Comparative study of protective activities of Neospora caninum bradyzoite antigens, NcBAG1, NcBSR4, NcMAG1, and NcSAG4, in a mouse model of acute parasitic infection

Abstract

Neospora caninum is an obligate intracellular protozoan parasite that causes severe neuromuscular diseases, repeated abortion, stillbirth, and congenital infection in livestock and companion animals. The development of an effective vaccine against neosporosis in cattle is an important issue due to the significant worldwide economic impact of this disease. We evaluated the immunogenicity of four bradyzoite antigens, NcBAG1 (first described in this study), NcBSR4, NcMAG1, and NcSAG4, using an acute infection mouse model to determine synergistic effects with the tachyzoite antigen as a candidate for vaccine production. Mice were inoculated with the recombinant vaccines (r-)NcBAG1, rNcBSR4, rNcMAG1, rNcSAG4, or phosphate-buffered saline (PBS) (adjuvant control group) in an oil-in-water emulsion with bitter gourd extract, a Th1 immune stimulator, or PBS alone as the infection control group. Mice inoculated with each vaccine developed antigen-specific IgG1 and IgG2a antibodies and isolated splenocytes from mice produced high levels of interferon-γ when infected with the N. caninum tachyzoite. The mice inoculated with rNcBAG1, rNcMAG1, or rNcSAG4 developed slight to moderate clinical symptoms but did not succumb to infection. In contrast, rNcBSR4 and both control groups developed severe disease and some mice required euthanasia. The parasitic burden in the brain tissues of vaccinated mice was assessed by N. caninum-specific real-time PCR at 5 weeks after infection. The parasite load in rNcBAG1-, rNcMAG1-, and rNcSAG4-inoculated mice was significantly lower than that in adjuvant and infection control mice. Therefore, these antigens may be useful for the production of a N. caninum-specific vaccination protocol.

A vaccine formulation combining rhoptry proteins NcROP40 and NcROP2 improves pup survival in a pregnant mouse model of neosporosis

“…These results could be explained by means of the immunodominant role of NcGRA7, as mentioned above, or by a lower stimulation of the immune cells in those groups immunized with in rhoptry proteins due to a lower number of replicating parasites. Nevertheless, several vaccine trials employing rNcSRS2 (Haldorson et al, 2005;Pinitkiatisakul et al, 2005Pinitkiatisakul et al, , 2007, rNcPDI (Debache et al, 2010, rNcMIC3 (Cannas et al, 2003b), rNcROP2 (Debache et al, 2008(Debache et al, , 2010, chimeric protein rNcMIC3-MIC1-ROP2 and bradyzoite proteins (rNcBAG1, rNcMAG1 and rNcSAG4; Uchida et al, 2013) have evidenced the ability of these antigens to reduce the cerebral parasite load in infected mice. However, in terms of parasite burden, our results remained consistent with our previous studies (AguadoMartínez et al, 2009;Jiménez-Ruiz et al, 2012).…”

“…Particularly, proteins located in micronemes, rhoptries and dense granules, organelles whose contents are involved in tachyzoite host cell adhesion and invasion, have emerged as promising vaccine targets to block parasite dissemination and foetal transmission during the acute phase of infection . Vaccine candidates have been traditionally selected based on their immunogenic capacities (Ellis et al, 2008), their role in tachyzoite to bradyzoite stage conversion (Aguado-Martínez et al, 2009;Jiménez-Ruiz et al, 2012;Uchida et al, 2013) and their implication in adhesion or invasion processes (Debache et al, 2009;Monney et al, 2011), with variable results in experimental mouse models. Recently, proteome expression changes in different isolates revealed that the rhoptry protein NcROP40 and the dense granule protein NcNTPase are more abundantly expressed in virulent isolates compared to isolates of low virulence (Regidor-Cerrillo et al, 2012).…”

Evaluation of recombinant Neospora caninum antigens purified from silkworm larvae for the protection of N. caninum infection in mice

“…Simultaneous immunization with recombinant antigens of N. caninum with DNA vaccines possessing the antigen genes of N. caninum may enhance the induction of immune responses to N. caninum in mice (18,26). In addition, the Th1 immune response can be induced by an oil-in-water emulsion with bitter ground extract (28).…”

On the application of reverse vaccinology to parasitic diseases: a perspective on feature selection and ranking of vaccine candidates

“…Other bradyzoite stage proteins in the known candidate list are MAG1 (a similar antigen to NcGRA1, NcGRA2, and NcGRA7 (Uchida et al, 2013) and SAG4. Both these antigens were predicted to be exposed with appropriate peptide-MHC binders.…”

“…Ortholog countnumber of orthologous genes as obtained from OrthoMCL DB version 5. (Uchida et al, 2013) Abbreviations: -= unknown; Ribi = Ribi Adjuvant System; FIA = Freund's incomplete adjuvants; VT = vertical transmission. a Vaccine candidates are from studies over the past 10 years that attempt to prevent infection, abortion or vertical transmission; b MIC1, MIC3, GRA2, GRA6 and SRS2 antigens were expressed in Brucella abortus strain RB51; c Four recombinant proteins of N. caninum (GRA1, GRA2, MIC10, and p24B), MIC10 and p24B provide best protection; d Protection = measured or described protection towards type of challenge, e UniProt F0VIH4 is an SRS domain-containing protein (Putative srs29b) but is not a complete sequence; f GRA6 sequence is 100% similar to GRA2; P24B is a possible novel protein; g Same protein as descrived in publication.…”