Comparison of the Immunogenicities and Cross-Lineage Efficacies of Live Attenuated Peste des Petits Ruminants Virus Vaccines PPRV/Nigeria/75/1 and PPRV/Sungri/96

Abstract

Peste des petits ruminants (PPR) is a severe disease of goats and sheep that is widespread in Africa, the Middle East, and Asia. Several effective vaccines exist for the disease, based on attenuated strains of the virus (PPRV) that causes PPR. While the efficacy of these vaccines has been established by use in the field, the nature of the protective immune response has not been determined. In addition, while the vaccine derived from PPRV/Nigeria/75/1 (N75) is used in many countries, those developed in India have never been tested for their efficacy outside that country. We have studied the immune response in goats to vaccination with either N75 or the main Indian vaccine, which is based on isolate PPRV/India/Sungri/96 (S96). In addition, we compared the ability of these two vaccines, in parallel, to protect animals against challenge with pathogenic viruses from the four known genetic lineages of PPRV, representing viruses from different parts of Africa, as well as Asia. These studies showed that, while N75 elicited a stronger antibody response than S96, as measured by both enzyme-linked immunosorbent assay and virus neutralization, S96 resulted in more pronounced cellular immune responses, as measured by virus antigen-induced proliferation and interferon gamma production. While both vaccines induced comparable numbers of PPRV-specific CD8⁺ T cells, S96 induced a higher number of CD4⁺ T cells specifically responding to virus. Despite these quantitative and qualitative differences in the immune responses following vaccination, both vaccines gave complete clinical protection against challenge with all four lineages of PPRV.IMPORTANCE Despite the widespread use of live attenuated PPRV vaccines, this is the first systematic analysis of the immune response elicited in small ruminants. These data will help in the establishment of the immunological determinants of protection, an important step in the development of new vaccines, especially DIVA vaccines using alternative vaccination vectors. This study is also the first controlled test of the ability of the two major vaccines used against virulent PPRV strains from all genetic lineages of the virus, showing conclusively the complete cross-protective ability of these vaccines.

Keywords: immune response; livestock disease; morbillivirus; protection; vaccines.

FIG 1

Phylogenetic tree of PPRV showing lineages I to IV. A phylogenetic tree showing the genetic distances between the available full-length PPRV genomes was calculated as described in Materials and Methods using MEGA6. The tree with the highest log likelihood is shown, with the percentage of trees in which the associated taxa clustered together in the bootstrap (500 replicates) shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site; the scale bar shows 0.02 substitutions per site. The labels at the ends of the branches show the accession number of the genome sequence and the country and year of the virus’s isolation. The clades considered as lineages I to IV are shown on the right.

FIG 2

Antibody response in goats to PPRV vaccines. Goats were vaccinated with N75 or S96, and serum samples were taken at 0, 7, 14, 21, and 28 dpv. Sera taken at all time points were assayed for antibodies to the PPRV N protein (a) and the PPRV H protein (b) using available cELISA kits. Sera taken at 14, 21, and 28 dpv were additionally assayed for neutralization of PPRV; neutralizing titers were determined against N75 vaccine virus (c) and S96 vaccine virus (d). (e) The differences in titers (titer against N75 virus – titer against S96 virus) for each individual serum was calculated, and these differences are plotted. Note that the manufacturer’s recommended calculation for the N protein cELISA kit (in panel a) gives values that decrease as the amount of anti-N antibody increases (percentage of negative control), whereas that for the H protein cELISA kit (in panel b) gives values that increase as the amount of competing antibody increases (percent inhibition of binding of MAb). The data are presented as box-and-whisker plots, in which the bars span the minimum and maximum values, and the box shows the range from the first to the third quartile. The central horizontal line in each box shows the median value. The numbers of samples assayed at each time point shown were as follows: 25/19/25/19/24 (a), 15/10/15/10/14 (b), and 15/15/15 (c to e).

FIG 3

T cell proliferative responses in goats after PPRV vaccination. Goats were vaccinated with N75 or S96 vaccine, and PBMCs were prepared from heparinized blood at 0, 7, 14, 21, and 28 dpv. Proliferative responses were measured by incorporation of [³H]thymidine as described in Materials and Methods. Proliferation was stimulated with protein from cells infected with N75 (a), protein from cells infected with S96 virus (b), heat-inactivated preparations of N75 virus (c), or heat-inactivated preparations of S96 virus (d). The stimulation index (SI) was measured as the proliferation relative to that seen in PBMCs incubated with protein from uninfected cells (a and b) or mock viral preparations from uninfected cells (c and d). The data are presented as a stacked bar plot showing the SI for each animal, using different shades to delineate the contribution of different animals to the cumulative SI for the group of vaccinates at that time point. Data from animals vaccinated with S96 (n = 15) are in blue shades; data from animals vaccinated with N75 (n = 14) are in red shades. To allow for the differing number of animals in each group, the values have been scaled before plotting by dividing by the number of animals in the group.

