Progress 09/01/23 to 08/31/24

Outputs
Target Audience:Scientists evaluating progress, including the National Program Leader for this project. Changes/Problems:During this time period, we encountered the problem of a lack of establishment of persistent high-grade S. suis infection of naïve weaned pigs, following experimental infection with different serotypes of clinically releventS. suis. The ability of a pathogen to establish high-grade infection in a susceptible natural host following an experimental infection-challenge is imperative for studies evaluating efficacy of new vaccines to protect against disease caused by the cognate pathogen. Given that our goal here is to develop a broadly protective S. suis vaccine, we worked to obviate this problem by adopting two strategies: The first, was to culture each of the S. suis serotype selected for infection challenge in the presence of multiple in vivo signals the pathogen encounters when it is actually infecting its host. This strategy of providing in vivo host environmental cues induces strong expression of a large set of proteins, including virulence factors that significantly enhance the pathogenic potential of the organism. This is the same strategy that was used by us to generate the native pCPs that are constituents of the multi-component protein subunit vaccine demonstrated to engender strong protection in pigs challenged with the homologous or heterologous S. suis serotypes. The other strategy, was to examine a co- infection model, i.e., by employing an infection inoculum that includes a mixture of the porcine reproductive and respiratory syndrome (PRRS) virus and the S. suis serotype, especially since pigs co-infected with a mix of these pathogens are reportedly more susceptible to S. suis infection and hence highly like to cause persistent high-grade infection. We determined that both strategies established high-grade infection of naïve weaned pigs, and all pigs were either succumbed to infection or had to be euthanized due to severe S. suis disease within five days following experimental infection. The establishment of high-grade infection following experimental S. suis, evidenced by the manifested clinical features, was further confirmed by gross and histopathological findings of various necropsied tissues, and by culturing the pathogen from such tissues. Going forward, based on the above observations, for challenge of immunized pigs,S. suis cultured in the presence of in vivo environmental cues will be used in vaccine efficacy studies. What opportunities for training and professional development has the project provided?These studies thus far have provided ample opportunities for professional development. Working collaboratively, both the vaccine development team and the veterinary team have been facilitated with a better understanding S. suis mechanics of infection, pathogenicity and host susceptibility to these pathogens. These insights will be further disseminated to the scientific community in the form of a peer-reviewed publication in a relevant scientific journal, following completion of this study and protection of intellectual property. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Pig Challenge studies for vaccine efficacy (in progress). All of the issues pertaining to pig infection-challenge protocols we had encountered thus far have been resolved; and hence, we have initiated the final task of Technical Objective #2, namely, evaluation of the efficacy of the multi-component protein subunit vaccine, especially its ability to confer protection against 15 disparate S. suisserotypes that cause pig disease in diverse geographical locations of the globe. Vaccine efficacy is being determined by the ability of the vaccine completely protect immunized pigs in the "Test" group from developing systemic disease or by its ability to significantly prolong time to death compared with unimmunized pigs in the "Control" group. Such vaccine efficacy pig studies will be performed in a staggered manner. Three to five serotypes will be tested each time in separate cohorts of pigs (one serotype per cohort). We anticipate a timeframe of 8-10 months to complete the studies, given that vaccine efficacy will be examined against 15 serotypes. We have requested a No-Cost-Extension of an additional 12 months to complete the study

