vaccine design. The figure summarises the different anti-
vaccine types developed to date. Live attenuated vaccines are obtained by successive parasite passages in splenectomised cattle or in vitro cultured parasites that are subsequently attenuated by different methods. Protein-based vaccines have been obtained by size separation of crude parasite extracts, recombinant expression in
and subsequent lysis and purification by affinity chromatography, or viral vectors engineered to express the protein if interest in vivo. Peptide-based vaccines are focused on the in silico identification of short regions containing B- or T-cell epitopes and subsequent chemical synthesis. Figure created with BioRender.com.
Immunisation with live attenuated
B. bovis microorganisms targeting this species has been used in Australia, Brazil, Israel, Mexico, Colombia, Argentina, South Africa and Uruguay. In addition to the aforementioned advantages, it must be stressed that this type of vaccine involves significant limitations related to its production and administration. For example, production of this type of vaccine requires fresh bovine erythrocytes and serum from specific donors requiring very strict production/maintenance conditions to ensure that they are pathogen-free (which can be difficult when the biological product is made in tick-endemic regions) and guaranteeing parasite viability from the beginning of preparation until the final product delivery. In vitro cultured parasite immunogenicity may be lost after long-term maintenance in such conditions; access to suitable laboratory equipment and the availability of trained personnel is necessary, given the complexity of maintaining a
B. bovis culture for large-scale vaccine production
[14][16]. Regarding administration, there are still difficulties related to standardising the dose required for the vaccination. There is also the potential risk of tick transmission of the vaccine strain that could recombine with the field strains, potentially leading to new virulent variants or virulent reversion
[14][15]. The ideal moment for immunisation must be taken into account; administration to weaning age calves being preferred because adult cattle can be highly susceptible to vaccine components inducing serious reactions, such as abortion
[24].
2.2. Protein-Based Vaccines
The main molecules activating an immune response are the parasite proteins expressed during the blood stages; there has thus been major interest in studying complete recombinant molecules or their fragments which can activate the cellular and the humoral immune responses controlling infection
[25][26]. This approach has been useful, given that the parasite proteins/fragments can be obtained individually, involving low production costs using different expression systems (
Escherichia coli being the most used), along with the relative ease of standardising the production and storage and a greater degree of safety compared to that for live vaccines
[27].
Affinity chromatography purification and identification of the antigens obtained from the
B. bovis crude extracts formed the first approach involving this type of vaccine. This assay involved the sequential vaccination/challenge experiments in adult cattle using crude parasite extracts systematically fractionated by chromatographic and electrophoretic procedures (
Figure 2); fractions eliciting a protective response were used for purifying the antigens by pull-down assays (i.e., 11C5 and 12D3), using the monoclonal antibodies (Ab) developed against these parasite fractions.
The rhoptry associated protein 1 (RAP-1) has been confirmed as an antigen containing the immunodominant and conserved T-cell epitopes in its N-terminal region which can induce a response from the IFN-γ-producing CD4
+ T-cells and increase IgG2 synthesis, thereby conferring partial protection in animals challenged with the parasite
[28][29][30]. Nevertheless, such a response did not guarantee protective immunity against challenge with the virulent
B. bovis strain, nor did it change the infection’s clinical course
[31].
Merozoite surface antigen-1 (MSA-1) has also been of interest given that it is mainly exposed to the immune system during natural infection. However, despite MSA-1 being able to induce neutralising Ab production in vitro
[32], it did not protect calves immunised with three doses of recombinant saponin-emulsified MSA-1 after they were challenged with the
B. bovis T2Bo strain
[33]. Such findings could be explained by limitations such as the brief period of exposure to Mrz in serum, thereby limiting accessibility to neutralising epitopes
[34], difficulties related to producing protective Abs (due to high MSA-1 variability inducing strain-specific responses and thus the lack of cross-protection), or the presence of other proteins from the
B. bovis variable merozoite surface antigen (VMSA) family functionally replacing MSA-1 proteins.
The apical membrane antigen-1 (AMA-1) is a very interesting molecule as its evaluation in
Plasmodium has shown that it plays an indispensable role in the host cell invasion. It has been found that the Ab production induced by the
B. bovis recombinant protein encoded by the conserved central region (
BbAMA-1 ectodomain I (DI)and II(DII)) significantly inhibited Mrz growth as well as the efficiency of in vitro Mrz invasion of the bovine erythrocytes by 70% after 6 h, suggesting that this region is a good candidate for inclusion in an anti-
B. bovis vaccine
[35].
