Image_1_Heterologous vaccine immunogenicity, efficacy, and immune correlates of protection of a modified-live virus porcine reproductive and respiratory syndrome virus vaccine.JPEG

Although porcine reproductive and respiratory syndrome virus (PRRSV) vaccines have been available in North America for almost 30 years, many vaccines face a significant hurdle: they must provide cross-protection against the highly diverse PRRSV strains. This cross-protection, or heterologous vaccine efficacy, relies greatly on the vaccine’s ability to induce a strong immune response against various strains—heterologous immunogenicity. Thus, this study investigated vaccine efficacy and immunogenicity of a modified live virus (MLV) against four heterologous type 2 PRRSV (PRRSV-2) strains. In this study, 60 pigs were divided into 10 groups. Half were MOCK-vaccinated, and the other half vaccinated with the Prevacent® PRRS MLV vaccine. Four weeks after vaccination, groups were challenged with either MOCK, or four PRRSV-2 strains from three different lineages—NC174 or NADC30 (both lineage 1), VR2332 (lineage 5), or NADC20 (lineage 8). Pre-and post-challenge, lung pathology, viral loads in both nasal swabs and sera, anti-PRRSV IgA/G, neutralizing antibodies, and the PRRSV-2 strain-specific T-cell response were evaluated. At necropsy, the lung samples were collected to assess viral loads, macroscopical and histopathological findings, and IgA levels in bronchoalveolar lavage. Lung lesions were only induced by NC174, NADC20, and NADC30; within these, vaccination resulted in lower gross and microscopic lung lesion scores of the NADC20 and NADC30 strains. All pigs became viremic and vaccinated pigs had decreased viremia upon challenge with NADC20, NADC30, and VR2332. Regarding vaccine immunogenicity, vaccination induced a strong systemic IgG response and boosted the post-challenge serum IgG levels for all strains. Furthermore, vaccination increased the number of animals with neutralizing antibodies against three of the four challenge strains—NADC20, NADC30, and VR2332. The heterologous T-cell response was also improved by vaccination: Not only did vaccination increase the induction of heterologous effector/memory CD4 T cells, but it also improved the heterologous CD4 and CD8 proliferative and/or IFN-γ response against all strains. Importantly, correlation analyses revealed that the (non-PRRSV strain-specific) serum IgG levels and the PRRSV strain-specific CD4 T-cell response were the best immune correlates of protection. Overall, the Prevacent elicited various degrees of efficacy and immunogenicity against four heterologous and phylogenetically distant strains of PRRSV-2.</p

Biological evaluation of transgenic founder animals.

<p>(A) Binding studies of LEA29Y to the CD80/CD86-positive porcine B cell line L23. Cells were incubated with serial dilutions of sera from the three founder animals as well as from wild-type controls. Binding of LEA29Y was assessed by using a goat anti-human IgG-FITC antibody. Labeled cells were analyzed by flow cytometry. The results are expressed as the mean fluorescence intensity. (B) Inhibition of human anti-pig T cell proliferation by serum from LEA29Y-tg pigs. 10⁵ human PBMC were stimulated with 2 x 10³ irradiated L23 cells. Cultivation was performed in the presence of sera taken from transgenic and wild-type control pigs. Proliferation was determined after 5 d by [³H]-TdR incorporation (ccpm, counts/min). The results are expressed as the mean ± SD of triplicate cultures.</p

T cell subpopulations in transgenic pigs.

<p>PBMCs were isolated from LEA pigs, 3-week-old wild-type (WT) piglets and 6-month old WT pigs and analyzed by flow cytometry for the phenotype of CD4⁺ T cells and production of IFN-γ and TNF-α. (A) CD4 and CD8α expression on lymphocytes. Total CD4⁺ T cells were gated (black gates, red population) and analyzed for CD8α expression (green gates). The numbers indicate percent CD8α⁺ cells within CD4⁺ T cells. (B) CD8α and CD27 expression on gated CD4⁺ T cells. (C) CD8α and CD45RC expression on gated CD4⁺ T cells. (D) IFN-γ and TNF-α production in gated CD4⁺ T cells following stimulation with PMA/Ionomycin for four hours as a representative flow cytometry plot (upper panel) and a graph comprising data of all animals as the mean value + standard deviation (lower panel). (E) CD4 and Foxp3 expression of lymphocytes. The numbers indicate the percent of gated CD4⁺Foxp3⁺ regulatory T cells within lymphocytes. The data are representative of two three-week-old WT pigs, two tg pigs and two six-month-old pigs.</p

Immunological profile of re-cloned #9908 pigs.

