A thermostable presentation of the live, attenuated peste des petits ruminants vaccine in use in Africa and Asia.

The research objective was to develop a thermostable vaccine against peste des petits ruminants (PPR), a morbilliviral disease of small ruminants targeted for eradication that is a major constraint on the livelihoods of the rural poor throughout much of Africa and Asia. Although existing PPR vaccines provide life-long immunity, they require continuous refrigeration. This limits their utility in developing countries. Methods for the lyophilization of a related morbillivirus, rinderpest (RP), resulted in vaccine that could be used in the field for up to 30days without refrigeration which was a major contribution to the global eradication of RP completed in 2011. The present research applied the rinderpest lyophilization method to the attenuated Nigeria 75/1 PPR vaccine strain, and measured thermostability in accelerated stability tests (AST) at 37°C. The shelf-life of the vaccine was determined as the time a vial retained the minimum dose required as a 25-dose presentation at the specified temperature. A lactalbumin hydrolysate and sucrose (LS) stabilizer was compared to stabilizers based on trehalose. PPR vaccine produced using the Xerovac drying method was compared to vaccine produced using the rinderpest lyophilization method in AST. LS vaccine was evaluated in AST at 37, 45 and 56°C and an Arrhenius plot was constructed for estimation of stability at temperatures not tested. Vaccines produced using LS and the rinderpest method of lyophilization were the most stable. The shelf-life of the Xerovac preparation was 22.2days at 37°C. The three LS vaccine batches had shelf-lives at 37°C of 177.6, 105.0 and 148.9days, respectively, at 37°C. At 56°C, the shelf-life was 13.7days. The projected half-life at 25°C was 1.3years. This is sufficient thermostability for use without a cold chain for up to 30days which will greatly facilitate the delivery of vaccination in the global eradication of PPR.

Molecular characterization of live Theileria parva sporozoite vaccine stabilates reveals extensive genotypic diversity.

The current Infection and Treatment Method of vaccination against East Coast fever comprises an inoculation of live Theileria parva sporozoites and simultaneous administration of oxytetracycline. Immunization with a combination of parasite types has been shown to provide broader protection than inoculation of individual strains. In this study, we used a high-throughput capillary electrophoresis system to determine the genotypic composition of the Muguga Cocktail, a widely used vaccine stabilate derived from three seed stabilates-Muguga, Serengeti-transformed and Kiambu 5. Five satellite markers were used to genotype the vaccine and reference stabilates from two commercial-scale preparations of the vaccine. In addition, 224 cloned cell lines established by infection of bovine lymphocytes with T. parva parasites from the component stabilates were genotyped. The results indicate that, for the recently prepared batch, there are at least eight genotypes in each of the Muguga and the Serengeti-transformed stabilates, while parasites from the Kiambu 5 stabilate showed no diversity at the five loci. The Serengeti-transformed stabilate contained parasites of the Kiambu 5 genotype and of two genotypes present in the Muguga stabilate, whereas there were no genotypes common to the Muguga and Kiambu 5 stabilates. When stabilates from the two vaccine batches were compared, no allelic variations were identified between the Muguga and Kiambu 5 parasites, while lack of sufficient clones prevented a full comparison of the Serengeti-transformed stabilates. The findings will facilitate examination of the extent to which the vaccine strains become resident in areas under vaccination, the identification of 'breakthrough' strains and the establishment of the quality assurance protocols to detect variations in the production of the vaccine. The cloned cell lines will be useful for further understanding the antigenic diversity of parasites in the vaccine.

Treatment of cattle with DNA-encoded Flt3L and GM-CSF prior to immunization with Theileria parva candidate vaccine antigens induces CD4 and CD8 T cell IFN-γ responses but not CTL responses.

Theileria parva antigens recognized by cytotoxic T lymphocytes (CTLs) are prime vaccine candidates against East Coast fever in cattle. A strategy for enhancing induction of parasite-specific T cell responses by increasing recruitment and activation of dendritic cells (DCs) at the immunization site by administration of bovine Flt3L and GM-CSF prior to inoculation with DNA vaccine constructs and MVA boost was evaluated. Analysis of immune responses showed induction of significant T. parva-specific proliferation, and IFN-γ-secreting CD4(+) and CD8(+) T cell responses in immunized cattle. However, antigen-specific CTLs were not detected. Following lethal challenge, 5/12 immunized cattle survived by day 21, whereas all the negative controls had to be euthanized due to severe disease, indicating a protective effect of the vaccine (p<0.05). The study demonstrated the potential of this technology to elicit significant MHC class II and class I restricted IFN-γ-secreting CD4(+) and CD8(+) T cells to defined vaccine candidate antigens in a natural host, but also underscores the need to improve strategies for eliciting protective CTL responses.

Characterization of the fine specificity of bovine CD8 T-cell responses to defined antigens from the protozoan parasite Theileria parva.

Immunity against the bovine intracellular protozoan parasite Theileria parva has been shown to be mediated by CD8 T cells. Six antigens targeted by CD8 T cells from T. parva-immune cattle of different major histocompatibility complex (MHC) genotypes have been identified, raising the prospect of developing a subunit vaccine. To facilitate further dissection of the specificity of protective CD8 T-cell responses and to assist in the assessment of responses to vaccination, we set out to identify the epitopes recognized in these T. parva antigens and their MHC restriction elements. Nine epitopes in six T. parva antigens, together with their respective MHC restriction elements, were successfully identified. Five of the cytotoxic-T-lymphocyte epitopes were found to be restricted by products of previously described alleles, and four were restricted by four novel restriction elements. Analyses of CD8 T-cell responses to five of the epitopes in groups of cattle carrying the defined restriction elements and immunized with live parasites demonstrated that, with one exception, the epitopes were consistently recognized by animals of the respective genotypes. The analysis of responses was extended to animals immunized with multiple antigens delivered in separate vaccine constructs. Specific CD8 T-cell responses were detected in 19 of 24 immunized cattle. All responder cattle mounted responses specific for antigens for which they carried an identified restriction element. By contrast, only 8 of 19 responder cattle displayed a response to antigens for which they did not carry an identified restriction element. These data demonstrate that the identified antigens are inherently dominant in animals with the corresponding MHC genotypes.

Theileria parva candidate vaccine antigens recognized by immune bovine cytotoxic T lymphocytes.

East Coast fever, caused by the tick-borne intracellular apicomplexan parasite Theileria parva, is a highly fatal lymphoproliferative disease of cattle. The pathogenic schizont-induced lymphocyte transformation is a unique cancer-like condition that is reversible with parasite removal. Schizont-infected cell-directed CD8(+) cytotoxic T lymphocytes (CTL) constitute the dominant protective bovine immune response after a single exposure to infection. However, the schizont antigens targeted by T. parva-specific CTL are undefined. Here we show the identification of five candidate vaccine antigens that are the targets of MHC class I-restricted CD8(+) CTL from immune cattle. CD8(+) T cell responses to these antigens were boosted in T. parva-immune cattle resolving a challenge infection and, when used to immunize naïve cattle, induced CTL responses that significantly correlated with survival from a lethal parasite challenge. These data provide a basis for developing a CTL-targeted anti-East Coast fever subunit vaccine. In addition, orthologs of these antigens may be vaccine targets for other apicomplexan parasites.