Performing Department
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Non Technical Summary
Non-technical SummaryCurrently, there are no efficacious vaccines to protect piglets against disease caused by the emerging zoonotic (transmissible from animals to humans) pathogen, Streptococcus suis. Porcine S. suis disease is a major cause of morbidity and mortality in weaned piglets and is a significant economic burden on pork producers globally. Losses to the pig industry globally are in the range of tens of millions to several hundred millions USD annually worldwide. Human S. suis disease can be serious and sometimes fatal, which places a heavy societal burden on public health. Infected pigs are the principal source of human infection; hence, an efficacious vaccine to thwart pig S. suis disease will not only increase revenues of pork producers but also significantly reduce the incidence of human infection, and thereby positively impact public health worldwide. A major impediment to development of highly efficacious vaccines to pig S. suis disease (porcine Streptococcosis) is the fact that different variants of this pathogen (i.e., many serotypes and multiple strains within each serotype) cause disease in different geographical regions of the globe. This means that a vaccine that provides exemplary protection against one variant prevalent in a particular region of the world may not necessarily protect against a variant that causes disease in another part of the world. Therefore, for a vaccine, to be effective, must be able to block infection caused by all / majority of the globally clinically relevant S. suis serotypes and strains (universal or broadly protective vaccines). Consequently, from the point of view of vaccine development, it is imperative that the vaccine be made up of components that are "shared" or "conserved" by the vast majority of S. suis capable of causing disease. An important first step toward such universal / broadly protective vaccines is to identify pathogen components (such as proteins) that are highly conserved across all / majority of S. suis with disease causing potential; however, this is easier said than done and presents a stiff challenge. This is because highly conserved proteins serve indispensable functions to the pathogen and consequently, are "hidden" by the pathogen by highly evolved "decoy" mechanisms that prevents the host from recognizing these conserved proteins during natural infection. Novel and innovative techniques are therefore required to identify such proteins.The proprietary protein antigen discovery tool (called Proteomics-based Expression Library Screening or PELS) proposed in this project is one such platform for identification of proteins conserved across diverse S. suis. The technology employs highly specific probes, including antibodies, to identify pathogen proteins. Here, PELS will be innovatively applied to interrogate the entire repertoire of proteins (the proteome) produced by individual S. suis variants comprising a group of 15 serotypes / strains, representative of those causing disease globally. Proteins that react with the antibodies are separated from the rest of the proteome of that particular variant and then identified using tandem mass spectrometry. Sophisticated computer-based algorithms (bioinformatics) will then be employed to compare the proteins identified from each variant to arrive at a panel of highly conserved proteins. A subset of these proteins will be further prioritized on the basis of characteristics that render them suitable as vaccine components. Finally, the prioritized proteins will be produced in a purified form and formulated as a vaccine. In this phase I SBIR project, efficacy of such a vaccine will be determined by the ability of the vaccine to protect weaned piglets from experimental disease caused by a S. suis variant with disease causing potential that is not part of the 15 serotypes / strains examined in this study. Further studies, such as those in phase II, will evaluate the ability of the vaccine to protect piglets against disease caused by each of the 15 S. suis serotypes / strains. It is anticipated that this work will ultimately lead to a highly efficacious universal protein subunit vaccine capable of conferring robust protection to weaned pigs against diverse S. suis that are prevalent and cause disease in different parts of the world. Such a vaccine will positively impact the global pork industry and international public health. Following successful completion of the phase I studies, an international patent application will be submitted to protect intellectual property, after which the results of the study will be disseminated to diverse audiences: to (i) the scientific community via oral and poster presentations in scientific meetings, publication in a peer-reviewed scientific journal and a NDA-covered, password-protected online database of the novel proteins identified in the study; (ii) major veterinary biologics companies via presentations at scientific meetings, scientific publications, and direct contact; and (iii) pork producers via presentations at scientific and think tank meetings and discussions, and also via online articles on websites focused on pig/hog industry.
