Compositions and methods of enhancing immune responses to streptococcus

Abstract

A vaccine composition for mammals / Fish against Streptococcus comprising an amino acid sequence selected from the group consisting of Sequence Number (ID): 1, Sequence Number (ID): 2, and Sequence Number (ID): 3, and a pharmaceutically acceptable carrier.

Description

[0001] COMPOSITIONS AND METHODS OF

[0002] ENHANCING IMMUNE RESPONSES TO STREPTOCOCCUS

[0003] INVENTOR(S): Sheryll Layton and Jeffrey W. Hall

[0004] FIELD OF INVENTION

[0005] The present invention is related to the fields of microbiology and vaccinology and more specifically concerns the development of a vaccine capable of conferring immunity to infection by Streptococcus.

[0006] BACKGROUND OF THE INVENTION

[0007] The bacterial genus Streptococcus is a of a large group of Gram-positive cocci that form long chains of cells. The genus is large and diverse with many important human and agricultural pathogens that cause a wide range of different diseases. The worldwide mortality, morbidity, and economic impacts of streptococcal infections are significant. While effective vaccines have been developed against S. pneumoniae (Pneumococcus), there is still great need for affordable and effective vaccines against other species of streptococci that infect humans and animals such as S. suis (swine and zoonotic pathogen), S. agalactiae (animal, human, and fish pathogen). As with many genera, classification of the Streptococcus genus has undergone many revisions. There are several methods of classification for streptococci (Lancefield, hemolysis, metabolism), but the leading and most comprehensive method to measure genetic relatedness is the use of 16s rDNA sequencing, with taxonomists classifying the genus into 6 major groups containing at least 75 distinct species. Further complicating classification, and ultimately successful immunity, is genome plasticity within the genus which may allow individual strains to develop new traits that allow it to evade an immune response or become more pathogenic. Strains within a species are often referred to as a sequence type (ST) or serotype to indicate their shared genetic or serological traits, respectively.

[0008] The capsular polysaccharide (CPS) is an extracellular cellular matrix that surrounds the bacterial cell made up of repeating linear or branched oligosaccharide units connected by amino acids linked to the hydroxyl groups of the sugar rings. The CPS limits the ability of a host’s immune system to engulf, destroy, and eradicate the bacterial cells. In most streptococci, the cps locus consists of eight genes that encode the biosynthetic pathway for CPS production. The CPS of streptococci and other pathogenic bacteria have been the target of vaccine research for decades as it is highly immunogenic, with success limited to S. pneumoniae. Unfortunately, the CPS is also highly variable owing to the fact that streptococci have a highly plastic genome, with high levels of genome recombination and gene switching. Recombination within the cps locus gives rise to many serotypes within each streptococcus species and makes developing a broadly effective vaccine against a particular species extremely difficult. In addition to the CPS, pathogenic streptococci encode an incredibly vast array of surface-associated or secreted components, regulatory genes, or metabolic pathways that all promote the ability to colonize at host and evade the immune system. For example, there are at least 100 known or putative factors within the S. su / s pangenome that confer pathogenic capabilities, but because of genome plasticity and recombination, they are not homogeneously distributed within the species leading to ineffective vaccination strategies. Targeting individual traits, serogroups, or sequence types is an ineffective vaccination strategy.

[0009] The advent of high-throughput genome sequencing has revolutionized scientists’ ability to classify and organize species. More importantly, it has allowed scientists to define core- and pan-genomes of bacteria; genes that are essential and found in all species of genera, and genes that are mobile and are found in certain lineages of a species, respectively. By analyzing and comparing core-genomes of streptococci it is possible to identify highly conserved essential genes that contain potentially antigenic and immunologically protective epitopes across species. These antigens have the potential to provide far greater efficacy because they train a host’s immune system to target protein(s) that are essential to the growth and replication of the bacterial cell and are conserved throughout streptococci, providing for broad protection across species.

[0010] Thus, there is clearly a need of a broadly protective streptococci vaccine which is antigenic and immunogenic in domestic crossbred commercial swine and provides Nile tilapia protection from infection with S. agalactiae. SUMMARY OF THE INVENTION

[0011] The instant invention includes a broadly protective streptococci vaccine which is antigenic and immunogenic.

[0012] DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 illustrates Enzyme-linked immunosorbent assays (ELISAs) detecting anti-Strep epitope antibodies in A) secretory IgA in saliva on study day 34, B) secretory IgA in intestinal mucosa on study day 35, C) IgG in serum on study day 34. Data is grouped by vaccination route. All data is relative to the non-treated saline control animals. Ratios greater than 1 indicate antibody levels greater than the saline control animals.

[0014] Figure 2 illustrates a Kaplan-Meier survival analysis of Nile tilapia challenged with virulence S. agalactiae. The IP-AV (positive control) and SV1 10% immersion and 1 % oral fed groups had significantly better survival than the unvaccinated controls (p < 0.05).

[0015] Figure 3 illustrates the Scheme of Assessment.

[0016] Figure 4 illustrates the slgA S / P ratio from saliva of pigs vaccinated with SV1 .

[0017] Figure 5 illustrates the IgG S / P ratio from serum of pigs vaccinated with SV1 . DETAILED DESCRIPTION

[0018] The present invention now will be described more fully hereinafter in the following detailed description of the invention, in which some, but not all embodiments of the invention are described. Indeed, this 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 so that this disclosure will satisfy applicable legal requirements.

