Assessment of Babesia bovis 6cys A and 6cys B as components of transmission blocking vaccines for babesiosis

Abstract

Background: Babesia bovis reproduces sexually in the gut of its tick vector Rhipicephalus microplus, which involves expression of 6cys A and 6cys B proteins. Members of the widely conserved 6cys superfamily are candidates for transmission blocking vaccines (TBV), but intricacies in the immunogenicity of the 6cys proteins in the related Plasmodium parasites required the identification of transmission blocking domains in these molecules for vaccine design. Hereby, the immunogenic efficacy of recombinant (r) B. bovis 6cys A and B proteins as a TBV formulation was studied.

Methods: The immunogenicity of r6cys A and 6cys B proteins expressed in a eukaryotic system was evaluated in a cattle immunization trial (3 immunized and 3 control calves). A B. bovis sexual stage induction in vitro inhibition assay to assess the ability of antibodies to block the production of sexual forms by the parasite was developed.

Results: Immunized cattle generated antibodies against r6cys A and r6cys B that were unable to block sexual reproduction of the parasite in ticks. Additionally, these antibodies also failed in recognizing native 6cys A and 6cys B and peptides representing 6cys A and 6cys B functional domains and in inhibiting the development of sexual forms in an in vitro induction system. In contrast, rabbit antibodies generated against synthetic peptides representing predicted B-cell epitopes of 6cys A and 6cys B recognized recombinant and native forms of both 6cys proteins as well as peptides representing 6cys A and 6cys B functional domains and were able to neutralize development of sexual forms of the parasite in vitro.

Conclusions: These data, combined with similar work performed on Plasmodium 6cys proteins, indicate that an effective 6cys protein-based TBV against B. bovis will require identifying and targeting selected regions of proteins containing epitopes able to reduce transmission.

Keywords: Babesia bovis; Neutralizing antibodies; Recombinant proteins; Rhipicephalus microplus; Sexual stages; Synthetic peptides; Tick; Transmission blocking vaccine.

Fig. 1

Immunoblot analysis of r6cys A and r6cys B proteins. Reactivity of rabbit polyclonal antibodies against 6cys A and 6cys B peptides, with r6cys A and r6cys B (a and c left panel), and with B. bovis blood cultured lysate antigen (lane 1: uninfected erythrocytes; lane 2: B. bovis in vitro culture lysate) (a and c right panel). Sera from a bovine chronically infected with B. bovis recognizes B. bovis recombinant protein RAP-1CT (b and d panels) but does not recognize r6cys A (b) and r6cys B (d). Protein size markers are indicated on the left and right ends in each panel

Fig. 2

Analysis of the immune responses in cattle immunized with r6cys A and r6cys B. a ELISA analysis for the pre-immune and immune serum after immunization with the r6cys A and r6cys B proteins. Y axis represents optical density readings of the ELISA at 450 nm and the X axis the calf numbers; I: immunized; c: control. b Western blot analysis of r6cys A and r6cys B protein with pre- and immune serum from the two experimental animal groups taken at different times after immunization

Fig. 3

Parasitemia (determined by DNA analysis) and clinical parameters (PCV reduction and temperature rise) in immunized and control animals following B. bovis challenge. a: Quantitative PCR analysis performed on DNA extracted from blood samples of immunized and control animals at days 9–12 post-challenge. The P value for the qPCR data was > 0.05 indicating that there was no significant difference among the samples. b: Measurements of temperature and PCV of experimental animals in the two groups from day 1 until day 12 post-infection [dpi]

Fig 4

Reactivity of the bovine anti-6cys A and 6cys B polyclonal antibodies against native 6cys proteins. a Immunoblot analysis of B. bovis merozoite stage (MS) and induced sexual stage (ISS) lysates. Antibodies and serum used are indicated above each lane. b Comparison of the in vitro neutralization activity of antibodies on the development of sexual stages, performed on the T2Bo B. bovis strain. The Y axis represents % of infected RBC “orange bar chart” or the % of sexual forms “blue bar chart.” The X axis represents the rabbit polyclonal antibodies against 6cys A and B peptides and sera from r6cys A and r6cys B immunized cattle. P values > 0.05 indicate no significant difference and P values < 0.05 indicates there is significant difference. The abbreviations used in the figures are explained in Additional file 1: Table S1

Fig 5

Mapping the reactivity of bovine r6cys A and r6cys B polyclonal antibodies against 6cys A and 6cys B synthetic peptides. a Schematic representation of the synthetic peptides selected from the 6cys protein A and B. Peptides A2 and A3 (yellow line), but not A1 (green line) selectively reacted with bovine anti-r6cys A and r6cys B antibodies. Peptide B1 (yellow line), but not B2 (green line) react with bovine anti-r6cys A and r6cys B antibodies. b ELISA analysis. The Y axis represents the average of the optical density (OD) values in ELISA at 450 nm. The X axis represents the serum samples tested (samples taken at 84 dpi for both animal groups). Each bar represents reactivity against distinct synthetic peptides and recombinant proteins, as indicated with different color codes shown at the upright of the chart