Bm86 protein

The Bm86 protein is a glycoprotein identified and isolated from epithelial cells of the gut of ticks (Rhipicephalus microplus), Yeerongpilly strain⁹. It contains 650 amino acids and a predicted molecular weight of approximately 80 kDa^2,4; three sites of function are recognized: 1) the N-terminal signal peptide (1-19aa), which is the chain that appears first when the polypeptide chain is synthesized and contains the information on the secretion efficiency, pathway and destination of the protein, 2) the Bm86 antigen or glycoprotein (20-627aa), which is the polypeptide chain of the mature protein after processing, 3) a propeptide (628-650aa), which is the part of the protein that is cleaved during maturation or activation. It is known that the mature protein contains a hydrophobic transmembrane region that encompasses 23aa, located in the region of the C-terminal end, the rest of the protein exhibits high hydrophilic potential. The main characteristic of the protein sequence is the repeated pattern of 6 cysteine residues, which suggests the presence of various domains of the EGF (Epithelial Growth Factor) type (Figures 1 and 2)^2,10-12.

The following are shown: signal peptide (blue arrow); Epithelial Growth Factor-type domains (EGF- brown arrows); the glycosylphosphatidylinositol anchor (GPI-green arrow); Cysteines for the formation of disulfide bridges (grey boxes-arrowhead); N-glycosylation sites (isolated arrowheads).

Figure 1 Primary structure of the Bm86 protein 

The Bm86 protein is formed by a polypeptide chain of 650 amino acids that contains a signal peptide (1-19), several domains of the Epithelial Growth Factor (EGF) type (20-600) and is associated with the membrane by a GPI anchor at the C-Terminal. The figure shows the 3D model generated by Alpha-Fold, obtained from the Uniprot database (https://www.uniprot.org/uniprotkb/P20736/entry#structure) and visualized with ChimeraX (https://www.cgl.ucsf.edu/chimerax/).

Figure 2 3D model of the Bm86 protein 

Bm86 polymorphism

Discontinuous genetic variations in the Bm86 gene have been reported, present in different populations of R. microplus ticks^13,14. Two or more of these variants common in a natural population are called polymorphisms (from Greek, many forms)¹⁵. The sequence of the Bm86 gene of three reference strains has been compared (Yeerongpilly, Australia and Camcord from Cuba: these have identical amino acid sequences, except for residue 111, which is isoleucine in the Camcord strain and threonine in the Australian strain)^16,17. Likewise, in several studies it has been observed that, in some genetic regions, the vast majority of strains present a series of mutations (residues 89, 109, 142, 206, 210, 226, 248, 257 and 272) that are conserved in the strains of continental lands of the Americas but that differ from the reference strains (Table 1)^16,17.

In Argentina, the Bm86 gene was cloned and named Bm95 (genbank with accession number AF150891.2), it has almost the same variants as the South American and Texan genes mentioned above¹⁸. On the other hand, variations of 3.1-7.4 % have been found among Mexican strains in the nucleotides of the Bm86 protein, finding 145 point polymorphisms along the gene¹⁹. Similarly, in a study conducted in Thailand, they sequenced Bm86 from 29 tick samples, obtaining similarities between 91.5-97.0 % between Thai strains and the reference sequence (Yeerongpilly)²⁰. According to the author, the phylogenetic analysis revealed geographical patterns among the Bm86 sequences from Thailand, which were classified into 5 or 9 groups depending on the sequences of nucleotides or amino acids. This contrasts with what has been reported in Mexico, where haplotypes are not limited to one region, which could be explained by the occurrence of gene flow between different regions, perhaps due to the mobility of infested animals that redistribute genotypes¹⁹.