[0019] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of 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," "an," and "the" are intended to include the plural forms as well as the singular forms, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0020] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one having ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0021] In describing the invention, it will be understood that a number of techniques and steps are disclosed. Each of these has individual benefit and each can also be used in conjunction with one or more, or in some cases all, of the other disclosed techniques. Accordingly, for the sake of clarity, this description will refrain from repeating every possible combination of the individual steps in an unnecessary fashion. Nevertheless, the specification and claims should be read with the understanding that such combinations are entirely within the scope of the invention and the claims.

[0022] Streptococcus is a genus of gram-positive coccus or spherical bacteria that belongs to the family Streptococcaceae, within the order Lactobacillales (lactic acid bacteria), in the phylum Firmicutes. In addition to streptococcal pharyngitis (strep throat), certain Streptococcus species are responsible for many cases of pink eye, meningitis, bacterial pneumonia, endocarditis, erysipelas, and necrotizing fasciitis (the 'flesh-eating' bacterial infections). However, many streptococcal species are not pathogenic, and form part of the commensal human microbiota of the mouth, skin, intestine, and upper respiratory tract.

[0023] The instant invention identified a Streptococcus epitope which was part of a protective antigen. An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. The part of an antibody that binds to the epitope is called a paratope. Although epitopes are usually non-self proteins, sequences derived from the host that can be recognized (as in the case of autoimmune diseases) are also epitopes.

[0024] The epitopes of protein antigens are divided into two categories, conformational epitopes and linear epitopes, based on their structure and interaction with the paratope. Conformational and linear epitopes interact with the paratope based on the 3-D conformation adopted by the epitope, which is determined by the surface features of the involved epitope residues and the shape or tertiary structure of other segments of the antigen.

[0025] • A conformational epitope is formed by the 3-D conformation adopted by the interaction of discontiguous amino acid residues.

[0026] • In contrast, a linear epitope is formed by the 3-D conformation adopted by the interaction of contiguous amino acid residues. o A linear epitope is not determined solely by the primary structure of the involved amino acids. Residues that flank such amino acid residues, as well as more distant amino acid residues of the antigen affect the ability of the primary structure residues to adopt the epitope's 3-D conformation. o 90% of epitopes are conformational.

[0027] A T-cell epitope is a small fragment of an antigen that is recognized by a T cell receptor (TCR). T cells are a type of white blood cell that play an important role in the adaptive immune response. They recognize and respond to foreign invaders, such as bacteria and viruses. T-cell epitopes are typically 8 to 11 amino acids long and are derived from proteins. They are presented to T cells on the surface of antigen- presenting cells (APCs). APCs are cells that have taken up foreign antigens and processed them into peptides. The peptides are then bound to MHC molecules, which are displayed on the surface of the APC.

[0028] When a TCR binds to a T-cell epitope, it triggers a series of events that lead to the activation of the T cell. Activated T cells can then release cytokines, which help to recruit other immune cells to the site of infection. They can also kill infected cells directly. The ability of a T cell to recognize a particular T-cell epitope is determined by the structure of the TCR. Each TCR has a unique binding site that is specific for a particular set of T-cell epitopes. The binding site is formed by a combination of amino acids in the TCR molecule.

[0029] A B-cell epitope is a small part of an antigen that is recognized by a B cell receptor (BCR). B cells are a type of white blood cell that produce antibodies. Antibodies are proteins that bind to antigens and help to neutralize them. B-cell epitopes are typically 7 to 11 amino acids long and are derived from proteins. They can be linear or conformational. Linear epitopes are short sequences of amino acids that are recognized by the BCR regardless of the three-dimensional structure of the antigen. Conformational epitopes are three-dimensional structures that are recognized by the BCR. The ability of a B cell to recognize a particular B-cell epitope is determined by the structure of the BCR. Each BCR has a unique binding site that is specific for a particular set of B-cell epitopes. The binding site is formed by a combination of amino acids in the BCR molecule.

[0030] T-cell epitopes and B-cell epitopes are each small fragments of an antigen that are recognized by T cells and B cells respectively. Each are important for understanding the immune response and for developing new vaccines and therapies. Epitope-based vaccines stimulate humoral and cellular immune responses using isolated B-cell or T- cell epitopes. These vaccines can use multiple epitopes to increase their efficacy. To find epitopes to use for the vaccine, in silico mapping is often used. Once candidate epitopes are found, the constructs are engineered and tested for vaccine efficiency.

[0031] A nucleotide codon optimization for Bacillus subtilis was utilized to derive the necessary nucleotide sequence for the DNA sequence and a recombinant synthetic DNA sequence was manufactured (Genscript Biotech Corporation - Piscataway NJ, USA). Bacillus subtilis is a safe, non-pathogenic bacterium that can be easily genetically modified and produced on a large scale. 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 produced on a large scale. This is important for the development of vaccines against emerging diseases, which may require the production of large quantities of vaccine in a short period of time.