Animal Health Component
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Research Effort Categories
Basic
100%
Applied
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Developmental
(N/A)
Goals / Objectives
The focus is on development of broadly protective or universal porcine vaccines against diverse capsular serotypes /strains of Streptococcus suis, an emerging zoonotic pathogen that causes disease in different geographical regions of the world. Toward this goal, this project includes two technical objectives.Technical objective #1. This objective involves identification of a panel of highly conserved pathogen proteins (immunologically cross reactive / conserved proteins or CPs) that have potential as components of a broadly protective porcine S. suis vaccine. An innovative and proprietary platform proteome mining tool for vaccine protein antigen discovery called Proteomics-based Expression Library Screening (PELS) will be employed to define a panel of S. suis proteins that are highly conserved across 16 S. suis capsular serotypes/strains representative of those causing disease all over the world. The PELS technology involves screening recombinant proteins expressed by genes within non-pathogenic E. coli (protein expression host) clones of an expression library constructed using genomic DNA of a pathogen of interest using specific probes. High affinity polyclonal antibodies (PAbs; "bait" PAbs), elicited experimentally against the recombinant proteome of one S. suis serotype, will be the probes used in PELS. Immobilized "bait" PAbs are then used to immunoaffinity capture recombinant proteins expressed by genomic DNA expression libraries of each S. suis capsular serotype/strain. Recombinant proteins, immunoaffinity captured by "bait" PAbs, will be subjected to one-dimensional SDS-PAGE and tandem mass spectrometry (Ge-LC-MS/MS), and then identified by interrogating public non-redundant S. suis sequence databases. In this project, a novel application of PELS will be used to define CPs: "bait" PAbs generated against one serotype will be used in PELS to probe the recombinant proteomes of the remaining 15 S. suis capsular serotypes/strains, a strategy called "Proteome Walking." Following identification of immunoaffinity captured cross reactive proteins of each of the 15 S. suis serotypes and strains via interrogation of public pathogen-specific databases with the output spectral data, bioinformatics will be used to both arrive at a panel of immunologically cross-reactive proteins, as well as prioritize a subset of CPs (prioritized CPs or pCPs).Technical objective #1 involves the following tasks:(i) Construction of "optimized" inducible genomic DNA expression libraries. The pET(30) abc expression vectors and three different E. coli expression hosts strains, BL21 [DE3] (Invitrogen), general high-level expression; Origami 2 [DE3] (EMD Biosciences), and C41[DE3] (Lucigen) for optimal expression of disulfide-bonded and membrane proteins, respectively) will be used to generate optimized expression libraries (Elibraries). Genomic DNA, isolated from each of the 16 S. suis capsular serotypes/strains will be used to construct three Elibraries per cognate strain. Recombinant proteins will be harvested from lysate and pellet fractions, following IPTG induction. (ii) Generation of S. suis capsular serotype 2 strain P1/7 (ATCC BAA 853; P1/7) anti-proteome PAbs in mice. Recombinant proteins from lysate and pellet fractions of the three Elibraries of P1/7 (a clinical isolate from blood from a pig with meningitis) will be pooled, formulated with a potent adjuvant and used to generate anti-proteome "bait" PAbs in mice for defining a panel of conserved proteins (below).(iii) "Proteome walking" and definition of a panel of CPs, and subset of pCPs. Anti-proteome PAbs generated against P1/7 will be used as "bait" to sequentially immunoaffinity capture proteins expressed from genes on inserts within clones of Elibraries of each of the remaining 15 representative S. suis capsular serotypes/strains. Following LC-MS/MS, immunoaffinity captured proteins of each S. suis capsular serotype/strain will be identified by querying S. suis genomic sequence data in non-redundant databases. A suite of bioinformatics will then be used to compare the panel of LC-MS/MS-identified proteins of each strain with those of the remaining strains. The result will be the definition of a core panel of conserved proteins (CPs) based on identity/near identity/partial identity or on the presence of three dimensional epitopes on disparate proteins. Between 35-40 CPs are anticipated. A second round of specialized bioinformatics will prioritize CPs (pCPs) based on cell-surface location, contribution to colonization/adherence to host epithelial cells, orthology to established virulence factors in diverse pathogens, and lack of homology to