<p>PBMCs isolated from animals re-cloned from #9908 primary cells (LEA, n = 2) were analyzed by flow cytometry for the frequency of major lymphocyte subpopulations and compared to PBMCs isolated from age-matched wild-type pigs (WT, n = 11). (A) Frequencies of NK cells (CD3^-CD8α⁺), B cells (CD79α⁺) and T cells (CD3⁺) as a percent of lymphocytes. (B) Frequencies of γδ (CD3⁺TCR-γδ⁺) and αβ T cells (CD3⁺TCR-γδ^-) as a percent of total T cells. (C). Frequencies of cytolytic T cells (CTL; CD3⁺TCR-γδ^-CD4^-CD8α^high), T-helper cells (Th; CD3⁺TCR-γδ^-CD4⁺Foxp3^-) and regulatory T cells (Treg; CD3⁺TCR-γδ^-CD4⁺Foxp3⁺) as a percent of αβ T cells. (D) Frequencies of regulatory T cells (Treg) as a percent of αβ T cells with a different scaling of the y-axis. Asterisks indicate significant differences between WT and LEA lymphocyte subpopulations (p < 0.05).</p

Lymph node histology in transgenic pigs.

<p>Developmental stage of lymph nodes of a 6-month-old CAG-LEA transgenic pig (B, E) in comparison with WT porcine lymph nodes; the age of the animals was 19 days (A, D) and 6 months (C, F). Immunohistochemistry using the PPT3 anti-CD3 antibody demonstrates nearly T cell-free follicles (Fo) near the trabecules (T) in all samples. Follicles in samples of 6-month-old WT animals were generally larger than in transgenic or young animals (compare C versus A and B). Follicles of 6-month-old WT porcine lymph nodes showed a distinct reaction center (F) with heavily proliferating centroblasts in the dark zone (DZ) and less proliferating cells in the pale zone (PZ)–comparing strong and weak Ki67 immunopositivity (proliferation marker) in DZ and PZ, respectively. In contrast, marked proliferation of lymphocytes could be detected in either the follicles of transgenic (E) nor 19-day-old WT animals (D). The amount of proliferating T cells did not seem to differ between the samples. Visualization of the immunoreaction diaminobenzidine—horseradish peroxidase (positive staining = brown), counterstaining hematoxylin, scale bar = 200 μm.</p

Genetic distance between mammalian CTLA4, B7.1/CD80 and B7.2/CD86.

<p>The percentage of amino acid identities between pairwise comparisons are shown as a matrix for CTLA4 (A), CD80 (B) and CD86 (C). The distribution of pairwise identities was calculated in segments of 4% (D) and illustrates higher amino acid conservation in CTLA4 compared to CD80 and CD86.</p

Localization of LEA29Y expression in F1 generation.

<p>Immunohistochemistry was performed using an antibody specific for the human IgG tail of LEA29Y. (A) LEA29Y was predominantly detected in endothelial cells including capillaries and interstitia, as well as in organ- and tissue-specific cell types such as pulmonary alveolar cells, exocrine pancreas cells, bile duct cells of the liver, or the stratum spinosum cell layer of the skin. Endocrine cells of the Langerhans islet (indicated by an arrow) as well as the thyroid gland exhibited strong staining. Additionally, intravascular serum stained positive for LEA29Y. (B) No immune staining could be detected in WT control tissue. Chromogen: DAB; nuclear staining: hemalum; scale bar = 200 μm. Inset in pancreas: Giemsa stain of the corresponding pancreas region to demonstrate Langerhans islet. Lymph node: L. tracheobronchialis medialis.</p

Establishment of CAG-LEA pigs.

<p>(A) The CAG promoter, containing a CMV enhancer element, the chicken beta-actin core promoter as well as a genomic element from the rabbit HBB gene, were used to drive ubiquitous expression of LEA29Y. A neomycin resistance cassette (neo) was used to achieve positive selection. Arrows indicate the position of primers used for genotyping the animals. The <i>Xba</i>I restriction site used for digesting genomic DNA for Southern Blot analysis is indicated as well as the neo-specific probe used for hybridization. (B) LEA29Y protein abundance was measured in mg/ml by an ELISA assay specific for the human IgG tail of LEA29Y in different tissues of three founder animals and a WT control.</p

Biological evaluation of F1 animals.

<p>(A) CD80/CD86-positive porcine B cell line L23 and human B cell line laz509 were incubated with serial dilutions of blood serum drawn from genetically modified F1 pigs and littermate controls. Labeled cells were analyzed for abundance of LEA29Y using a goat anti-human IgG-FITC antibody. The results are expressed as the mean fluorescence intensity. (B) 10⁵ human PBMCs were stimulated with irradiated L23 cells, Laz509 cells or allogeneic PBMCs and 1:32 dilutions of serum from either CAG-LEA or wild-type animals. Proliferation was determined after 5 d by [³H]-TdR incorporation (ccpm, counts/min). The results are expressed as the mean ± SD of triplicate cultures.</p