The Development of a Multivalent Capripoxvirus-Vectored Vaccine Candidate to Protect against Sheeppox, Goatpox, Peste des Petits Ruminants, and Rift Valley Fever.

Capripoxviruses are the causative agents of sheeppox, goatpox, and lumpy skin disease (LSD) in cattle, which cause economic losses to the livestock industry in Africa and Asia. Capripoxviruses are currently controlled using several live attenuated vaccines. It was previously demonstrated that a lumpy skin disease virus (LSDV) field isolate from Warmbaths (WB) South Africa, ORF 005 (IL-10) gene-deleted virus (LSDV WB005KO), was able to protect sheep and goats against sheeppox and goatpox. Subsequently, genes encoding the protective antigens for peste des petits ruminants (PPR) and Rift Valley fever (RVF) viruses have been inserted in the LSDV WB005KO construct in three different antigen forms (native, secreted, and fusion). These three multivalent vaccine candidates were evaluated for protection against PPR using a single immunization of 104 TCID50 in sheep. The vaccine candidates with the native and secreted antigens protected sheep against PPR clinical disease and decreased viral shedding, as detected using real-time RT-PCR in oral and nasal swabs. An anamnestic antibody response, measured using PPR virus-neutralizing antibody response production, was observed in sheep following infection. The vaccine candidates with the antigens expressed in their native form were evaluated for protection against RVF using a single immunization with doses of 104 or 105 TCID50 in sheep and goats. Following RVF virus infection, sheep and goats were protected against clinical disease and no viremia was detected in serum compared to control animals, where viremia was detected one day following infection. Sheep and goats developed RVFV-neutralizing antibodies prior to infection, and the antibody responses increased following infection. These results demonstrate that an LSD virus-vectored vaccine candidate can be used in sheep and goats to protect against multiple viral infections.

Baseline analysis of Mycoplasma mycoides subsp. mycoides antigens as targets for a DIVA assay for use with a subunit vaccine for contagious bovine pleuropneumonia.

BACKGROUND: Mycoplasma mycoides subsp. mycoides (Mmm) is the causative agent of contagious bovine pleuropneumonia in cattle. A prototype subunit vaccine is being developed, however, there is currently no diagnostic test that can differentiate between infected cattle and those vaccinated with the prototype subunit vaccine. This study characterized Mmm proteins to identify potential antigens for use in differentiating infected from vaccinated animals. RESULTS: Ten Mmm antigens expressed as recombinant proteins were tested in an indirect ELISA using experimental sera from control groups, infected, and vaccinated animals. Data were imported into R software for analysis and drawing of the box and scatter plots while Cohen's Kappa assessed the level of agreement between the Mmm antigens. Two vaccine antigens (MSC_0499 and MSC_0776) were superior in detecting antibodies in sera of animals vaccinated with the subunit vaccines while two non-vaccine antigens (MSC_0636 and LppB) detected antibodies in sera of infected animals showing all clinical stages of the disease. Sensitivity and specificity of above 87.5% were achieved when the MSC_0499 and MSC_0636 antigens were tested on sera from vaccinated and infected animals. CONCLUSIONS: The MSC_0499 and MSC_0776 antigens were the most promising for detecting vaccinated animals, while MSC_0636 and LppB were the best targets to identify infected animals. Further testing of sera from vaccinated and infected animals collected at different time intervals in the field should help establish how useful a diagnostic test based on a cocktail of these proteins would be.

Potential link of single nucleotide polymorphisms to virulence of vaccine-associated field strains of lumpy skin disease virus in South Africa.

South Africa is endemic for lumpy skin disease and is therefore reliant on various live attenuated vaccines for the control and prevention of the disease. In recent years, widespread outbreaks of vaccine-like strains of lumpy skin disease virus (LSDV) were reported internationally, leading to an increase in the generation of full genome sequences from field isolates. In this study, the complete genomes of six LSDVs submitted during active outbreaks in the 1990s in South Africa were generated. Based on phylogenetic analysis, the six viruses clustered with vaccine strains in LSDV Subgroup 1.1 and are subsequently referred to as vaccine-associated. The genetic differences between the phenotypically distinct vaccine and vaccine-associated strains were 67 single nucleotide polymorphisms (SNPs). This study characterized the location and possible importance of each of these SNPs in their role during virulence and host specificity.

Characterisation of putative immunomodulatory gene knockouts of lumpy skin disease virus in cattle towards an improved vaccine.