host proteins. A subset of 10-12 pCPs are anticipated for further evaluation. Technical objective #2. Evaluation of protective efficacy of an experimental vaccine, constituted by pCPs identified in the previous step, for cross-protection of weaned pigs challenged with a pathogenic S. suis serotype 3 (isolated from the brain of a pig with meningitis; not part of the 15 S. suis capsular serotypes/strains subjected to proteome "walking" to identify the vaccine components.Technical objective #2 involves the following tasks: (i) Purification of pCPs shortlisted in the previous step to homogeneity/near homogeneity. Genes encoding each of the 10 - 12 pCPs (above) will be cloned devoid of the native signal sequence in the inducible expression plasmid vector, pET30b, in translational frame downstream of a hexa-histidine tag. Proteins will be expressed in the E. coli BL21 (DE3], the recommended host strain, and purified via immobilized metal ion affinity chromatography (IMAC) and sequential chromatography. Purified proteins will be quantitated, and equal concentrations pooled for use as a vaccine.(ii) Pig challenge studies to determine vaccine efficacy. Purified proteins will be pooled, formulated with the adjuvant, Quil A (InvivoGen). One ml of this formulation will be administered intramuscularly to 4-week old "Test" (vaccine formulation) and "Control" pigs (no vaccine, physiological saline and adjuvant only) on days 0, 21and 42. Pigs in both groups will be challenged intranasally ~14 days following the last immunization with 2 x 109 CFU of wild type S. suis serotype 3. Vaccine efficacy of the vaccine will be determined by the ability of the vaccine to completely protect pigs in the "Test" group from developing systemic disease or by its ability to significantly prolong time to death compared with pigs in the "Control" group.
Project Methods
MethodsToward broadly protective / universal porcine S. suis vaccines, the objectives of this project are to (i) define a panel of pathogen proteins that have potential for inclusion in such a vaccine; and (ii) evaluate efficacy of an experimental vaccine constituted by a subset of the identified pathogen proteins to protect piglets against challenge with a heterologous S. suis capsular serotype. The innovative methods to achieve each of these two objectives are as follows:Methods to accomplish Technical Objective #1. The proprietary PELS proteome mining tool will be employed in the novel proteome "walking" strategy to define a panel of immunologically cross-reactive proteins (CPs) of clinically relevant S. suis capsular associated with porcine Streptococcosis. Following identification of such proteins, bioinformatics will be employed to prioritize a subset of interesting CPs prioritized (prioritized CPs or pCPs) for evaluation of vaccine potential in pig studies. The PELS platform technology involves screening of genomic DNA expression libraries (Elibraries) of a pathogen of interest using "bait" probes, such as specific antibodies ("bait" PAbs). Immunoaffinity captured proteins are subjected to tandem mass spectrometry and then identified by interrogating public pathogen-specific databases with the output spectral data. PELS involves the following methods:(i) Construction of "optimized" inducible genomic DNA expression libraries. The pET (30) abc expression vectors and three different E. coli expression hosts strains, BL21 [DE3] (Invitrogen), Origami 2 [DE3] (EMD Biosciences), and C41[DE3] (Lucigen), will be used to generate optimized expression libraries (Elibraries) using genomic DNA isolated from each of the 16 serotypes/strains representative of S. suis causing disease all over the world.(ii) Generation of S. suis serotype 2 strain P1/7 (ATCC BAA 853; P1/7) anti-proteome PAbs in mice. High titer and high affinity polyclonal antibodies (PAbs) were generated in mice against recombinant proteins expressed from the Elibrary of S. suis serotype 2 strain P1/7 (ATCC BAA 853; P1/7), an isolate from blood of a pig with meningitis. These will serve as "bait" PAbs in PELS-based proteome walking (below). (iii) Proteome "walking" and definition of a panel of CPs, and subset of pCPs. The "bait" PAbs generated above will be used to sequentially immunoaffinity capture recombinant proteins expressed from individual Elibraries of each of the 15 S. suis serotypes/strains. Immunoaffinity captured proteins will then be subjected to tandem mass spectrometry (GeLC-MS/MS), and then identified by querying translated genome sequences of S. suis serotype/strains available thus far at NCBI Genome Project database. Comparative bioinformatics will result in a core panel of 35-40 immunologically cross-reactive proteins (CPs) conserved across the 15 S. suis serotypes/strains, which will be culled by a subsequent round of bioinformatics to 10-12 CPS (prioritized or pCPs).Methods to accomplish Technical Objective #2. The methods are as follows for evaluation of potential of a vaccine constituted by a subset of purified pCPs to protect weaned pigs from heterologous challenge with S. suis serotype 3, an isolate from the brain of a pig with meningitis.