Structural and immunological characterization of an epitope within the PAN motif of ectodomain I in Babesia bovis apical membrane antigen 1 for vaccine development

Abstract

Background Bovine babesiosis caused by Babesia bovis (B. bovis) has had a significant effect on the mobility and mortality rates of the cattle industry worldwide. Live-attenuated vaccines are currently being used in many endemic countries, but their wide use has been limited for a number of reasons. Although recombinant vaccines have been proposed as an alternative to live vaccines, such vaccines are not commercially available to date. Apical membrane antigen-1 (AMA-1) is one of the leading candidates in the development of a vaccine against diseases caused by apicomplexan parasite species. In Plasmodium falciparum (P. falciparum) AMA-1 (PfAMA-1), several antibodies against epitopes in the plasminogen, apple, and nematode (PAN) motif of PfAMA-1 domain I significantly inhibited parasite growth. Therefore, the purpose of this study was to predict an epitope from the PAN motif of domain I in the B. bovis AMA-1 (BbAMA-1) using a combination of linear and conformational B-cell epitope prediction software. The selected epitope was then bioinformatically analyzed, synthesized as a peptide (sBbAMA-1), and then used to immunize a rabbit. Subsequently, in vitro growth- and the invasion-inhibitory effects of the rabbit antiserum were immunologically characterized. Results Our results demonstrated that the predicted BbAMA-1 epitope was located on the surface-exposed α-helix of the PAN motif in domain I at the apex area between residues 181 and 230 with six polymorphic sites. Subsequently, sBbAMA-1 elicited antibodies capable of recognizing the native BbAMA-1 in immunoassays. Furthermore, anti-serum against sBbAMA-1 was immunologically evaluated for its growth- and invasion-inhibitory effects on B. bovis merozoites in vitro. Our results demonstrated that the rabbit anti-sBbAMA-1 serum at a dilution of 1:5 significantly inhibited (p < 0.05) the growth of B. bovis merozoites by approximately 50–70% on days 3 and 4 of cultivation, along with the invasion of merozoites by approximately 60% within 4 h of incubation when compared to the control groups. Conclusion Our results indicate that the epitope predicted from the PAN motif of BbAMA-1 domain I is neutralization-sensitive and may serve as a target antigen for vaccine development against bovine babesiosis caused by B. bovis.

Immunization of Cattle With Recombinant Structural Ectodomains I and II of Babesia bovis Apical Membrane Antigen 1 [BbAMA-1(I/II)] Induces Strong Th1 Immune Response

“…Due to the fact that the vaccines currently being administered are neither fully safe nor effective, researchers have extensively searched for a candidate vaccine antigen that could be used to develop an effective vaccine against B. bovis (4). As has been found with BbAMA-1, studies of its immunogenicity demonstrated its vaccine potential (12,36,37). However, a lack of in-depth knowledge and understanding of the immunological characterization of the response may have implications for the design and assessment of AMA-1-based vaccines for bovine babesiosis.…”

“…In the present study, the ectodomains I and II region of BbAMA-1 has been successfully expressed as a doublet band corresponding to around 48 kDa. Because BbAMA-1 encompasses the extracellular cysteine-rich domains I and II ( 12 , 36 , 37 ). It is likely that cysteine oxidation and formation of intra-molecular disulfide bonds is responsible for the doublet formation in Laemmli SDS-PAGE.…”

“…The process was run with 100 V for 1 h. The SDS-PAGE slab gels were then stained with Coomassie blue R-250 (Sigma-Aldrich, St. Louis, MO, United States) for protein band detection. Moreover, the proteins obtained from the SDS-PAGE gel were electrically transferred onto a nitrocellulose membrane (Merck Millipore™, Merck KGaA, Darmstadt, DEU) at 15 V for 1 h. The membrane was then incubated with previously produced rabbit polyclonal anti-sBbAMA-1 serum (1:50 dilution) at 4°C overnight ( 36 ). Subsequently, the membrane was probed with horseradish peroxidase (HRP)-conjugated goat anti-bovine immunoglobulin (Ig) G antibody (1:2,000 dilution; KPL, Gaithersburg, MD, United States).…”

“…The purified recombinant protein was dialyzed by gradually decreasing the urea concentration. The rBbAMA-1(I/II) protein was concentrated using Amicon R centrifugation filters (30 kDa MWCO; Merck KGaA, Darmstadt, Germany) and concentration was then measured using the BCA protein assay (36). Subsequently, the membrane was probed with horseradish peroxidase (HRP)-conjugated goat anti-bovine immunoglobulin (Ig) G antibody (1:2,000 dilution; KPL, Gaithersburg, MD, United States).…”

“…Importantly, a strong negative or purifying selection across the whole of the gene was established, especially in domain I, indicating strong functional constraints on this gene ( 35 ). Interestingly, B. bovis AMA-1 (BbAMA-1) is recognized by the antibodies against the epitopes that are mainly situated within domain I ( 12 , 36 ) and domain II ( 37 ). These antibodies could inhibit the in vitro growth and invasion of erythrocytes by B. bovis merozoites.…”