FIG 4

Gating strategy for identifying PPRV-specific CD4⁺ and CD8⁺ T cells. PBMCs were stimulated with live virus or antigen for 18 h and then for an additional 6 h in the presence of the Golgi transport inhibitor Golgi-Plug. The cells were then labeled with anti-CD4 and anti-CD8 MAbs and Live/Dead stain, after which they were fixed/permeabilized and labeled with anti-IFN-γ. The numbers of labeled cells were determined by flow cytometry using a MACSQuant, and the data were analyzed with FlowJo X. An example data set is shown for PBMCs taken from an animal vaccinated with S96 at 14 dpv and stimulated with S96 virus. The cells were gated successively for singlets (a), lymphocytes (b), live cells (c), and then by the presence or absence of the surface markers CD8 and CD4 (d). The percentages of IFN-γ⁺ cells was obtained for CD8⁺ cells (e), CD4⁺ cells (f), and CD4^– CD8^– cells (g) are depicted.

FIG 5

PPRV-specific IFN-γ-producing CD4⁺ and CD8⁺ T cells in goats following PPRV vaccination. Goats were vaccinated with N75 or S96 vaccine and PBMCs were prepared from heparinized blood at 0, 7, 14, 21, and 28 dpv. T cell subtypes responding to live PPRV virus or inactivated PPRV antigen by producing IFN-γ were determined in triplicate by intracellular labeling of IFN-γ and flow cytometry as described in Materials and Methods. The gating strategy for identifying IFN-γ⁺ CD8⁺ or CD4⁺ T cells was as shown in Fig. 4. The percentages of total CD4⁺ T cells (a and c) or CD8⁺ T cells (b and d) that responded to PPRV were calculated by subtracting the percentages observed after stimulation with mock antigen or virus preparations. The graphs show the mean specific percentages of IFN-γ-producing cells observed in response to the indicated stimulation of PBMCs from five N75 (a and b) and five S96 (c and d) vaccinates. Error bars show the standard errors of the mean.

FIG 6

Rectal temperatures and clinical scores in experimental animals after challenge with wild-type viruses. The rectal temperatures (a to e) and clinical scores (f to j) of each experimental animal were recorded daily after challenge with the indicated wild-type virus. Data from individual unvaccinated animals are shown in black, data from S96 vaccinates are shown in blue, and data from N75 vaccinates are shown in red.

FIG 7

Detection of PPRV genome by RT-qPCR in unvaccinated controls after challenge. EDTA-blood was collected every second day after challenge, and 1-ml aliquots were frozen for later analysis. PPRV RNA was detected using the one-tube RT-qPCR technique (see Materials and Methods). Points are the means of duplicate PCRs, results are displayed as “45 – C_T” values. Graphs depict the results for mock-vaccinated controls infected with PPRV/Nigeria/76/1 (a), PPRV/Ghana/78 (b), or PPRV/Sudan/Sennar/72 (c). None of the vaccinated animals tested had detectable PPRV RNA in any blood sample taken postchallenge (data not shown).

FIG 8

Immune responses in goats after challenge with PPRV. Goats vaccinated with live PPRV vaccines were challenged 28 dpv, along with unvaccinated controls. Heparinized blood and serum was collected from each animal at 0, 7, and 14 days postinfection (dpi). (a and b) Serum samples were assayed for antibodies to the PPRV N protein (a) and the PPRV H protein (b) using available cELISA kits. The N protein cELISA was applied to all samples from all five challenge studies; the H cELISA was only applied to samples from animals challenged with PPRV/Ivory Coast/89, PPRV/Nigeria/76/1, and PPRV/Iran/2011. The numbers of serum samples assayed at the time points shown were as follows: 25/25/20 for vaccinates and 8/7/4 for controls (a) and 14 at each time point for N75, 15 at each time point for S96 vaccinates, and 6/6/3 for controls (b). The data in panels a and b are presented as box-and-whisker plots, in which the bars span the minimum and maximum values and the boxes shows the ranges from the first to the third quartile. The central horizontal line in each box shows the median value. (c and d) PBMCs were prepared from the heparinized blood, and the proliferation of cells in response to PPRV antigen was assayed as described for Fig. 3. Proliferation was stimulated with protein from cells infected with N75 (c) or protein from cells infected with S96 virus (d). The SI was measured as the proliferation relative to that seen in PBMCs incubated with protein from uninfected cells. The data are presented as stacked bar plots showing the SI for each animal, using different shades to delineate the contributions of different animals to the cumulative SI for the group of vaccinates at that time point. Data from individual animals vaccinated with N75 are in red shades, data from individual animals vaccinated with S96 are in blue shades, and data from individual unvaccinated animals are in gray shades. To allow for the various number of animals in each group, the values have been scaled before plotting by dividing by the number of animals in each group. Data were obtained for samples from animals challenged with PPRV/Nigeria/76/1 and PPRV/Sudan/Sennar/72; data were also obtained for the vaccinates challenged with PPRV/Iran/2011, but not from unvaccinated animals infected with this virus, whose white cell counts at 7 dpi were too low for the assay to be carried out and were euthanized before 14 dpi. The numbers of PBMC samples assayed at each time were 14/9/12 for N75 vaccinates, 15/10/14 for S96 vaccinates, and 4/4/4 for unvaccinated animals. Note that the data for 0 dpc are the same as that shown in Fig. 2 and 3 for 28 dpv and are included here for comparison.