Immunological evaluation of DNA vaccines against Haemonchus contortus and Toxoplasma gondii in sheep

Plasmid DNA vaccines which encode selected antigenic proteins direct protein expression in mammalian cells after vaccination. The use of DNA vaccines has been shown to be a promising approach to induce specific humoral and cellular immune responses against various pathogens, such as parasites. In this study, three experiments were carried out to evaluate the immune responses of DNA vaccines to Haemonchus contortus (H. contortus) and Toxoplasma gondii (T. gondii) in sheep. In the first experiment on H. contortus, sixty 12-month old ewe lambs were divided into six groups and vaccinated twice intramuscularly over a four week interval, using plasmid DNA, either pSecTag2-15 or pSecTag2-24 or a combination of both or neither. Sheep were given an oral challenge infection of 50,000 H. contortus L3 larvae four weeks after the second immunisation. Liveweights were measured and faecal and blood samples were taken and all sheep were slaughtered for worm burden determination at the end of the trial. Vaccination with pSecTag2-15 tended to reduce the mean FEC (by 58%) and mean adult worm burdens (by 65%) compared with the PBS controls. Significantly higher levels of ES-specific IgA and IgG2 were found in this group. In the group treated with pSecTag2-15, FEC was negatively correlated with IgG2 level to adult H. contortus, after challenge. The pSecTag2-24 DNA vaccine did not contribute to protection. The efficacy of the DNA cocktail vaccine (pSecTag2-15 + pSecTag2-24) was less than injection with pSecTag2-15 alone. In the second experiment on H. contortus, the same gene fragments encoding H. contortus antigens, 15 kDa, 24 kDa and the pepsin-like aspartyl proteases, were cloned into a different vector-pVAX1 to produce pVAX15, pVAX24 and pVAXHcPEP1, respectively. These purified plasmids were co-administrated with a plasmid encoding sheep GM-CSF or CpG or the adjuvant emulsigen. One hundred and twenty ten-month old ewe lambs were divided into 12 groups and vaccinated twice intramuscularly (4 week interval) with plasmid DNA or primed/boosted with plasmid/recombinant protein and challenged using 20,000 L3 larvae 4 weeks after the 2nd injection. All animals were slaughtered to assess worm burdens 7 weeks after challenge. Blood samples, faecal samples and live weights were taken weekly, except for the two injection and challenge weeks. Vaccination with the above three antigens, used as DNA or recombinant protein, did not resulted in protection as no reduction of FEC and worm burdens were found in the treated groups compared with controls. Significantly high levels of IgG1 and IgG2 were produced by the DNA prime/protein boost strategy used. In the experiment on T. gondii, gene fragments encoding T. gondii antigens, SAG1 and ROP1, were cloned into vector pVAX1 to produce pVAXSAG1 and pVAXROP1. Sixty sheep divided into 12 groups were injected with the purified plasmids, pVAXSAG1 and pVAXROP1, and/or recombinant SAG1, SAG2 and GRA1 proteins, in combination with plasmid encoding GM-CSF, CpG and liposome as adjuvants. The injections were applied twice intramuscularly with a six weeks interval. Blood samples were taken weekly. Administration of the plasmid encoding the ROP1 gene significantly enhanced IFN-γ production from peripheral blood cells cultured in vitro in response to native Toxoplasma antigen and ROP1-specific IgG1 and IgG2 antibody levels present in serum. Immunisation with recombinant GRA1 protein also stimulated IFN-γ production and GRA1-specific IgG2 antibody levels. However, injection with SAG1 (given as recombinant protein or as plasmid), did not stimulate IFN-γ production. These results indicate that ROP1 given as plasmid DNA and GRA1 given as recombinant protein are potential vaccine candidates for the development of protection against T. gondii infection in sheep. In summary, vaccination with plasmid DNA encoding H. contortus antigens, 15 kDa, 24 kDa and aspartyl protease, did not provide protection against experimental challenge as measured by FEC and worm burdens. In order to develop a successful commercial vaccine against H. contortus, further work should be focused on recombinant versions of Haemonchus protective antigens. For T. gondii, the results from this study indicate for the first time that sheep respond immunologically to DNA vaccines encoding T. gondii antigens. Further experiments are needed to identify more effective vaccine candidates, test the efficacy of other adjuvants, such as IFN-γ and IL-12, and evaluate the protective efficiency of vaccination after challenge