Impacts
What was accomplished under these goals? Two objectives in this proposal. Technical Objective #1 (completed), pertains to the generation of the multi-valent protein subunit vaccine constituted by 28 vaccine proteins for pig immunization, followed by optimization of vaccine formulation and the immunization regimen. We had identified these 28 "prioritized conserved proteins" (pCPs) in Phase I, through the strategy of "proteome walking" using our proprietary platform proteomics-based proteome mining tool called Proteomics-based Expression Library Screening (PELS). For preparation of this vaccine, lysates of whole-cells of three Serotype 2, virulent S. suis strains, P1/7 (ATCC BAA-853), 91-1804 (ATCC 700796) and 958242 (ATCC 700794), isolated from infected pig blood, pig brain and human cerebrospinal fluid, respectively, were cultured in Brain Heart Infusion (BHI) broth the presence of in vivo cues encountered by the pathogen during an actual process of infection of pig / human hosts. During Phase I studies, we determined that when cultured in such conditions, S. suis elaborates a repertoire of in vivo-expressed (IVE) proteins, i.e., those proteins that are differentially expressed during host infection and help pathogen establish itself and cause disease. These IVE proteins included increased expression of the majority of the 28 pCPs, which were then enriched several-fold using sequential fractionation / chromatography from individual whole-cell lysates for evaluation of potential to protect broadly against disparate clinically relevant serotypes that cause pig disease across North America and other parts of the world. Quality control, i.e., the presence of each of the 28 pCPs in sufficient amounts in lot / batch of the vaccine preparation is confirmed by tandem mass spectrometry. The vaccine prepared in this manner is stored at -80oC until further use in upcoming pig vaccine evaluation studies. As a prelude to studies comprising Technical Objective #2, which involves evaluation of cross protective efficacy of the multi-component subunit vaccine against disparate clinically relevant S. suis serotypes implicated in pig disease worldwide, preliminary infection challenge studies were performed to determine whether virulent serotypes used to challenge immunized pigs could consistently establish robust / persistent high-grade infection. These studies were deemed imperative because inferences drawn from our Phase I studies indicated that not all S. suis serotypes / strains, even those freshly isolated, consistently infected naive pigs, following simultaneous intranasal, despite pre-treatment 1 hour before challenge with 1 ml 5% acetic acid in each nostril, and intraperitoneal challenge with1 x 109 C.C.U. Given that robust infection of pigs by challenge is central to evaluation of vaccine efficacy. An initial round of infection challenge studies was unsuccessful in that a challenge of four cohorts of 7-week old pigs (mixed gender; n = 3 / cohort), with 1 x 109 C.C.U. of clinically relevant Serotypes 1, 2, 1/2 and 3, cultured in Todd Hewitt Broth, supplemented with 10% FCS, was inconsistent: pigs in each cohort and across all cohorts were not uniformly infected, and the few animals that appeared to be infected demonstrated only mild, low-grade infection. There was an absence of overt clinical disease manifestations in the majority of animals across all cohorts, which was confirmed by microbiological culture of swabs from joints, peritoneal surface, pleural surface, and meningeal surface from each pig (inconsistent growth / low numbers of bacteria isolated from necropsied tissues from these different anatomical sites), gross examination of organs, and histopathological examination of brain, liver, spleen, heart, meninges, joints, synovial membranes, lungs. Given that establishment of robust infection by diverse S. suis serotypes is central to evaluation of vaccine efficacy, a second round of pig infection-challenge studies with S. suis strains with enhanced potential for host infection was undertaken. For these studies, we provided a culture of a fresh clinical isolate of serotype 2 S. suis that had previously failed in pig infection challenge experiments with multiple in vivo signals likely encountered by the pathogen when it is actually engaged in the process of infecting a host. These in vivo signals included supplementing BHI broth culture medium with 200 µmolar of 2,2' Bipyridyl, and growing the pathogen in BHI broth medium statically at 37oC for 24 hours under microaerophilic conditions (5 -10% CO2). This strategy of providing in vivo host environmental cues induced strong expression of a large set of proteins, including virulence proteins that significantly enhance the pathogenic potential of the organism. This is the same strategy that was used by us to generate the native pCPs that are constituents of the multi-component protein subunit vaccine demonstrated to engender strong protection in pigs challenged with the homologous or heterologous S. suis serotypes. We also simultaneously examined a co-infection model, i.e., by employing an infection inoculum that includes a mixture of the porcine reproductive and respiratory syndrome (PRRS) virus and the S. suis serotype, especially since pigs challenged in this manner are reportedly more susceptible to S. suis infection and hence highly like to cause persistent high-grade infection. Two groups of pigs (n = 4, group I; n = 6 group 2)were examined in the second round of pig infection-challenge studies: Group 1 pigs were challenged with S. suis only. Group 2 pigs were challenged with S. suis followed by a virulent PRRSv challenge. For all groups, the challenge was administered intraperitoneally (6ml) and intranasally (2ml/nostril) on Day 0. Prior to challenge administration, all pigs were administered 2 mls (1ml/nostril) of acetic acid intranasally. The pigs were observed for changes in behavior and locomotion daily. In addition to daily clinical observations, the body temperatures were recorded daily. By Day 5 post-challenge, all pigs in both groups had died or were euthanized. At necropsy, a swab for culture was from the joints, peritoneal surface, pleural surface and meningeal surface.After collection of the swabs, tissue samples of lung, brain, spleen, liver, and joint synovium were collected and placed in formalin for histopathological evaluation. Infected pigs in both groups demonstrated clinical features of S. suis infection / disease. Features of gross necropsy findings indicated pigs demonstrated all / majority of the following: severe fibrinous peritonitis, congested lungs, pleuritis and fibrin strands on pleural surfaces, pericarditis and pericardial effusion, congested meningeal vessels, and swollen joints. Histological examination indicated evidence of peritonitis, pleuritis and interstitial pneumonia, suppurative meningitis, synovitis and multi-organ congestion. microbiology of blood and necropsy tissue specimens were confirmatory of S. suis infection / disease.