[0032] The recombinant synthetic DNA sequence, complete with the homologous restriction sites, was amplified using traditional PCR with sequence specific primers resulting in an amplification product. The amplification product was purified by gel extraction techniques, concentrated, digested with Bam HI and Xbal overnight, and repurified. Bam HI is a restriction enzyme isolated from the bacterium Bacillus amyloliquefaciens. It is a type II restriction endonuclease, which recognizes a specific sequence of DNA and cuts the DNA within that sequence. BamHI recognizes the sequence GGATCC and cuts the DNA between the G and the A. This results in two DNA fragments, each with a sticky end. Sticky ends are complementary to each other, so they can be ligated (joined) together. BamHI is a very versatile restriction enzyme as it can be used to cut DNA from a variety of sources, including bacteria, viruses, and plants. It is also easy to use and it’s inexpensive, making it a cost-effective choice. Xbal is a restriction enzyme isolated from the bacterium Xanthomonas badrii. It is a type II restriction endonuclease, which recognizes a specific sequence of DNA and cuts the DNA within that sequence. Xbal recognizes the sequence TACTAGA and cuts the DNA between the T and the A. Xbal is a very versatile restriction enzyme as it can be used to cut DNA from a variety of sources, including bacteria, viruses, and plants. Like BamHI, it is also easy to use and it’s inexpensive, making it a cost-effective choice. The Bacillus expression plasmid pHT10 was digested with BamHI and Xbal, purified, concentrated, and treated with rSAP. The digested synthetic insert and plasmid were then mixed into a T4 DNA ligase reaction overnight at room temperature. T4 DNA ligase is an enzyme that joins together two pieces of DNA by catalyzing the formation of a phosphodiester bond between the 5'-phosphate group of one DNA strand and the 3'- hydroxyl group of the other DNA strand. This reaction is essential for DNA replication, repair, and recombination.

[0033] The T4 DNA ligase reaction takes place in the following steps:

[0034] 1 . The T4 DNA ligase enzyme binds to the two DNA strands to be joined.

[0035] 2. The enzyme breaks the phosphodiester bonds between the 5'-phosphate group of one DNA strand and the 3'-hydroxyl group of the other DNA strand.

[0036] 3. The enzyme then reforms the phosphodiester bonds between the two DNA strands, joining them together.

[0037] 4. The T4 DNA ligase enzyme releases the two DNA strands, which are now joined together.

[0038] The ligation reaction was transformed into E. coli DH5a (New England Biolabs - Ipswich, Massachusetts, USA) and transformants were screened for the gene insert on LB agar with ampicillin (100 pg / ml, LBAmP). The ligation reaction is a process that joins together two pieces of DNA. The resulting DNA molecule is then transformed into E. coli DH5a cells. The cells are then grown on a plate containing an antibiotic that kills any cells that do not contain the cloned gene. The cells that survive are then cultured and the cloned gene is harvested.

[0039] The new plasmid, pStrep, was purified from E. coli and transformed into B. subtilis. Transformants were selected on tryptic soy agar with chloramphenicol (5 pg / ml, TSACm), creating the recombinant Bacillus subtilis stain, Strep Vaccine 1 , (SV1 ).

[0040] A protective subunit is then produced during fermentation of SV1 at between 28 and 37 °C and induction with Isopropyl (3-D-1 -thiogalactopyranoside (IPTG) at concentration ranging from 0.1 to 0.5 mM. The culture is inactivated with formalin. To produce the Test Vaccine and iterations thereof, the inactivated SV1 culture was mixed with a naturally occurring polysaccharide, methyl cellulose (encapsulation media) which acts as the vehicle and protectant for the antigen.

[0041] EXAMPLES

[0042] Example 1 : Immunogenicity study of streptococcus epitope in weanling pigs:

[0043] Title: Pilot study determining the humoral immune response in three-week-old piglets after vaccination by various routes with an experimental Streptococcus vaccine.

[0044] Justification: Streptococcus suis and streptococcal-associated diseases are a significant disease and economic burden on intensive, indoor swine operations. Diseases associated with streptococcal infections include meningitis, septicemia, arthritis, polyserositis, valvular endocarditis, myocarditis, pericarditis, and abortion. Streptococcus suis is now the most common cause of meningitis in pigs submitted to veterinary diagnostic laboratories. The vaccines currently available fail to induce adequate cross-protection between the many serotypes of S. su / s thus, a vaccine that induces a strong protective and cross-serotype reactive immune response is needed.

[0045] Materials and Methods: Thirty pigs at 3 weeks of age will be weaned from a high health farm with no previous S. s / s concerns, or other disease confounders (PRRS, Mycoplasma hyopneumoniae, PEDv, APP, etc). The pigs will be tagged, weighed, and randomized into one of 10 groups (2 replicates x 5 groups (3 pigs / group)) and allowed to acclimate for 2 to 3 days. On Day 0 of the study, serum samples from each pig will be collected and the first dose of vaccine or saline will be administered as indicated in Table 1 . The second dose is administered 14 days later as indicated in Table 1 .

[0046] Treatment groups are separated. Vaccination: The vaccine will be administered in 2 doses at 2 mL per dose either through an intramuscular injection (IM) behind the ear in the neck, oral gavage, or nasal spray (1 mL / nostril). Saline administration will be by oral gavage.

[0047] The study timeline and samples to be collected are outlined in Table 2.

[0048] Table 1 : Route of administration for vaccine

[0049] Table 2: Study timeline and sample collection The results are illustrated in Figure 1 in which Enzyme-linked immunosorbent assays (ELISAs) detecting anti-Strep epitope antibodies in A) secretory IgA in saliva on study day 34, B) secretory IgA in intestinal mucosa on study day 35, C) IgG in serum on study day 34. Data is grouped by vaccination route. All data is relative to the non-treated saline control animals. Ratios greater than 1 indicate antibody levels greater than the saline control animals.

[0050] Example 2: Survival of Nile tilapia after vaccination with SV1 and challenge with virulent Streptococcus agalactiae.

[0051] Experimental Design, Sampling and Vaccination: A total of 180 fingerlings were acclimatized for two days and distributed in four treatments, all in triplicate (3 tanks / treatment, 15 fish / tank). The juveniles were distributed in 12 experimental units of 80 L, totaling 15 fish per tank. Fish were administered treatments as described (Table 1 ) below and grown for 54 days.