(i) Purification of pCPs shortlisted in the previous step to homogeneity/near homogeneity. pCPs will be purified to homogeneity/near homogeneity using immobilized metal ion chromatography (IMAC) and formulated as a vaccine.(ii) Pig challenge studies to determine vaccine efficacy. Sixteen three-week old pigs, free of S. suis, PRRSV, Mycoplasma hyopneumoniae, and transmissible gastroenteritis virus, will be acclimated for 7 days, and randomly placed in three groups: No Treatment Group (n = 2); Control Group (n =7; unimmunized, but challenged); Test Group (n = 7, immunized and challenged). One day prior to immunization, pre-immune blood and sera will be collected for bacteriology and serology from the jugular vein using single-use blood collection system with and without EDTA (Vacutainer; Becton Dickinson, USA). (iii) Immunization and challenge. Four-week-old pigs (Control and Test groups) will be immunized intramuscularly with 1 ml of either the experimental vaccine, containing 200 µg of each purified pCP in sterile physiological saline, admixed with 400 µg of Quil A adjuvant (Test group) or 400 µg of adjuvant alone with 1 ml of sterile physiological saline (Control group). Pigs will be immunized on day 0 (primary) and then administered two booster doses on days 14, and 28. Immune sera will be collected and immune responses against vaccine proteins assessed via immunoblotting. Challenge will be as follows: Two weeks following the second boost (on day 42), pigs in Test and Control groups will be challenged intranasally (heterologous challenge) with 1 ml (2 x 109 CFU; LD100) of a log-phase culture of S. suis serotype 3 strain. (iv) Post-challenge monitoring of pigs, and post mortem procedures. Following challenge, pigs in both groups will be monitored twice per day, and rectal temperatures recorded every 12 h for 10 days. Clinical signs characteristic of involvement of the respiratory system, central nervous system, and musculoskeletal system will be monitored daily. A clinical score from 0 to 8 will be used to assess allocated based on the degree of involvement of each of these systems. Protection from disease/prolongation of time to death in pigs in the Test group will be construed as an attestation of the protective efficacy of the vaccine. Postmortem specimens from dead and surviving pigs (unchallenged; protected by the vaccine) will be subjected to bacteriological, pathological and histological analysis to confirm the pathogen. A kinetic ELISA will be used to measure titers of vaccine-specific total IgG and IgG subclasses, IgG1 and IgG2 in pig sera, and to establish correlates of immunity.(v) Statistical analysis of results. Vaccine efficacy calculated as follows: (given by the formula [ARU x ARV / ARU] x 100: ARU, attack rate in unvaccinated; ARV, attack rate in vaccinated. Comparison of means for CFU counts of bacteriological analysis will be done using the Mann Whitney test. Survival distributions/curves will be plotted using the Kaplan-Meier method, and the significance of difference will be tested using the log rank test. The Fisher exact test will be applied to compare S. suis recovered from post mortem tissues. For ELISA results, comparison of geometric means will be performed with the independent-sample Studentttest or with the Mann-Whitney U test. P-value of <0.05 will be used to indicate statistically significant differences. GraphPad Prism5 v.5 will be used for statistical analysis.Efforts to deliver results/knowledge gained from the study, following protection of intellectual property, will be via poster and oral presentations at local, national and scientific meetings. The results will also be disseminated via a publication in a peer-reviewed journal. Furthermore, a password-protected database of the proteins identified in this study will be made available to the scientific community upon execution of an NDA/MTA. Additionally, direct communication methods will be employed to aggressively spread the information gleaned from this project to major companies in the field of veterinary vaccines for licensing and SBIR phase II partnering opportunities. Pork producers will be made aware of a potential vaccine via online publications/articles on relevant pig/hog management websites.Evaluation of a successful outcome of this phase I study will be by demonstrating the protective efficacy of the experimental vaccine developed in this project. Specifically, the ability of the vaccine to protect piglets against disease following a challenge with a heterologous S. suis serotype will be construed as a highly successful outcome.