add secondary antibody to each well at the following concentration: 100 µl / well.

[0083] • Anti-porcine IgA HRP 1 µg / ml (A100-102P, Bethyl laboratory) • Anti-pig IgG HRP 0.25 µg / ml (A100-205P, Bethyl Laboratory) Incubate at room temperature for 1 hour.

[0084] 8) Wash the plate three times with TBS-T (200 µl / well).

[0085] 9) Add TMB single-component substrate (E102, Bethyl laboratory) (100 µl / well) and incubate at room temperature for 3 minutes.

[0086] 10) Use 100 µl / well of 0.1 M Sulfuric acid terminates the reaction.

[0087] 10) Use a visible spectrophotometer to read the optical density of each pore at λ = 450 nm.

[0088] Statistical analysis After determining the homoscedasticity, sphericity, and normality of the groups, analysis of variance was performed on the time-varying factors of the two measurements using IBM SPSS Statistic 25 software.

[0089] Experimental Design A schematic diagram outlining the experimental design is shown in Figure 3.

[0090] result Two doses of the candidate SV1 vaccine induced antigen-specific secretory IgA and serum IgG in piglets as young as three days old. Determination of the S / P ratio of salivary secretory IgA (sIgA). The results are shown in Table 6 and Figure 4. An increase in the antigen-specific salivary sIgA S / P ratio was observed in some of the evaluated groups over several weeks, with no significant differences observed when compared between groups weekly (weeks 1, 3, 4, and 6). At week 8, pigs receiving two 2 mL doses of SV1 orally or IM, followed by an oral booster, had significantly higher S / P ratios compared to other groups, with no significant difference between the 2 mL oral / oral and 2 mL IM / oral groups. These data suggest that the SV1 candidate vaccine is immunogenic and capable of inducing antigen-specific secretory IgA in pigs as early as 3 days of age; in particular, the 2 mL / dose regimen of oral or IM / oral administration provided a robust sIgA response.

[0091] Table 6

[0092] Serum IgG S / P ratio determination The results are shown in Table 7 and Figure 5. An increase in the antigen-specific serum IgG S / P ratio was observed for several weeks in some of the evaluated groups, with no significant differences observed when compared between groups weekly (weeks 1, 3, and 4). At weeks 6 and 8, pigs receiving two 2 mL doses of SV1 orally or IM followed by an oral booster had significantly higher S / P ratios compared to other groups. At 6 weeks of age, the mean S / P ratio in the 2 mL IM / oral group was significantly higher than that in the 2 mL oral / oral group, but this difference was no longer present at 8 weeks of age. These data indicate that the SV1 candidate vaccine is immunogenic and capable of inducing antigen-specific serum IgG in pigs as early as 3 days of age; in particular, the 2 mL / dose regimen administered orally or IM / oral provided a robust serum IgG response.

[0093] Table 7 Age of the animal at the time of sampling

[0094] in conclusion These data demonstrate that the candidate vaccine SV1 is both immunogenic and antigenic, and importantly, it protects fish (in this example, Nile tilapia) from attack by virulent Streptococcus agalactiae. Furthermore, the SV1 vaccine induced robust antigen-specific salivary sIgA and serum IgG responses in piglets, with the first dose administered as early as 3 days of age. Administration routes of two oral doses or one intramuscular dose followed by an oral booster dose provided similar sIgA responses, while IM / oral administration induced a higher and faster serum IgG response, as expected based on scientists' understanding of immune responses and activation. However, the responses at 8 weeks of age were similar, suggesting that the new antigen-specific baseline antibody response may not be significantly different. Although the optimal antibody response volume in pigs is 2 mL, this is ultimately irrelevant, as the antigen / mL concentration can be adjusted so that 1 mL of vaccine can have the same antigen concentration as a 2 mL dose.

[0095] Any method described herein may be incorporated into this application and any design element contained in any other document / application incorporated herein by reference.

[0096] This invention may be embodied in other forms without departing from its spirit and essential attributes, and therefore the scope of the invention should be determined by reference to the appended claims rather than the foregoing description. The invention illustratively disclosed herein may be suitably practiced in the absence of any elements not specifically disclosed herein.

[0097] Sequence list information: DTD Version: V1_3 File name: SEQUENCE LISTING 3113.005.xml Software Name: WIPO Sequence Software version: 2.3.0 Date of creation: 2023-05-12 General information: Current application / Intellectual Property Office: US Current application / applicant document reference: 3113.005 Applicant Name: Ventano Corporation Applicant's Name / Language: English Inventor's name: S. Layton Inventor's name / language: English Invention Title: Compositions and Methods for Enhancing Immune Responses Against Streptococcus Spp. Total number of sequences: 3 sequence: Serial Number (ID): 1 Length: 49 Molecular type: AA Feature location / qualifier: -Source, 1..49 > mol_ type, protein Biological organisms, synthetic constructs residues:

[0098] Serial Number (ID): 2 Length: 878 Molecular type: AA Feature location / qualifier: -Source, 1..878 > mol_ type, protein Biological organisms, synthetic constructs residues:

[0099]

[0100] Serial Number (ID): 3 Length: 2643 Molecular type: AA Feature location / qualifier: -Source, 1..2643 > mol_ type, protein >Biosomes, synthetic constructs residues:

[0101]

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[0103] Finish

Claims

1. A method targeting Streptococcus spp. ( Streptococcus The vaccine composition comprises: The amino acid sequences selected from sequence numbers (ID):1, (ID):2 and (ID):3; and Pharmaceutically acceptable carrier.

2. The vaccine composition of claim 1, wherein the vaccine is derived from a protective antigen, the protective antigen being nucleotide-optimized against Bacillus subtilis (…). Bacillus subtilis ) is optimized to obtain the nucleotide sequence of the DNA sequence.

3. The vaccine composition according to claim 1, further comprising one or more immunostimulatory polypeptides.

4. The vaccine composition according to claim 1, wherein the vaccine composition induces an immune response.

5. The vaccine composition according to claim 4, wherein the immune response comprises an antibody response.

6. The vaccine composition according to claim 1, wherein the vaccine composition is administered by a method selected from oral, intranasal, and parenteral administration.

7. A method for enhancing an immune response against Streptococcus spp., comprising administering to a subject an amount of the vaccine composition according to claim 1 that effectively enhances the subject's immune response to one or more Streptococcus spp. strains.

8. The method of claim 7, wherein the vaccine composition induces an immune response.

9. The method of claim 7, wherein the immune response comprises an antibody response.

10. The method of claim 7, wherein the vaccine is administered by a method selected from oral, intranasal, and parenteral administration.

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