[0052] Fish were vaccinated with the SV1 via immersion (imer) and orally (oral) via coating the food pellets. Currently, there are only interparental injectable vaccines commercially available to prevent streptococcosis, thus a commercial S. agalactiae vaccine (AquaVac) was used as a positive control treatment (Table 3). Table 3: Treatment groups, routes of administration and number of replicates per treatment.

[0053] Replicates

[0054] Treatments Challenged Route of Administration (15 fish / tank)

[0055] T1|p_Apyes AquaVac intraperitoneal (IP)3injection

[0056] T2. Immersion 10% Yes 2 baths (study day 0 and day 45) 3

[0057] T3. Oral 10% Yes Oral (fed days 30 / 31 and 45 / 46) 3

[0058] T4. Control Yes No treatment 3

[0059] Intraperitoneal

[0060] The application of the vaccines intraperitoneally was carried out on the 41 st day of the experiment, 14 days before the infection challenge. A commercial vaccine, AQUAVAC® Strep SA, MSD, was administered according to the label instructions in the positive control group. The commercial vaccine (AV) was administered at 50 pL per fish.

[0061] Immersion

[0062] In the immersion application, the baths occurred at two distinct days of the experiment. The first bath was performed at the beginning of the experiment (day zero) and the second bath at 45 days (d 45) of experimentation. A dose of 500 mL of the SV1 in 4.5 L (10% v / v) in which the fish remained immersed for 20 minutes.

[0063] Oral

[0064] In the oral route, the SV1 was mixed directly on the feed. The fish received the feed containing the SV1 for two consecutive days, at two different times during the period. The first feeding containing SV1 was offered on days 30 and 31 and the second on days 45 and 46 of the experiment. The inclusion of SV1 in the diet was performed 30 minutes before food management. The dose was defined as 100 pL of the vaccine per gram of feed (10% v / w).

[0065] Experimental challenge: On the 55th day of the experiment, fish of all groups were challenged with the bacterial pathogen S. agalactiae strain S13 (ST-552, type 1 b) isolated from a streptococcosis outbreak in Nile tilapi in Brazilian state of Parana (Facimoto et al., 2017).

[0066] A 50% lethal dose experiment was initially conducted to determine the concentration of the bacteria capable of killing 50% of the unvaccinated and challenged population (LDso). For this dosage trial, 40 Nile tilapia were distributed in 8 experimental units of 30 L, with aeration and controlled temperature, totaling five animals per experimental unit. The LDso was performed in duplicate, and the concentration of bacteria tested were: 1 * 105; 1 x 106; 1 * 107; 1 *108CFU mL’1(data not shown). The LDso of S. agalactiae S13 by intraperitoneal injection was determined to be x Q7CFU mL’1and this concentration was used in the subsequent vaccination and challenge period described below.

[0067] The fish were fed for 54 days until they reached the approximate weight of 50 g, a weight in which the animals are more susceptible to diseases (Mian et al., 2009). After this fattening period, each fish was challenged with 1 x 107CFU mL’1of S. agalactiae S13 by intraperitoneal injection. The bacteria were pelleted and washed 3x in cold, sterile, PBS prior to injection. The fish underwent an anesthetic procedure with eugenol to perform the procedure.

[0068] Clinical signs were observed daily for 7 days post-challenge, and all dead fish were removed daily from the tanks and recorded in the trial file. Any fish that was unable to stay submerged or maintain proper orientation was humanely euthanized.

[0069] Results:

[0070] Table 4: Growth parameters (mean ± standard deviation) of Nile tilapia after 54 days of experiment. Different letters indicate significant difference among the treatments (p< 0.05).

[0071] Immersion 10% IP-AV Oral 10% Control

[0072] Weight (g) 58.62 ± 12.48ab 59.31 ± 9.78ab 62.17 ± 11.08a 43.10 ± 0.10c

[0073] Length (cm) 14.39 ± 1.13b 14.41 ± 0.41 b 15.13 ± 0.84a 12.94 ± 0.16

[0074] Survival analysis of Nile tilapia challenged with virulent S. agalactiae:

[0075] The results are illustrated in Figure 2 which shows a Kaplan-Meier survival analysis of Nile tilapia challenged with virulent S. agalactiae. The IP-AV (positive control) and SV1 10% immersion and 10% oral fed groups had significantly better survival than the unvaccinated controls (p < 0.05).

[0076] Example 3: Determination of the dose volume, route of administration, and kinetics of the humoral immune response in pigs vaccinated with candidate Strep Vaccine 1 (SV1 ). Objective

[0077] Evaluate the humoral immune response in pigs vaccinated with the candidate streptococcal vaccine formulation using different dose volumes and administration routes in order to determine the optimal vaccine presentation based on the level of specific antibodies determined by antigen-specific immunoassay tests.

[0078] Material and method

[0079] Animals and Accommodation

[0080] “Las Chirus” is a full-cycle confined pig farm. It has 450 breeders, generating 20 deliveries per week. It is located in French, district of 9 de Julio in the province of Buenos Aires, Republic of Argentina.

[0081] In farrowing and post-weaning, work was done with an all-in-all-out system by rooms and the finishing fattening of the animals is done in a continuous flow system.

[0082] A total of 105 animals (5 animals / group) were used. They were genetic Agroceres Pig Improvement Company (PIC) females and males obtained from the crossing of females Camborough 1050 with male Terminal 337.

[0083] They were housed in rooms with a full slat floor, with a floodable pit. The density was 0.33 animals per m2at termination of fattening phase.

[0084] Ventilation by means of curtains (natural ventilation). Food, water and usual handling of animals.