The construction of a cladogram with some sequences of the Bm86 gene obtained from reports available in GenBank allows to observe that there is a large genetic polymorphism in specimens from different regions of the world (Figure 3). The cladogram was made using the Neighbor-joining algorithm, its arrangement shows the evolutionary direction and the relationship of sequences of the Bm86 gene. Three major clades, A, B and C, can be distinguished, each of which presents a polyphyletic arrangement. Likewise, a common ancestor seems to give rise to the outgroup formed by the only sequence of R. appendiculatus and the different branches that form clade A, it shows the terminal dichotomies: China-Yeerongpilly (Australian vaccine strain TickGARD®) and Mozambique-XM, in addition to a pair of unique terminal sequences, corresponding to sequences from Texas. One of the ramifications of the common ancestor for all sequences also gives rise to the putative ancestor for clades B, C and for the only loose terminal corresponding to one of the Mexican isolates, and which fits in a different evolutionary direction from clades B and C. In clade B, unique terminals are observed for 3 of the Mexican sequences and a dichotomy for 2 of the Texan sequences. Clade C shows the dichotomies Mexico-Zapata and Texas-Texas, as well as unique terminals such as Zapata 5, Zapata 1 and Argentina. Although some branches are supported by relatively low bootstrap values, the polyphyletic arrangement of the cladogram helps explain the polymorphism of the Bm86 gene which could have a direct impact on the efficacy of the commercial vaccine in Mexico, in addition, reports for the Bm86 gene from Mexico and Texas show relatively close relationships, which could be due to the commercial relationship of cattle and the natural migration of wild animals between the two countries¹⁶.

The cladogram shows the phylogenetic relationship between different sequences of the Bm86 gene. The strain of Rhipicephalus appendiculatus homologous to Bm86 (Ra86-2) was used as an outgroup. The numbers on the nodes indicate bootstrap values. The boot support percentage of 10,000 pseudo-replicates is shown next to the branches, which are based on evolutionary distances calculated with Jukes-Cantor. Evolutionary analyses were performed in MEGAX²².

Figure 3 Neighbor-joining cladogram of the Bm86 gene of Rhipicephalus microplus 

Commercial vaccines

Anti-tick vaccines began to be developed around the 1980s, when animals were immunized with proteins from crude extracts of semi-engorged females^23,24. It was observed that the proteins affect the midgut of the tick and cause permanent damage to both epithelial cells and basement membrane. This has negative effects on the metabolism, digestive system and reproductive performance of ticks^23,24. In this context, the protein of interest was isolated from the midgut and identified as a membrane glycoprotein, which was purified, characterized and named Bm86⁹.

Based on the above, the first vaccines against ticks in cattle were registered between 1993 and 1997 in Latin America (Gavac®) and Australia (TickGARD®)^4,6,25. The TickGARD® vaccine was developed by the GCSIRO (Commonwealth Scientific and Industrial Research Organization) industry in collaboration with Biotech Australia Pty. Ldt., coming to market in 1994 by Hoechst Animal Health. To prepare it, they isolated the Bm86 protein gene from the gut of the Rhipicephalus microplus tick and cloned it in Escherichia coli²⁶. In 1995, they expressed Bm86 in Pichia pastoris and it was released on the market as TickGARD®PLUS²⁷. The reason was that Bm86 demonstrated improved expression in P. pastoris due to glucan residues added during protein maturation in yeast (reviewed by Willadsen in a report of the industry)²⁸.

In the first four years after its launch, it became the best-selling treatment in that country. However, near the end of the decade, its sale ceased due to the separation and closure of the companies in charge of its production. Years later it was reintroduced to the market by Intervet Australia Pty. Ltd., but after a short period its public sale was ceased⁶.

The results of the application of the TickGARD® vaccine (Table 2) were first reported in 1995, obtaining a significant reduction in the use of acaricides and a 72 % decrease in the reproductive capacity of ticks²⁹. Another study reported 56 % reductions in the number of ticks in the field over one generation and a 72 % reduction in the reproductive capacity of R. microplus females under laboratory conditions³⁰. On the other hand, the Cuban group developed the Gavac® vaccine at the Center for Genetic Engineering and Biotechnology in Havana Cuba by Heber Biotec SA., which was registered in Cuba in 1993, in Colombia and the Dominican Republic in 1994 and in Brazil, Paraguay and Bolivia in 1995^6,31,32. They used the rBm86 protein expressed in the methylotrophic yeast Pichia pastoris, because it has been shown to be a suitable host for the industrial production of heterologous proteins³³. It was estimated that the amount of rBm86 secreted by P. pastoris is approximately 1.5-2.0 g/L, higher than that obtained in fermentations of E. coli^34,35. Its vaccination schedule consists of three primary applications of 2 ml each (100 μg rBm86), intramuscularly (IM), with intervals of three weeks in each application, with a booster every 6 mo³⁶. The effectiveness of the vaccine has been determined using several parameters such as: the number of adult females; the effect on the weight of semi-engorged ticks; the survival of adult female ticks; the number of eggs and their fertility³².