Lumpy skin disease virus (LSDV) is responsible for causing severe economic losses to cattle farmers throughout Africa, the Middle East, and more recently, South-Eastern Europe and Russia. It belongs to the Capripoxvirus genus of the Poxviridae family, with closely related sheeppox and goatpox viruses. Like other poxviruses, the viral genome codes for a number of genes with putative immunomodulatory capabilities. Current vaccines for protecting cattle against lumpy skin disease (LSD) based on live-attenuated strains of field isolates passaged by cell culture, resulting in random mutations. Although generally effective, these vaccines can have drawbacks, including injection site reactions and/or limited immunogenicity. A pilot study was conducted using a more targeted approach where two putative immunomodulatory genes were deleted separately from the genome of a virulent LSDV field isolate. These were open reading frame (ORF) 005 and ORF008, coding for homologues of an interleukin 10-like and interferon-gamma receptor-like gene, respectively. The resulting knockout constructs were evaluated in cattle for safety, immunogenicity and protection. Severe post-vaccinal reactions and febrile responses were observed for both constructs. Two calves inoculated with the ORF008 knockout construct developed multiple lesions and were euthanised. Following challenge, none of the animals inoculated with the knockout constructs showed any external clinical signs of LSD, compared to the negative controls. Improved cellular and humoral immune responses were recorded in both of these groups compared to the positive control. The results indicate that at the high inoculation doses used, the degree of attenuation achieved was insufficient for further use in cattle due to the adverse reactions observed.

A lumpy skin disease virus deficient of an IL-10 gene homologue provides protective immunity against virulent capripoxvirus challenge in sheep and goats.

Sheep and goat pox continue to be important livestock diseases that pose a major threat to the livestock industry in many regions in Africa and Asia. Currently, several live attenuated vaccines are available and used in endemic countries to control these diseases. One of these is a partially attenuated strain of lumpy skin disease virus (LSDV), KS-1, which provides cross-protection against both sheep pox and goat pox. However, when used in highly stressed dairy cattle to protect against lumpy skin disease (LSD) the vaccine can cause clinical disease. In order to develop safer vaccines effective against all three diseases, a pathogenic strain of LSDV (Warmbaths [WB], South Africa) was attenuated by removing a putative virulence factor gene (IL-10-like) using gene knockout (KO) technology. This construct (LSDV WB005KO) was then evaluated as a vaccine for sheep and goats against virulent capripoxvirus challenge. Sheep and goats were vaccinated with the construct and the animals were observed for 21days. The vaccine appeared to be safe, and did not cause disease, although it induced minor inflammation at the injection site similar to that caused by other attenuated sheep and goat pox vaccines. In addition, no virus replication was detected in blood, oral or nasal swabs using real-time PCR following vaccination and low levels of neutralising antibodies were detected in both sheep and goats. Leukocytes isolated from vaccinated animals following vaccination elicited capripoxvirus-specific IFN-γ secretion, suggesting that immunity was also T-cell mediated. Following challenge with virulent capripoxvirus, vaccinated sheep and goats were found to be completely protected and exhibited no clinical disease. Furthermore, real-time PCR of blood samples at various time points suggested that viremia was absent in both groups of vaccinated animals, as opposed to capripoxvirus-related clinical disease and viremia observed in the unvaccinated animals. These findings suggest that this novel knockout strain of LSDV has potential as a vaccine to protect livestock against sheep pox and goat pox.

Peste des petits ruminants virus tissue tropism and pathogenesis in sheep and goats following experimental infection.

Peste des petits ruminants (PPR) is a viral disease which primarily affects small ruminants, causing significant economic losses for the livestock industry in developing countries. It is endemic in Saharan and sub-Saharan Africa, the Middle East and the Indian sub-continent. The primary hosts for peste des petits ruminants virus (PPRV) are goats and sheep; however recent models studying the pathology, disease progression and viremia of PPRV have focused primarily on goat models. This study evaluates the tissue tropism and pathogenesis of PPR following experimental infection of sheep and goats using a quantitative time-course study. Upon infection with a virulent strain of PPRV, both sheep and goats developed clinical signs and lesions typical of PPR, although sheep displayed milder clinical disease compared to goats. Tissue tropism of PPRV was evaluated by real-time RT-PCR and immunohistochemistry. Lymph nodes, lymphoid tissue and digestive tract organs were the predominant sites of virus replication. The results presented in this study provide models for the comparative evaluation of PPRV pathogenesis and tissue tropism in both sheep and goats. These models are suitable for the establishment of experimental parameters necessary for the evaluation of vaccines, as well as further studies into PPRV-host interactions.

Capripoxvirus-vectored vaccines against livestock diseases in Africa.

Five different viral diseases of livestock, lumpy skin disease (LSD), sheep pox (SPP), goat pox (GTP), Rift Valley fever (RVF) and peste des petits ruminants (PPR), circulate in the same regions of Africa, imposing a major burden on economic activity and public health. While commercial vaccines against these viruses are available, the cost of implementing regular vaccination regimens against multiple diseases is prohibitive for most African farmers. A single, affordable multivalent vaccine that simultaneously protects against all 5 diseases would therefore be of significant benefit to the livestock sector in Africa. It could also serve as a platform for the development of new vaccines of significance to other developing countries around the world. In this paper, we present an overview of the economic importance of livestock in Africa, the pathogens responsible for RVF, PPR, SPP, GTP and LSD and the vaccination strategies currently used to combat them. We then review experience with the development of attenuated capripoxviruses as vaccines against LSD, SPP and GTP and of recombinant capripoxvirus-vectored vaccines against RVF and PPR. We conclude the article by presenting the rationale for a single, multivalent capripoxvirus-vectored vaccine that would protect against all 5 diseases of livestock, and describe the approach being taken by a consortium of Canadian and South African researchers to develop such a vaccine.

