Performing Department
(N/A)
Non Technical Summary
The U.S pork industry produces an estimated 2.2 metric tons of pork annually, valued at $23.4 billion, of which 26% is exported to other countries (Queck-Matzie, 2019). Despite significant growth, pork production continues to be hampered by viruses, such as PCV2, with rapid evolution and high levels of genetic and antigenic variation. As new strains emerge, the relevance of existing vaccines is lost (Constans, 2015; Meng, 2012). The primary goals of this proposal are (1) to develop an improved PCV2 vaccine candidate that stimulates broadened protective immunity and (2) to assess the accuracy of a novel bioinformatic tool (ANATOPE) in the design of immune refocused vaccines. Our proposed work is concerned with improving our nation's food security and animal health and wellbeing. Current PCV2 vaccines stimulate protective immunity to PCV2a and 2b serotypes but reduced protection against evolving and emerging strains such as PCV2d, 2g, and 2h. We propose to use Immune Refocusing Technology (IRT) to reduce the immunogenicity of epitopes that stimulate subtype-restricted immunity and refocus towards more broadly protective epitopes. Our first-generation IRT candidate demonstrated improved protection against PCV2d compared to a commercially available vaccine. We have designed two second-generation candidates to increase the breadth of protection towards additional subtypes. The vaccine candidates are based on an attenuated strain which will reduce the cost of the vaccines and include a marker epitope for distinguishing between immunized and infected swine. The project will be led by long-time collaborators at Biological Mimetics, Inc. and NDSU. BMI will design the IRT candidates and perform serological analyses of swine sera including cross-neutralization of PCV2d, 2g, and 2h viruses. Dr. Ramamoorthy at NDSU will produce the recombinant viruses and the challenge stocks. Dr. Pillai at SDSU will perform the swine immunization and challenge experiment and analyze tissues by pathology and histopathology. We aim to identify an improved PCV2 vaccine that stimulates broadened cross-subtype protection that can be further developed in a Phase II project.
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Goals / Objectives
The overall goal of the project is to design, construct, and test a novel PCV-2 vaccine candidate that stimulates improved cross-protective immunity against heterologous virus strains than current products. We are using an immune refocusing approach which entails identifying and then mutating key amino acids in epitopes that stimulate stain-restricted immunity. A successful outcome would be a vaccine that protects against heterologous subtypes such as PCV2d and others. We have used a variety of methods to identify these amino acids which are characterized loosely as (1) evolutionarily variable between strains, (2) surface exposed, (3) not directly participating in receptor binding, (4) hydrophilic, (5) charged, and other features. In prior projects, we have observed that reducing the immunogenicity of strain-restricted epitopes allows the recognition of more highly conserved regions that were previously not immunodominant.The short-term goals are:1. Re-analyze PCV2 immunogens and confirm the amino acids that we planned to target when preparing our application proposal.2. Design cloning strategies to introduce the mutations into plasmid DNAs that bear the parental proviral clone. This clone directs the expression of PCV2 when transfected into mammalian cells and allows rescue of an attenuated marker virus vaccine candidate.3. Produce modified proviral clones. Transfect into mammalian cells and rescue recombinant virus.4. Assess the viruses for successful infection of target cells to ensure that they are functional and can enter cells (i.e., we have not reduced receptor interactions).5. Produce and characterize vaccine lots of the attenuated candidates. Test for neutraliation using sera from animals immunized with a variety of PCV2 vaccines/viruses.6. Immunize pigs with the candidates, characterize their immune responses, and determine level of protection when challenged with heterologous PCV2 strains. Examine pigs for signs of infection and disease after challenge. After euthanasia, titer organs for virus load and examine organs by histopathology to observe cytopathic effects of the virus.7. Report findings in publications. Discuss findings with major manufacturers of current vaccines.8. If warranted, use data to apply for a Phase 2 project.Future studies will likely include expanded testing against additional heterologous PCV2 strains, development of production methods for the attenuated vaccine including the analytical package, and investigation of the regulatory roadmap.Further studies will include pre-commercialization and commercialization activities to include regulatory approvals, manufacturine scale-up, and investigations of potential partnerships with existing PCV2 vaccine suppliers.
