Vaccination of mice with recombinant NcROP2 antigen reduces mortality and cerebral infection in mice infected with Neospora caninum tachyzoites

Rhoptry antigens are involved in a variety of cellular functions related to host cell invasion, formation of the parasitophorous vacuole and parasite-host cell interplay. The cDNA sequence of one of these antigens, NcROP2 was identified from Neospora caninum expressed sequence tags (ESTs), amplified by reverse transcription-PCR, expressed in Escherichia coli as a (His)6-tagged recombinant protein (recNcROP2) and purified over Ni2+-affinity chromatography. Both recNcROP2 and antibodies directed against recNcROP2 had a negative impact on N. caninum tachyzoite host cell invasion in vitro, indicating that this protein participates in the host cell entry process. Subsequently, the protective efficacy of NcROP2 as a potential vaccine candidate was evaluated in a C57BL/6 mouse cerebral disease model. Mice were vaccinated three times at 2-week intervals with recNcROP2 emulsified either in Freund's incomplete adjuvants (FIA) or saponin, and control groups were treated with adjuvants alone (adjuvants control) or PBS (infection control). Subsequently, mice were challenged with 2 × 106N. caninum tachyzoites. Nine mice, all belonging to the infection control or adjuvants control groups, exhibited clinical signs of cerebral neosporosis and succumbed to infection, whilst no clinical signs were noted for recNcROP2-vaccinated mice. For all other animals, the experiment was terminated 35 days p.i. Cerebral parasite burdens were assessed by quantitative PCR in all mice, and were revealed to be significantly reduced in the recNcROP2-vaccinated mice. ELISA of sera revealed IgG1 to be elevated in recNcROP2-saponin vaccinated mice, whilst IgG2a was higher in recNcROP2-FIA vaccinated animals. This shows that, depending on the adjuvants used, vaccination with NcROP2 induces a protective Th-1- or Th-2-biased immune response against experimental N. caninum infection.