For example, in a controlled study, the effectiveness of the Gavac® vaccine against different strains of R. microplus was evaluated and in different breeds of cattle, it was found that the efficacy was >50 %³². The vaccine has also been shown to be effective in controlling artificial infestations of B. annulatus, B. decoloratus, and strains of B. microplus, whether sensitive or resistant to chemicals in Australia, Africa, the Americas, and Iran (Table 2)³. In addition, in a cost-effectiveness analysis, the vaccine demonstrated a 60 % reduction in the number of acaricidal treatments, as well as the control of tick infestations and babesiosis transmission, which resulted in savings of 23.4 dollars per animal/yr³⁷. Similarly, in controlled field trials in Cuba, 588,573 dairy cows were vaccinated, which showed an 87 % reduction in the use of acaricides over 8 yr (1995-2003)³⁸.

In addition, in a study, cattle were inoculated with TickGARDPLUS® and Gavac® and the animals were challenged with a Brazilian strain of R. microplus “Campo Grande”, the results showed an effectiveness of 46.4 and 49.2 %, respectively³⁹. Considering the above, variations in the efficacy of vaccines can be observed, which can be explained by the genetic diversity (polymorphism) of tick populations and host factors⁸. Some authors suggest that the variation in the sequence of the Bm86 locus (> 3 % in the amino acid sequence) is one of the main reasons why commercial vaccines have limited efficacy in certain R. microplus populations¹³.

In 2018, a vaccine was developed in Mexico from the germplasm of the R. microplus strain “Media Joya”, it was cloned in the expression vector Pichia pastoris. The vaccine was registered under the name Bovimune Ixovac® (Lapisa S.A., Guadalajara, Mexico), with registration SAGARPA B-2083-03, and is being used in the state of Guerrero where there are populations of multidrug-resistant ticks⁴⁰. Its vaccination schedule consists of three primary applications of 2 ml each (120 ± 20 μg of rBm86), IM, at weeks 0, 4 and 7, followed by biannual revaccination (at a single dose)⁴¹.

The treatment has made it possible to maintain infestations of less than 10 ticks per animal, and to reduce the application of tick baths from 14 to 3 per year, in addition to reducing fertility of female ticks by 86 %⁴². So far there is no more information published on the sequence and effectiveness of the vaccine in other regions of Mexico.

Similarly, in Colombia, the LIMOR laboratory developed the Go-Tick vaccine against R. microplus with registration No. 6774-BV of the Colombian Agricultural Institute, based on the Bm86 antigen extracted from tick larvae. The same vaccine is marketed under the brand name Tick-Vac by the TECNOQUÍMICAS laboratory. Its vaccination schedule consists of three initial vaccinations of 2 ml each, subcutaneously or IM, on days 0, 20 and 60, followed by biannual revaccination⁴³. In a field trial in Colombia, the vaccine showed a combined effect of 80 % protection⁷. Whereas in clinical field studies, it has demonstrated protection from 64 to 96 %, reducing parasite load in production systems located in different national agroecosystems in Colombia⁴⁴.

Humoral immune response against Bm86 antigen

Anti-tick vaccines stimulate the humoral immune response of cattle through the production of protective antibodies, which allows a sustained immune response for a period of time after the application of the vaccine, even when the natural Bm86 antigen of the ectoparasite never comes into contact with the host^48,49. When cattle are inoculated with an initial dose of vaccine containing the Bm86 antigen, antigen-presenting cells (APCs), such as dendritic cells and macrophages⁵⁰, capture, process and present the antigen in the lymph node via bovine leukocyte antigen (BoLA)⁵¹. Activated dendritic cells interact with naive T cells that differentiate into effector helper T cells. These cells migrate to the lymphoid germinal centers, where they interact with naive B cells, which leads to the formation of plasma and memory B cells. Plasma cells migrate to the bone marrow⁵² and induce the production of antigen-specific antibodies, while memory B cells are found in the spleen and lymph nodes^53,54, where they await subsequent inoculation (booster dose) of the antigen (Figure 4).