[0085] Food

[0086] Birth-15 days of life: Colostrum and milk

[0087] 15 days to weaning (21 d): milk and pre-starter food

[0088] 21-30 days of life: food F1 (starter)

[0089] 30-40 days of life: food F2

[0090] 40-55 days of life: food F3 (with Amoxicillin 400ppm in the food)

[0091] 55-70 days of life: food F4 (with Tiamulin 100ppm + Chlortetracycline 300ppm in the food)

[0092] Water

[0093] Water was provided ad libitum.

[0094] Number of pacifiers: 1 for every 10 animals housed.

[0095] Other handling during the experimentation period.

[0096] Upon admission to weaning boxes (approx. 21 days of age) piglets received a vaccination against PCV2 and Mycoplasma hyopneumoniae.

[0097] 42 days of life: Pigs received Mycoplasma revaccination.

[0098] Application of injectable Ceftiofur or injectable amoxicillin in animals showing nervous system signs as a result of potential infection by Streptococcus suis or Haemophilus parasuis. Treatments

[0099] Groups

[0100] Vaccine candidate evaluated:

[0101] • SV1 : Candidate vaccine against Streptococcus spp (Ss)

[0102] Table 5: PO: (oral administration); IM (intramuscular)

[0103] Administration date

[0104] First dose: Within the first 3 days of life

[0105] Second dose: 14-18 days after the first vaccination.

[0106] Vaccination Management

[0107] Oral administration: the above indicated volume was administered using a dispenser or an automatic syringe.

[0108] Intramuscular administration: The above indicated volume was administered with an automatic syringe on the neck region just behind and below the ear, but in front of the shoulder. Preparation of vaccines in the laboratory

[0109] 10 microliters were taken from a cryovial from the SV1 working seed, which was plated on TSA plates with chloramphenicol (CP5: 5 mg / ml). The plates were incubated at 37 °C ON. The following day, a colony of the culture was cultured in TSB with CP5. The pre-culture (PC) was incubated at 37 °C overnight. The following day, a certain volume of TSB + CP5 was seeded into a certain large volume of sterile TSB. The culture was incubated for 2 hours at 37 °C, then a certain volume of 0.5 M IPTG (1 ml / L) was added to induce antigen production, and incubation was continued for another 6 hours.

[0110] After a total of 8 hours, samples were taken for colony forming unit calculations. Subsequently the culture was inactivated by the addition of a certain volume of formaldehyde and the culture was incubated at room temperature overnight.

[0111] The Excipient was prepared following the usual preparation process.

[0112] The formulations for the 1 ml and 2 ml dose groups were mixed in equal proportions of inactivated culture (SV1 ) to excipient (1 :1 ).

[0113] Formulations were packed in 10 ml vial, labeled and stored between 2 - 8 °C until use.

[0114] Samples and parameters evaluated.

[0115] Sampling Date

[0116] Samples were taken on the following days: • 1stsampling: Primary vaccination day (Week 1 )

[0117] • 2ndsampling: Booster Day (Week 3)

[0118] • 3rdsampling: Week 4

[0119] • 4thsampling: Week 6

[0120] • 5thsampling: Week 8

[0121] Sampling and processing:

[0122] Serum Samples

[0123] Samples of blood were taken from the jugular vein with 18G x 1 .5” needle for pigs and 20G x 1” or 1 .5” for piglets. Around 5 ml of blood were taken without anticoagulant in a 15 ml conical tube. To extract serum from blood, the serum is allowed to coagulate in a tube at an angle of at least 60 degrees. Once the clot is formed, it is detached from the glass with the help of a needle. To obtain maximum serum, tubes were heated to 37 °C for a minimum of 30 minutes to a maximum of 1 hour and then placed at 4 °C overnight. The next morning, the tubes were centrifuged at 3000 rpm for 30 minutes and the serum (supernatant) was directly extracted with a pipette. The amount of serum collected was between 500 pl to 1 ml. Serum were stored at -20°C until analysis.

[0124] Saliva Samples

[0125] IgA determination from Saliva samples were taken as follows:

[0126] Oral swabs: Sterile cotton swabs were used to collect saliva from the pigs, the swabs were placed in the pig’s mouth until soaked in saliva. The saliva-soaked swabs were then submerged in 1 ml of sterile PBS, pH 7.4 in a 15 ml conical tube and stored at - 20°C until analysis.

[0127] Analysis

[0128] ELISA for antibody determination

[0129] ELISA S / P ratios were used to determine IgA and IgG antibody response to SV1 with the following formula:

[0130] Sample - negative control / positive control - negative control

[0131] ELISA protocol:

[0132] 1 ) Coating: BioGreiner plates were coated with 5 pg / ml / well (100 pl / well) of the synthetic Streptococcus peptide antigen covalently linked to BSA and then incubated at room temperature overnight.

[0133] 2) Plates were washed 3X with TBS-T (Tris-buffer saline with 0.1 % Tween-20) (200 pl / well)

[0134] 3) The plates were then blocked with 200 pl / well of blocking buffer (Bethyl lab, Cat.

[0135] E104) and incubated 1 hour at room temperature.

[0136] 4) Plates were washed 3X with TBS-T (200 pl / well)

[0137] 5) Test Samples were diluted as follows:

[0138] • Serum: 1 :100 dilution in sample buffer (50 m / W Tris buffered saline, pH 8, 1 % bovine serum albumin (BSA), 0.05% Tween-20), 100 pl / well

[0139] • Saliva: undiluted, 100 pl / well

[0140] The plate was incubated for 1 hour at room temperature. 6) Plates were washed 3X with TBS-T (200 pl / well). Secondary antibodies were added to each well, 100 pl / well, at the following concentrations.

