Definition^{[1][2][3][4][5][6][7][8][9][10][11][12]}

1. Approaches  to identifying Cocktail vaccine antigens

1.1. Single-Antigen Vaccine Efficacy

Often the selection of cocktail anti-tick vaccines is based on two factors (1) prior efficacy of the single antigens. (2) the vaccine-effect on target tick protein. Presumably the rationale is that a combination of vaccine A and B (with prior efficacy of 45% and 55% respectively) could lead to enhanced protection efficacy. Additionally, the cocktail vaccine could induce a synergistic effect on the different proteins in the same [13] or different tick species. Although the approach is logical, it does not take into account the immunological short comings of combining antigens which could lead to cocktail vaccine low efficacy.

1.2. Antigen Serum immuno-cross reactivity

Cocktail vaccine-antigens can be selected based on the ability of sera induced against one antigen to cross-react against a protein of heterologous tick species. The method has been used to identify recombinant GST-based cocktail vaccines [14] and a tick-cement- based potential broad-spectrum vaccines [15]. However, the approach can only be applied while selecting homologous tick antigens.

1.3. Antigen Discovery Approaches

Cocktail vaccine-antigens could be selected using the methods that are used to identify single tick-antigens. The methods include RNA interference [16], expression library immunization (ELI), evaluation of expression sequence tags [17], interactomics [18], proteomics [17], and transcriptomics [18]. However, although RNAi could be used, the method is best suited for examining the potential roles of target genes in tick physiology [19], as such the gene-coding proteins may not be immunogenic ^[20][21][20-21]. Currently, the commonly used approach is transcriptomics and has been used in combination with proteomics [22] and metabolomics [23]. Analogous to single anti-tick vaccine antigens, this could enhance the efficiency and accuracy of cocktail antigen discovery, hence hasten the formulation of cocktail vaccines.

1.4. Antigen Serum-induce effect

In addition to the conventional method (use of animal models), in vitro or artificial tick-feeding assays could be used for cocktail vaccine antigen selection. So far, there are three established methods: capillary tubes ^[24][25][24-25], glass tube [26], membrane feeding ^[27][28][29][27-29]. The approaches have been used to (1) maintain tick colonies ^[28][29][30][28-30]. (2) examine novel acaricide molecules [31] and (3) study the proteins involved in tick-pathogen transmission [32]. (4) identifying candidate tick antigens ^[26][33][26,33] The limitation of capillary in vitro feeding is blood-clotting which leads to blood clogging. This can be resolved by blood-defibrination, the addition of blood-anticoagulants and the use of glass tubes. Lew-Tabor [26] have resolved the question whether anti coagulants can affect the tick physiology, hence interfering with the experiment.

To illustrate the relevance of in vitro assays, Trentelman [13] examined the effect of cocktail Bm86 and subolesin anti-serum against Rhipicephalus australis larvae. It was found that the cocktail anti-serum inhibits larvae feeding [13] which suggests that antigens are suitable candidates for formulating a cocktail vaccine. Finally, the assays could also be used to determine the appropriate concentration of  cocktail vaccine components.

21.5. Status of cocktail anti-tick vaccine formulation

Currently, although numerous researchers have embraced the concept to cocktail tick vaccine; see compiled list [34 Ndawula and Tabor 2020], the anticipated outcome is yet to be attained under field conditions. However, the reasons are still unclear. Discussed below are the probable reasons behind the ineffectiveness of cocktail anti-tick vaccines are still unclear.

21.16. Antigenic competition

The efficacy of a vaccine is directly proportional to the induced humoral immune response. On the contrary, when cocktail anti-tick vaccines are formulated, there is reduction of antibodies induced against each cocktail antigen ^[34][35][36][34-36]. This phenomenon is referred to as antigenic competition [37]. The reduction in humoral immune response is due intra-molecular and inter-molecular competition between determinants of the same or different immunogens respectively [38]. Although the mechanisms of antigenic competition have been extensively investigated in human vaccines [39], with regard to cocktail anti-tick vaccines, these are still unknown.

21.27. Antigen concentration

As with other vaccines [40], the concentration of the anti-tick antigen concentration influences the host humoral immune response. Therefore, while constituting cocktail anti-tick vaccines it is tempting to use high concentration of cocktail antigens. To put it figuratively, suppose that 100 μg of vaccine A and B independently induced 45% and 55% efficacy, 100 μg of A and B may be combined to make a cocktail vaccine. Although this approach is mathematically logical, but it may trigger undesired immune responses which include (1) Antigen competition which is pronounced in determinants of different immunogens ^[38][42][43][38, 42-43]. (2) immunotolerance which could be triggered by high or low antigen concentrations ^[44][45][44-45].

21.38. Antigen- Adjuvant interaction

To ensure that cocktail vaccines mount a substantial humoral immune response, antigens are combined with an adjuvant. In this case, the adjuvants act as immunopotentiators or delivery systems ^[46][47][46-47]. A combination of adjuvants Freund’s complete adjuvant (FCA) and Freund‘s incomplete adjuvant (FIA) with a cocktail recombinant antigens showed a higher humoral immune response [48]. However, considering that Freunds’ adjuvants are not recommended for use in large animals [49], Montanide (‘oil-in-water’) adjuvants have been adopted [34 Ndawula and Tabor, 2020].

21.49. Animal genetics

The immunogenicity of a vaccine is influenced by animal genetics ^[50][51][50-51] but it is rarely scrutinized. The influence is high among inbred animals ^{[52][53][54][55]}[52-55]. For instance, antigenic competition is shown to vary with animal genetic factors [56]. In comparison to other models (sheep and mice), the major histocompatibility complex (MHC) gene diversity among different cattle breeds could significantly influence the immunogenicity of a cocktail vaccine. For instance, subolesin has been shown to induce varying immune responses between Bos indicus and Bos indicus and Bos taurus crossbred cattle [57].

21.510. Protein expression system

Expression of anti-tick vaccines is regularly undertaken in bacterial systems (i,e. Escherichia coli) [34 Ndawula and Tabor 2020]. This could be because bacterial expression systems present numerous advantages over other systems [58]. However, bacteria- expressed proteins may be misfolded, hence lacking in conformational epitopes that induce humoral immune responses [59]. Such proteins are less immunogenic. For instance, Bm86 protein expressed in E. coli was shown to be less immunogenic than the Bm86 expressed in yeast [2] or insect cells [60]. This is likely to influence the efficacy of cocktail vaccines.

32. Conclusion

As a proof of concept the efficacy of single anti-tick vaccines could be enhanced through the formulation of cocktail vaccines [61]. Antibodies induced against the cocktail antigens could induce a synergistic benefit by interfering with the functionality of proteins in the same [13] or different tick species. Noteworthy, while formulating cocktail tick vaccines, researchers ought to take into account factors discussed [34 Ndawula and Tabor 2020]. There is optimism that effective cocktail anti-tick vaccines can be formulated which should boost the effort toward control of ticks under field conditions. 

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