1) The vaccine contains the recombinant protein Bm86. 2) It is inoculated intramuscularly (IM). 3) The T-cell receptor (TCR) recognizes Bm86 epitopes bound to Major Histocompatibility Complex (MHC II) class II molecules and expresses itself on the surface of antigen-presenting cells (APCs). 4) Helper CD4+ T lymphocytes (TL) recognize the MHC II + peptide complex and, activated by cytokines, they begin the humoral immune response. 5) The antigen binds to a B lymphocyte (BL) receptor, stimulating proliferation and differentiation into plasma cells, antibody-producing cells and memory cells⁵¹. Created with BioRender.com

Figure 4 Mechanism of action of immunity induced by the Bm86 antigen in vaccinated animals 

Anti-Bm86 antibodies present in the blood of immunized cattle reach the gut of the tick when they feed on vaccinated animals. The antibodies (IgM and IgG) bind to the Bm86 antigen and activate the complement system (classical pathway), culminating in the lysis of enterocytes, disrupting their function of digesting and absorbing nutrients from the blood, thus affecting the gut and general health of the tick^55,56.

Immunofluorescence and immunohistochemistry studies have shown that antibodies recognize the plasma membrane of the enterocyte; site associated with the Bm86 protein⁵⁷. In other experiments, feeding ticks with serum from cattle inoculated with Bm86 demonstrated inhibition in endocytosis, an effect that precedes detectable enterocyte damage⁵⁸.

Related to the above, in a study they used the plasmid pBMC2 encoding the Bm86 antigen of the strain of the Colombian tick R. microplus to perform immunization tests in mice and cows. Mice immunized with pBMC2 showed high levels of anti-Bm86 antibodies. When evaluating immunogens, significant levels of IgG2a and IgG2b were observed in mice immunized with 50 mg of pBMC2. They also assessed the persistence of the antibody response to vaccination as serum antibody levels must remain high for the vaccine to be effective. Antibody responses were present 56 d after the first immunization in mice and 70 days in cattle. In addition, cattle immunized with 2 mg (1 ml) of pBMC2 had a mean tick count below 50, while other groups (immunized with PBS or 1 mg of pBMC2) exceeded this level⁵⁹. Similarly, the recombinant form of the Bm86 antigen of the Campo Grande strain (rBm86-CG) of R. microplus has been used to stimulate humoral immunity in cattle. Immunoblot results showed that vaccinated cattle developed significant levels of antibodies against rBm86-CG (P<0.05), which peaked 21 d after the third vaccination⁶⁰.

The Bm86 protein as a hidden antigen

Hidden antigens are components of remarkable physiological importance for ectoparasites, which do not usually participate in host-parasite interaction and in their native form have no contact with the host during natural infestation, so, naturally, no immune response of any kind develops against them. However, they have been shown to be able to trigger a protective immune response when the host is exposed to them artificially, by inoculating them into a vaccine⁶¹. The Bm86 antigen is found naturally in the gut wall of the R. microplus tick; this protein has a transmembrane sequence that keeps it bound to the epithelial cells of the tick gut, so that, in its natural form, it never has contact with the immune system of the tick host⁵⁵. Nevertheless, when this protein is used in its recombinant form as a vaccine antigen, it causes a humoral immune response in the animal; Bm86-specific immunoglobulins bind to the antigen presented on tick enterocytes, causing lysis of the tick’s gut wall, which interferes with blood digestion and nutrient absorption, which in turn reduces egg production (Figure 5). The use of these antigens in vaccine design has the hypothetical advantage of avoiding parasitic evasion mechanisms, that is, thanks to the lack of contact between the hidden antigens and the immune system, the tick cannot develop a strategy to escape the animal’s immune action⁶¹. Nonetheless, since a large polymorphism of this gene has been demonstrated, this supposed advantage of Bm86 as a hidden antigen is compensated, therefore it is necessary to consider this polymorphism in the development of vaccines based on the recombinant protein Bm86. Additionally, one of the disadvantages of Bm86 as a hidden antigen is that it induces a permanent response. Therefore, the protective immunity induced by Bm86 derivatives in vaccines is short-lived^2,62 and requires booster doses at 6-mo intervals⁶³.

Figure 5 Effect of the bovine immune response on the tick 

The anti-Bm86 antibodies present in the blood of the immunized cattle penetrate through the mouth of the tick until they reach the gut. The antibodies bind to the Bm86 antigen and activate the complement system (classical pathway)⁵¹. Complement activation begins when two or more Fc (fragment crystallizable) fragments of the antibody react with the C1 component (subcomponents C1q, C1r and C1s)⁶⁴. Cell damage is mediated by the membrane attack complex (MAC), which consists of a polymer of components C5b, C6, C7, C8 and a polymer of C9. MACs cause lytic damage to cells, facilitating the separation of lipids from the membrane and creating pores through which water, ions and macromolecules can enter and exit^64,65.