Identification of Mycoplasma mycoides subsp. mycoides small colony genes coding for T-cell antigens.

Genes of the Mycoplasma mycoides subsp. mycoides small colony biotype (MmmSC) coding for proteins capable of eliciting protective T-cell memory responses have potential for incorporation into a recombinant subunit vaccine against contagious bovine pleuropneumonia (CBPP). Here we used lymphocytes from cattle that had completely recovered from infection to screen products of MmmSC genes for recognition by CD4(+) effector memory (Tem) and central memory (Tcm) T lymphocytes. Six MmmSC genes (abc, gapN, glpO, lppA, lppB, and ptsG) were expressed as histidine-tagged recombinant polypeptides, or synthetic overlapping peptides, before inclusion in proliferation and gamma interferon (IFN-gamma) assays. Only two MmmSC antigens, LppA and PtsG, consistently induced recall proliferation from immune CD4(+) T cells and IFN-gamma production in all animals tested. Moreover, LppA and PtsG were shown to possess epitopes recognized by both short-lived CD4(+) Tem and long-lived CD4(+) Tcm cells.

Identification of genes coding for B cell antigens of Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC) by using phage display.

BACKGROUND: Contagious bovine pleuropneumonia (CBPP) is a mycoplasmal disease caused by Mycoplasma mycoides subsp. mycoides SC (MmmSC). Since the disease is a serious problem that can affect cattle production in parts of Africa, there is a need for an effective and economical vaccine. Identifying which of the causative agent's proteins trigger potentially protective immune responses is an important step towards developing a subunit vaccine. Accordingly, the purpose of this study was to determine whether phage display combined with bioinformatics could be used to narrow the search for genes that code for potentially immunogenic proteins of MmmSC. Since the production of IgG2 and IgA are associated with a Th1 cellular immune response which is implicated in protection against CBPP, antigens which elicit these immunoglobulin subclasses may be useful in developing a subunit vaccine. RESULTS: A filamentous phage library displaying a repertoire of peptides expressed by fragments of the genome of MmmSC was constructed. It was subjected to selection using antibodies from naturally- and experimentally-infected cattle. Mycoplasmal genes were identified by matching the nucleotide sequences of DNA from immunoselected phage particles with the mycoplasmal genome. This allowed a catalogue of genes coding for the proteins that elicited an immune response to be compiled. Using this method together with computer algorithms designed to score parameters that influence surface accessibility and hence potential antigenicity, five genes (abc, gapN, glpO, lppB and ptsG) were chosen to be expressed in Escherichia coli. After appropriate site-directed mutagenesis, polypeptides representing portions of each of these proteins were tested for immunoreactivity. Of these five, polypeptides representing expression products of abc and lppB were recognised on immunoblots by sera obtained from cattle during a natural outbreak of the disease. CONCLUSION: Since phage display physically couples phenotype with genotype, it was used to compile a list of sequences that code for MmmSC proteins bearing epitopes which were recognised by antibodies in the serum of infected animals. Together with the appropriate bioinformatic analyses, this approach provided several potentially useful vaccine or diagnostic leads. The phage display step empirically identified sequences by their interaction with antibodies which accordingly reduced the number of ORFs that had to be expressed for testing. This is a particular advantage when working with MmmSC since the mycoplasmal codon for tryptophan needs to be mutated to prevent it from being translated as a stop in E. coli.

SNAMA, a novel protein with a DWNN domain and a RING finger-like motif: a possible role in apoptosis.

We have characterized SNAMA a hitherto uncharacterized Drosophila protein that appears to play a role in apoptosis. SNAMA (something that sticks like glue) is a 1231 amino acid protein with a conserved 76 residue N-terminal domain called Domain With No Name (DWNN). The DWNN domain was first identified in cytotoxic T Cell-resistant CHO cells using promoter trap mutagenesis to screen for genes involved in apoptosis. Subsequently, this domain was identified in other eukaryotic organisms including animals and plants. The SNAMA transcript is abundant early in embryogenesis but reduced in older embryos and in adult males and females. Human and mouse homologues of SNAMA are known to bind to p53 and to the retinoblastoma protein (Rb) suggesting a role in transcriptional regulation and cell cycle control. We took advantage of a P-element insertion line in which the P-element is inserted in the first intron, to investigate the biological function of the gene. These mutants are lethal when homozygous. Apoptosis appears early during embryogenesis and is observed virtually throughout the gastrula. The DWNN domain has a ubiquitin-like fold and may interact with a subset of cellular proteins. There is also a conserved RING finger-like motif along the sequence of SNAMA following a C2HC zinc finger.