Project Methods
Aim 1. Design, production, and characterization of immune-refocused PCV2 vaccine candidates (technical objectives 1, 2, and 3). We propose to use the 1st generation rPCV2-vac virus as the parental strain upon which to engineer additional IRT modifications because of its partial success in expanding the breadth of cross-protective immunity. We have used both manual and automated (ANATOPE) methods to analyze the surface epitopes. Figure 4A and 4B in the proposal show 3-D and topological surface map of the capsid with 1st- and 2nd-generation IRT mutations in Red or Yellow. Figure 4C shows the amino acid mutations and how they affect the immunogenicity scores. The 2nd-generation variants will be built with both 1st-generation mutations in Epitopes A and B. Note that Epitope B is visible from the inside of the capsid while Epitopes A and C are surface-accessible. The map in Figure 4B identifies the amino acids to mutate in Epitope C as protruding the "highest" from the surface of the assembled capsid. The naturally-occurring amino acids, Lys-K and Arg-R, are highly charged and are frequently found in immunodominant, strain-restricted epitopes which is why they were targeted for immune refocusing. The two amino acids substituted in rPCV2-Vac2 dampen these charges. The one amino acid substituted in rPCV2-Vac2 reduces one charge and introduces a N-linked glycosylation site (NTT) which is also predicted to reduce the immunogenicity in this site.The amino acid substitutions will be introduced into the cloned PCV2b genomic DNA which, following sequence confirmation, will be transfected into PK-15 cells to rescue the recombinant viruses. Cells infected with the rescued viruses will be stained to confirm capsid and marker peptide expression (Kolyvushko, 2019).Aim 2. Analysis of second-generation IRT vaccine candidates (Technical objectives 4, 5, 6, and 7). Twenty-five 3-to-4-week pigs will be pre-tested as negative for PCV2 by PCR and ELISA and then shipped from the high health herd at the NDSU swine research facility to the Animal Resource Wing at SDSU for immunization and challenge studies which will be overseen by Dr. Viju Pillai, a board-certified pathologist. All animal experimentation will be carried out in compliance with the SDSU IACUC protocols. Animals will be humanely treated and euthanized in accordance with AVMA and USDA guidelines. After a 7-day acclimation period, the animals will be pre-bled for serum, tagged, and immunized on Days 0 and 14 with one of the following five immunogens: (1) vehicle control (no antigen), (2) rPCV2-Vac, (3) rPCV2-Vac2, (4) rPCV2-Vac3, or (5) Zoetis Fostera Gold PCV MH bivalent vaccine. The Zoetis vaccine has been selected as the most stringent comparator because it contains PCV2a and PCV2b antigens and may stimulate more broadened responses than monovalent vaccines. For administration of the attenuated vaccine candidates, pigs will be inoculated with 2 ml at 1 x 104 TCID50/ml both intramuscularly (IM) and intranasally (IN) to ensure adequate immunization. The Zoetis vaccine will be administered IM as per label instructions. 14 days after the boost, serum will be collected, and the pigs challenged both IM and IN using 2 mL of a stock of PCV2d at 104TCID50/mL at each site. Pigs will be monitored daily for signs of disease such as wasting, respiratory distress, jaundice, inappetence, diarrhea or weight loss. Pigs will be humanely euthanized 21 days following the challenge and tissues collected for analysis. The study read outs will be obtained as follows:Binding Ab responses: PCV2-specific antibody levels to PCV2 the in serum will be measured using commercial kits [SERELISA® PCV2 Ab ELISA, Synbiotics Corp.] according to manufacturer's instructions at the SDSU Veterinary Diagnostic lab. Antibody responses to the marker peptide will be assessed as described previously at BMI (Rakibuzzaman, 2020).Cross virus neutralization (VN) responses will be assessed by conventional virus neutralization assays in a fluorescent inhibition assay format as described previously using homologous and heterologous PCV2 subtypes; PCV2a (40895 and NC16846), PCV2b (NDSU41513), PCV2d (JX535296.1) plus more recently emerged 2g and 2h subtypes at BMI (Rakibuzzaman 2020).Assessment of post-challenge protection in vaccinated animals: Post-challenge weight loss and loss of bodily condition will be assessed every other day. On the 21st day after challenge, assessment of gross pathological lesions will be performed at SDSU by Dr. Pillai, a board-certified veterinary pathologist, and scored using established scales. Sections of major organs including lung, liver, kidney, spleen, ilium, tonsils, and tracheobronchial and mesenteric lymph nodes will be fixed in 10% buffered formalin, transferred to 70% ethanol, embedded and sectioned. The tissues will be stained using H&E for microscopic lesions and a PCV2-specific antibody for immunohistochemistry (IHC) for viral antigens as described previously (Kolyvushko, 2019; Rakibuzzaman, 2020; Ramamoorthy, 2009)Viral loads: Replication of the vaccine virus (pre-challenge) and challenge virus will be assessed using the serum collected by qPCR at the SDSU veterinary diagnostic lab.Rigor of experimentation and data analysis: A variety of statistical analyses will be performed by BMI's biostatistician, Dr. David MacLeod, member of the American Statistical Association. All in vitro experiments for Aim 1 will be conducted with a minimum of three biological replicates, each with triplicate or quadruplicate technical replicates. For the in vitro analysis of samples collected from pigs, each pig will serve as a biological replicate and each sample will be assessed in triplicate for virus load and quadruplicate (or more) for virus neutralization assays. A minimum of six microscopic fields will be evaluated for each tissue. Statistical analysis of data will be carried out by ANOVA with Duncan's multiple comparison tests applied where values are significant. Student's T tests or non-parametric tests such as Kruskal Wallis will be used as appropriate.The above studies are designed to identify a lead candidate for follow-up studies which will include animal studies with additional challenge subtypes and those designed to assess the immunization protocol and regimen. Also in the future, studies will be performed for pre-commercialization, such as scale-up, SOP validation, and analytics development and for obtaining regulatory approvals