In-silico analysis of Bm86

The identification of B-cell epitopes in target antigens is a crucial step for the development of vaccines based on these, immunodiagnostic tests, antibody production and disease therapy⁶⁴. Vaccines against R. microplus containing the Bm86 antigen are based on synthetic subunits or peptides, whose mechanism focuses on epitopes that are recognized by B and T cells and bovine leukocyte antigen (BoLA) with which a supposed specific immune response is predicted⁶⁵. Since experimental methods for mapping B-cell epitopes are time-consuming, expensive and laborious; in-silico (computer design) methods for predicting both linear and conformational B-cell epitopes⁶⁴ are a versatile tool that allows researchers to easily manage variables in a DNA sequence, such as: select promoter sequences; terminator sequences; genes; untranslated regions; signal peptides; insert or remove restriction sites that facilitate manipulation in the laboratory⁶⁶. This tool allows evaluating which regions of the proteins of interest could be candidates for the creation of new vaccines or treatments⁶⁷. This was the case of three peptides (SBm4912, SBm7462 and SBm19733) generated by in-silico trials of the Bm86 glycoprotein from the gut of R. microplus, with which they immunized cattle and evaluated the immune response by detecting anti-Bm86 antibodies. The results revealed an efficacy greater than 80 % in cattle immunized with the synthetic peptide SBm7462. Likewise, they observed a decrease in the number, weight and oviposition capacity of engorged females in the tick population that had fed on immunized animals⁶⁸. In one study, they used the synthetic peptide BD86 (a Bm86 ortholog of Rhipicephalus decoloratus) to immunize mice. The results showed that the antibodies generated were able to recognize Bm86 orthologs in four species: R. microplus, R. decoloratus, Hyalomma anatolicum anatolicum and R. appendiculatus, and bound to the ticks’ gut⁶⁹.

Globally, three antigenic determinants of protein sequences homologous to Bm86 were identified. Phylogenetic analysis of isolated epitopes and the Bm86-CG protein (GenBank: ACA57829) revealed geographic patterns between R. microplus tick isolates and those collected from the database. The authors propose the possibility of achieving effective vaccination against ticks in cattle using a single universal antigen based on Bm86. Therefore, as genomic technologies continue to be developed, it will be possible to identify new candidate vaccines against different tick species worldwide⁷⁰. On the other hand, there are publications that propose a reduction in the efficacy of the vaccine based on the Bm86 protein due to the natural polymorphism of the gene in open tick populations, which suggests that strategies should be used that cover a greater variety of antigens derived from the polymorphism of target populations of ticks⁷¹.

Bm86 alleles and orthologous genes

The high degree of polymorphism of Bm86 has led to the identification of multiple alleles of the protein. Recent studies have discovered new proteins that share structural similarities with Bm86, and that could be good candidate vaccines against cattle ticks. For example, an allele of the Bm86 gene identified in Argentina and that has been used as a vaccine is Bm95. This allele was cloned and expressed in P. pastoris yeasts, and the recombinant protein was shown to induce protection in cattle against strains resistant and sensitive to the GAVAC® vaccine⁷². Likewise, controlled immunization trials have shown that vaccines containing Rhipicephalus microplus Bm86 also protect against related tick species, such as; Rhipicephalus annulatus and Rhipicephalus decoloratus^73,74.

In this sense, some authors suggest that the efficacy (Table 3) of a vaccine based on Bm86 may be improved when the orthologous recombinant Bm86 antigen (genes that share the last common ancestor and whose divergence is due to speciation) is used⁷⁵. This is the case of the Bm86 orthologs of R. annulatus (Ba86) and R. decoloratus (Bd86) that were used to immunize rabbits. The results showed cross-immune reactivity of epitopes of orthologous Bm86 proteins⁴⁸. These trials provide a starting point for the development of improved vaccine formulations, incorporating several tick-protective antigens¹¹.

Likewise, the Bm86 ortholog of R. appendiculatus (Ra86) has been used to vaccinate cattle. The results showed a significant decrease in the molting success of nymphs, which suggests that repeated vaccinations with Ra86 could reduce tick populations in successive generations and could be part of comprehensive tick control strategies⁷⁶.