Publications

Progress 09/01/22 to 08/31/23

Outputs
Target Audience:Scientists evaluating progress, including the National Program Leader for this project. Changes/Problems:The abiltity of a pathogen to establish high-grade infection in a susceptible natural host following an experimental infection challengeis imperative for studies evaluating efficacy of newvaccines against the cognate pathogen. In this SBIR Phase II project, our goal is to develop a multi-component protein subunit pig vaccine that is capable of engendering broad protection against the majority of the clinically relevant S. suis serotypes causing disease in diverse geographical regions of the globe.As a prelude to vaccine efficacy studies, based on observations gleaned from SBIR Phase I studies, we determined the ability of four model serotypes(1, 2, 1/2 and 3) to establish consistent and robust/ high-grade pig infection following infection challenge pf cohorts of 3 pigs ( n = 3 / cohort). Individual S. suis serotypes were cultured in Hewitt broth supplemented with 10% FCS , harvested in mid-log phase, concentrated and then 1 x 109CCU / ml each of a particular serotype were used to challenge pigs. Each pig was simultaneously challengedboth intranasally (pre-treated with 1 ml of 5 % acetic acid 1 hour prior to challenge) and intraperitoneally. The pigs were monitored, euthanized and necropsied tissue specimens were examined as stated above. Results indicated thatinfection of pigs within a cohort and across cohorts was inconsistent, and when present, of a low-grade: the majority of pigs did not manifest clinical disease, which was further confirmed by gross examination of necropsied organs, microbiological culture, and histopatholgical examination. Given this, the following alternate approacheswill be examined: The first, is to culture each of the four S. suis serotypes selected for infection challenge in the presence of multiple in vivo signals the pathogen encounters when it is actually in the process of infecting a host. This strategy of providing in vivo host environmental cues induces strong expression of a large set of proteins, including viulence factors that significantly enhance the pathogenic potential of the organism. This is the same strategy that was used by usto generate the native pCPs that are constitutents of the multi-component protein subunit vaccine demonstrated to engender strong protection in pigs challenged with the homologous or heterologous S. suis serotypes. The other alternate strategy, should the first approach be found to be suboptimal, will be to examine the co-infection model,i.e., by employing an infection inoculum that includes a mixture of the porcine reproductive and respiratory syndrome (PRRS) virus and the S. suis serotype, especially since pigs in this scenario are reportedly more susceptible toS. suis infection and hence highly like to cause persistent high-grade infection. What opportunities for training and professional development has the project provided?These studies thus far have provided ample opportunities for professional development. Working collaboratively, both the vaccine develpment team and the veterinary team have beenfacilitated with a betterunderstanding S. suismechanics of infection, pathogenicityand host susceptibilty to these pathogens.These insights will be further disseminated to the scientific community in the form of a peer-reviewed publication in a relevant scientific journal, following completion of this study and protection of intellectual property. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?The immediate plan to continue with batches of vaccine stock preparations, evaluations of each batch of stock to facilitate vaccine trials. Also, a central focus is on enhancing the infection / pathogenic potential of each of above fourS. suis serotypesto facilitate consistent high-grade systemic infection of pigs, which will then allow for immediate studies to evaluate vaccine efficacy. Such vaccine efficacy studies are anticipated to commence in early 2024 and completed by the end of this SBIR Phase II grant.