[0141] • Anti-Pig IgA HRP 1 pg / ml (A100-102P, Bethyl laboratories)

[0142] • Anti-Pig IgG HRP 0.25 pg / ml (A100-205P, Bethyl laboratories)

[0143] Incubated for 1 hour at room temperature.

[0144] 8) Plates were washed 3X with TBS-T (200 pl / well).

[0145] 9) TMB One component Substrate (E102, Bethyl laboratories) was added (100 pl / well) and incubated 3 minutes at room temperature.

[0146] 10) The reaction was stopped with 100 pl / well of 0.1 M sulfuric acid.

[0147] 10) The optical density of each well was read at 450 nm using a visible light spectrophotometer.

[0148] Statistical analysis

[0149] An analysis of variance was applied for two factors measured over time using the IBM SPSS Statistic 25 software after determining homoscedastic of variances, sphericity, and normality of the groups.

[0150] Experimental Design

[0151] The schematic diagram outlining experimental design is illustrated in Figure 3.

[0152] RESULTS

[0153] Two doses of candidate SV1 vaccine induces antigen-specific secretory IgA and serum IgG in piglets as young as three days of age. Saliva secretory IgA (slgA) S / P ratio determination.

[0154] Results are shown in Table 6 and in Figure 4. An increase in the antigen-specific salivary slgA S / P ratios in pigs is observed throughout the weeks in some of the groups evaluated, when the comparison between groups is made within each week, no significant differences are observed (weeks 1 , 3, 4 and 6). At week 8, the pigs that received two 2 mL doses of SV1 orally or IM followed by an oral booster, had significantly higher S / P ratio relative to the other groups, with no significant difference between the 2 mL oral / oral and 2 mL IM / oral group. These data indicate 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 a 2 mL / dose regimen given orally or IM / orally provides a robust slgA response.

[0155] Table 6

[0156] Serum IgG S / P ratio determination.

[0157] Results are shown in Table 7 and in Figure 5. An increase in the antigen-specific serum

[0158] IgG S / P ratios is observed throughout the weeks in some of the groups evaluated, when the comparison between groups is made within each week, no significant differences are observed (weeks 1 , 3, and 4.). At weeks 6 and 8, the pigs that received two 2 mL doses of SV1 orally or IM followed by an oral booster, had significantly higher S / P ratio relative to the other groups. At 6 weeks of age, the 2 mL IM / Oral group had significantly higher mean S / P ratio than the 2 mL Oral / Oral group, but by 8 weeks of age this difference was no longer present. 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 a 2 mL / dose regimen given orally or IM / orally provides a robust serum IgG response.

[0159] Age of animal at time of sampling

[0160] Table 7

[0161] Conclusion

[0162] These data indicate that candidate vaccine SV1 is immunogenic and antigenic and importantly, the vaccine can protect fish, in this example nile tilapia, from a challenge with virulent Streptococcus agalactiae. Further, the SV1 vaccine induces a robust antigen-specific salivary slgA and serum IgG response in piglets with the first dose of vaccine administration as early as 3 days of age. The route of administration of either two doses orally or one dose intramuscularly and the second booster dose given orally provide similar responses for slgA while the IM / oral induces a higher and quicker serum IgG response as would be expected based on scientists understanding of the immune response and activation, but the response at 8 weeks of age was similar suggesting the new antigenic specific baseline antibody response may not differ. While the volume providing the best antibody response in pigs was 2 mL, it is ultimately irrelevant as the concentration of antigen / mL can be adjusted so that one mL of vaccine could have the same concentration of antigen as a 2 mL dose.

[0163] Any method described herein may incorporate any design element contained within this application and any other document / application incorporated by reference herein.

[0164] The present invention may be embodied in other forms without departing from the spirit and the essential attributes thereof, and, accordingly, reference should be made to the appended claims, rather than to the foregoing specification, as indicating the scope of the invention. The invention illustratively disclosed herein suitably may be practiced in the absence of any element which is not specifically disclosed herein.

[0165] Sequence Listing Information:

[0166] DTD Version: V1_3

[0167] File Name: SEQUENCE LISTING 3113.005.xml

[0168] Software Name: WIPO Sequence

[0169] Software Version: 2.3.0

[0170] Production Date: 2023-05-12

[0171] General Information:

[0172] Current application / IP Office: US

[0173] Current application / Applicant file reference: 31 13.005

[0174] Applicant name: Ventanco SA

[0175] Applicant name I Language: en

[0176] Inventor name: Sherry Layton

[0177] Inventor name / Language: en

[0178] Invention title: COMPOSITIONS AND METHODS OF ENHANCINGIMMUNE

[0179] RESPONSES TO STREPTOCOCCUS ( en )

[0180] Sequence Total Quantity: 3

[0181] Sequences:

[0182] Sequence Number (ID): 1

[0183] Length: 49

[0184] Molecule Type: AA

[0185] Features Location / Qualifiers:

[0186] - source, 1 ..49

[0187] > mol_type, protein

[0188] > organism, synthetic construct

[0189] Residues:

[0190] DPVLVGQEAV AIGELLAESD PKKADYYCQN AATLEGKGPK LADKYSPIF 49

[0191] Sequence Number (ID): 2

[0192] Length: 878

[0193] Molecule Type: AA

[0194] Features Location / Qualifiers:

[0195] - source, 1 ..878

[0196] > mol_type, protein

[0197] > organism, synthetic construct

[0198] Residues:

[0199] MLKATSKTFV TQHTAFSYTA QRFGLKQLGI AGVSEEEPSP PLTKVPGLED MEAGQGIDEA 60

[0200] SLYDPHTWDP VLVGQEAVAI GELLAESDPK KADYYCQNAA TLEGKGPKLA

[0201] DKYSPIFSSS 120

[0202] SSMSGKGPAI GIDLGTTYSC VGVFQHGKVE IIANDQGNRT TPSYVAFTDT

[0203] ERLIGDAAKN 180

[0204] QVAMNPTNTI FDAKRLIGRK YDDPTVQSDM KHWPFRVVNE

[0205] GGKPKVQVEY KGEMKTFFPE 240

[0206] EISSMVLTKM KEIAEAYLGK KVETAVITVP AYFNDSQRQA TKDAGTITGL NVMRIINEPT 300 AAAIAYGLDK KGTRAGEKNV LIFDLGGGTF DVSILTIEDG IFEVKSTAGD THLGGEDFDN 360 RMVNRFVEEF KGKHKRDNAG NKRAVRRLRT ACERARRTLS SSTQASIEID SLFEGIDFYT 420 SITRARFEEL NADLFRGTLE PVEKALRDAK LDKGQIQEIV LVGGSTRIPK IQKLLQDFFN 480 GKELNKSINP DEAVAYGAAV QAAILMGDKS ENVQDLLLLD VTPLSLGIET AGGVMTALIK 540 RNTTIPTKQT QTFTTYSDNQ SSVLVQVYEG ERAMTKDNNL LGKFDLTGIP PAPRGVPQIE 600 VTFDIDANGI LNVSAVDKST GKENKITITN DKGRLSKDDI DRMVQEAEKY KAEDEANRDR 660 VGAKNSLESY TYNMKQTVED EKLKGKISDQ DKQKVLDKCQ EVISSLDRNQ MAEKEEYEHK 720 QKELEKLCNP IVTKLYQGAG GAGAGGSGGP TIEEVDSSSS SSDPVLVGQE AVAIGELLAE 780 SDPKKADYYC QNAATLEGKG PKLADKYSPI FPLTKVPGLE DMEAGQGIDE ASLYDPHTWL 840 KATSKTFVTQ HTAFSYTAQR FGLKQLGIAG VSEEEPSP

[0207] 878

[0208] Sequence Number (ID): 3

[0209] Length: 2643

[0210] Molecule Type: AA

[0211] Features Location / Qualifiers:

[0212] - source, 1 ..2643

[0213] > mol_type, protein

[0214] > organism, synthetic construct

[0215] Residues:

[0216] ATGTTAAAGG CTACATCCAA GACTTTTGTT ACACAGCACA

[0217] CGGCTTTCAG TTATACAGCG 60

[0218] CAACGGTTTG GCTTGAAGCA GTTAGGGATT GCAGGTGTGA

[0219] GTGAAGAGGA ACCGTCCCCT 120

[0220] CCATTAACAA AAGTCCCTGG GTTGGAAGAC ATGGAGGCCG

[0221] GGCAAGGGAT TGATGAAGCC 180

[0222] TCACTCTATG ATCCTCATAC TTGGTTGGAC CCGGTGCTTG

[0223] TCGGTCAGGA GGCGGTAGCC 240

[0224] ATCGGAGAGC TTTTGGCTGA ATCAGATCCG AAGAAAGCTG

[0225] ATTATTACTG CCAAAACGCG 300

[0226] GCTACTTTAG AGGGTAAAGG GCCAAAGCTC GCTGACAAGT

[0227] ATTCACCGAT ATTTTCATCG 360

[0228] AGTTCATCAA TGAGCGGAAA GGGCCCTGCC ATTGGAATCG

[0229] ATCTGGGCAC AACATACAGC 420

[0230] TGTGTGGGAG TATTTCAGCA CGGCAAGGTT GAAATCATTG CAAATGACCA AGGCAACAGA 480 ACAACGCCGT CATATGTCGC TTTTACAGAT ACTGAACGCC

[0231] TCATCGGCGA CGCTGCCAAA 540

[0232] AACCAAGTGG CGATGAATCC GACAAATACG ATTTTCGACG

[0233] CGAAACGTCT CATTGGACGG 600

[0234] AAATACGATG ATCCAACAGT ACAATCTGAT ATGAAACATT

[0235] GGCCGTTTCG GGTCGTCAAC 660

[0236] GAAGGAGGAA AGCCCAAAGT GCAGGTCGAG TATAAAGGAG

[0237] AAATGAAAAC TTTCTTTCCC 720

[0238] GAAGAGATAA GTTCCATGGT TTTAACAAAA ATGAAGGAAA

[0239] TTGCCGAGGC TTATTTAGGA 780

[0240] AAAAAGGTGG AAACGGCGGT GATCACCGTG CCGGCATATT

[0241] TTAATGATAG CCAGCGCCAG 840

[0242] GCAACCAAGG ATGCGGGAAC CATTACAGGC TTAAATGTTA

[0243] TGAGAATTAT TAATGAACCT 900

[0244] ACTGCCGCAG CAATCGCTTA CGGACTGGAT AAAAAAGGAA

[0245] CAAGAGCAGG CGAAAAGAAT 960

[0246] GTACTGATTT TTGATTTAGG GGGAGGTACC TTCGACGTGT

[0247] CCATTCTTAC TATAGAAGAC 1020

[0248] GGCATCTTTG AAGTGAAATC AACAGCAGGA GATACACACT

[0249] TGGGCGGTGA GGATTTTGAC 1080

[0250] AACCGGATGG TCAATAGATT CGTCGAAGAA TTTAAAGGAA

[0251] AGCATAAACG CGACAATGCC 1140

[0252] GGGAACAAAA GGGCTGTACG ACGGCTGAGG ACTGCCTGTG

[0253] AAAGAGCGCG GCGTACGCTC 1200

[0254] TCCTCCTCCA CACAGGCTTC AATTGAGATA GATTCGCTTT

[0255] TCGAAGGCAT TGACTTCTAC 1260

[0256] ACTAGCATTA CTCGTGCGCG CTTTGAAGAA CTGAATGCGG

[0257] ATCTGTTTCG AGGCACGCTG 1320

[0258] GAGCCGGTTG AAAAAGCGCT CAGAGATGCA AAGTTGGACA

[0259] AAGGCCAGAT TCAAGAAATC 1380

[0260] GTACTGGTTG GAGGCAGCAC ACGCATTCCT AAAATCCAAA

[0261] AGCTTCTCCA GGATTTTTTT 1440

[0262] AACGGCAAAG AGCTGAATAA ATCGATCAAC CCGGATGAAG

[0263] CGGTTGCATA TGGCGCCGCC 1500

[0264] GTTCAAGCGG CGATCCTGAT GGGTGATAAA AGCGAAAACG

[0265] TCCAGGACCT TCTGCTTTTA 1560

[0266] GATGTTACGC CTTTATCACT GGGGATTGAA ACAGCCGGCG

[0267] GGGTCATGAC GGCTTTGATT 1620

[0268] AAACGAAATA CGACCATACC GACGAAGCAG ACACAAACAT

[0269] TTACGACGTA CAGCGATAAT 1680

[0270] CAGTCATCGG TACTCGTCCA GGTTTATGAA GGAGAACGTG

[0271] CAATGACAAA AGATAACAAC 1740

[0272] CTTTTGGGGA AATTTGATCT CACAGGGATT CCGCCTGCAC

[0273] CAAGGGGTGT GCCTCAAATT 1800

[0274] GAAGTAACCT TTGATATCGA CGCCAACGGG ATCTTGAATG

[0275] TTAGTGCTGT TGATAAGAGT 1860 ACCGGCAAGG AAAATAAAAT TACAATAACA AACGATAAAG

[0276] GCAGACTATC TAAAGACGAT 1920

[0277] ATTGACCGCA TGGTGCAAGA GGCTGAAAAG TACAAAGCAG

[0278] AGGATGAAGC AAACCGTGAC 1980

[0279] CGTGTAGGAG CAAAAAACAG CTTAGAAAGC TATACATATA

[0280] ATATGAAACA AACAGTGGAA 2040

[0281] GATGAGAAAC TGAAGGGGAA AATAAGCGAT CAAGACAAAC

[0282] AGAAAGTTCT TGATAAATGC 2100

[0283] CAGGAAGTCA TCTCATCTTT AGATAGAAAT CAAATGGCGG

[0284] AGAAAGAAGA ATATGAGCAT 2160

[0285] AAGCAAAAAG AGCTGGAAAA ATTATGCAAT CCAATCGTTA

[0286] CGAAACTTTA TCAAGGAGCC 2220

[0287] GGTGGTGCAG GTGCTGGGGG TTCTGGCGGC CCGACCATTG

[0288] AGGAAGTTGA CAGCTCATCT 2280

[0289] TCTTCCTCTG ACCCTGTCCT TGTCGGACAG GAAGCCGTGG

[0290] CAATTGGTGA ATTGCTGGCA 2340

[0291] GAATCTGATC CAAAAAAAGC GGACTATTAT TGTCAAAATG

[0292] CAGCCACGCT TGAAGGTAAA 2400

[0293] GGTCCGAAAC TAGCCGATAA ATACTCTCCT ATCTTCCCAT

[0294] TAACAAAAGT CCCTGGGTTG 2460

[0295] GAAGACATGG AGGCCGGGCA AGGGATTGAT GAAGCCTCAC

[0296] TCTATGATCC TCATACTTGG 2520

[0297] TTGTTAAAGG CTACATCCAA GACTTTTGTT ACACAGCACA

[0298] CGGCTTTCAG TTATACAGCG 2580

[0299] CAACGGTTTG GCTTGAAGCA GTTAGGGATT GCAGGTGTGA GTGAAGAGGA ACCGTCCCCT 2640

[0300] TAA 2643

[0301] END

Claims

CLAIMS1 . A vaccine composition against Streptococcus comprising: an amino acid sequence selected from the group consisting of Sequence Number (ID): 1 , Sequence Number (ID): 2, and Sequence Number (ID): 3; and a pharmaceutically acceptable carrier.

2. The vaccine composition of claim 1 wherein the vaccine is derived from a protective antigen on which nucleotide optimization was used to optimize for Bacillus subtilis to derive the nucleotide sequence for the DNA sequence.

3. The vaccine composition of claim 1 further comprising one or more immunostimulatory polypeptides.

4. The vaccine composition of claim 1 wherein the vaccine composition induces an immune response.

5. The vaccine composition of claim 4 wherein the immune response includes an antibody response.

6. The vaccine composition of claim 1 wherein the vaccine composition is administered by a method selected from the group consisting of oral, intranasal, and parenteral.

7. A method of enhancing the immune response against Streptococcus comprising administering to the subject the vaccine composition of claim 1 in an amount effective to enhance the immune response of the subject to one or more strains of Streptococcus.

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

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

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

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