Impacts
What was accomplished under these goals? Two objectives in this proposal. Toward Objective #1, in Year I of this SBIR Phase II grant, preparation of multiple lots / batches of the multi-component protein subunit vaccine for upcoming pig studies in Year II is underway. Specifically, three Serotype 2, virulentS. suis strains, P1/7 (ATCC BAA-853), 91-1804 (ATCC 700796) and 958242 (ATCC 700794), isolated from infected pig blood, pig brain and human cerebrospinal fluid, respectively, are being used to prepare the vaccine. To prepare the vaccines, thesestrains are cultured inBrain Heart Infusion (BHI) broth, and provided within vivo cues encountered by this pathogen when it is engaged in an actual process of infection of the pig or human hosts. In Phase I studies, we determined that when cultured in such conditions, S. suis elaborates a repertoire of in vivo-expressed (IVE) proteins, including increased expression ofthe majority of the 28 prioritized conserved proteins (pCPs)we had identified in Phase I, through the strategy of "proteome walking" usingour proprietary platform proteomics-based proteome mining toolcalled Proteomics-based Expression Library Screening (PELS). The28 native pCPS are then enriched several-foldusing sequential fractionation / chromatograpy from individual whole-cell lysates for evaluation of potential to protect broadly against disparate clinically relevant serotypes that cause pig disease across North America and other parts of the world. Quality control, i.e., the presence of each of the 28 pCPs in sufficient amounts in lot /batch ofthe vaccine preparation is confirmed by tandem mass spectrometry. The vaccine prepared in this manner is stored at -80oC until further use. As a prelude to studies comprising Objective 2 (to be performed in Year 2), which involves evaluation of cross protective efficacy of the multi-component subunit vaccine against disparate clinically relevant S. suis serotypes implicated in pig disease worldwide, a set of preliminary infection challenge confirmation studies are ongoing to determine whether virulent serotypes used to challenge immunized pigs can establish robust infection reliably and consistently.These studies are imperative because inferences drawn from our Phase I studiesindicate that not all strains, even those freshly isolated, fail to consistently cause infection of naive pigs, following simultaneous intranasal, despitepre-treatment 1 hour before challenge with 1 ml 5% acetic acid in each nostril, and intraperitoneal challenge with1 x 109C.C.U. Getting such strains to robustly infect pigs is central to evaluation of vaccine efficacy. We have completed thefirst round of infection challenge studies, in which four cohorts of 7-week old pigs (mixed gender; n = 3/ cohort) were challenged with 1x 109C.C.U. of four different clinically relevant S. suis serotypes: Serotypes 1, 2, 1/2 and 3. Following challenge, pigs were monitored every 12 hours for 14 days, after which pigs were euthanized and necropsied. We found that challenge infection caused by these serotypes, cultured in Todd Hewitt Broth, supplemented with 10% FCS, was inconsistent: pigs in each cohort and across all cohorts were not uniformly infected, and the few animals that appeared to be infecteddemonstrated only mild,low grade infection. This was evidenced by the absence of overt clinical disease manifestations in the majority of animals across all cohorts, which was confirmed by microbiological culture of swabs from joints, peritoneal surface, pleural surface, and meningeal surface from each pig (inconsistent growth / low numbers of bacteria isolated from necropised tissues from these different anatomical sites), gross examination of organs, and histopathological examination of brain, liver, spleen, heart, meninges, joints, synovial membranes, lungs. Given that establishment of robust infection by diverse S. suis serotypes is central to evaluation of vaccine efficacy, we are planning pig infection challenge studies with S. suis strains with enhanced potential for host infection (see below).

Publications