2.3. Recombinant Viral Vector Vaccines
Recombinant viral vector vaccines seek to enhance humoral and cellular immune responses by using non-pathogenic viral vectors as expression platforms
[36]. For example, the highly attenuated modified vaccinia virus Ankara (MVA) is a non-replicative vector used to express single or multiple immunodominant antigens in vivo (
Figure 2). Once MVA has infected host cells, recombinant proteins are expressed and exhibited to the immunological system to trigger responses, mainly Th1-type
[37].
2.4. Antigenic Peptide-Based Vaccines
The chemical synthesis of short sequences containing the T- and the B-cell recognition sites has been established as a strategy for vaccine development (
Figure 2) as only these molecule’s fragments can induce an immune response
[38]. This approach has been studied with proteins participating in the parasite-erythrocyte interaction and is based on evidence related to their antigenic role during natural infection.
Several peptides containing RAP-1, MSA-2c and AMA-1 B-cell epitopes were able to trigger neutralising Ab in cattle, inhibiting in vitro invasion by 34.7% on average (using RAP-1 P1 and P4), 6.28% (with AMA-1 P2) and 10.34% (MSA-2c P3 and P4) when used individually and 52.37% when evaluated as a pool. Interestingly, MSA2c P3 and AMA-1 P2 induced gamma interferon (IFN-γ) production in PBMCs from the vaccinated cattle after one year, thus providing a long-lasting Th1 immune response
[39]. It has been shown that a recombinant epitope derived from the
BbAMA-I-DI surface-exposed α-helix (specifically in the PAN motif) was able to elicit Ab in rabbits which significantly inhibited the in vitro parasite invasion by 60% within 4 h of incubation and Mrz growth by 50–70% on days 3 and 4 of culture
[40].
3. Functional Approach
Knowledge about the biology of the parasites belonging to the
Theileria, Plasmodium, Toxoplasma and
Babesia genera has advanced with different intensity,
Plasmodium being one of the most studied due to its significant impact on human health worldwide
[41].
Plasmodium falciparum and
Plasmodium vivax are the most important species causing the highest mortality and morbidity rates worldwide, whilst
Plasmodium ovale,
Plasmodium malariae and
Plasmodium knowlesi have the lowest prevalence
[42]. Different strategies have also been studied for developing a vaccine as the main disease control measure. However, a new and robust functional approach has been proposed during the last few years; it has used
P. falciparum as a malaria disease model. Rather than identifying variable regions, it is based on selecting critical protein-derived conserved regions related to parasite adhesion
[43]. These regions were initially identified in
P. falciparum proteins by screening the erythrocyte binding properties of 20-residue-long, non-overlapping peptides spanning the entire molecules. A sensitive and specific radio-iodination-based binding assay was used.
This approach has been used and adapted for identifying minimal regions involved in
P. vivax binding to the reticulocytes (
Figure 3)
[44]. Omic study results have been compiled as a strategy for filtering those regions expressed during the parasite’s life-cycle, that have the potential of participating in target cell entry. Bioinformatics tools are used to prioritise the candidate selection based on their in silico characteristics (such as the signal peptides, the membrane anchor sequences, the functional domains, the stage-specific expression profiles and the specific location pattern) and intra- and inter-species conservation level, the latter determined by natural selection analysis
[45]. The protein’s target cell binding role is then evaluated using only 20-residue-long, non-overlapping peptides derived from the regions under functional constraint by the protein-cell interaction assays, like those performed for
P. falciparum. This has enabled finding
P. vivax cHABPs to target cells; their role in cell invasion must be confirmed in vitro and in vivo assays using modified peptides in the latter to improve their antigenicity. Unlike results obtained by the classical approach to designing vaccines based on proteins and peptides, in vitro results obtained by the functional approach are expected to be consistent with those in vivo, as confirmed for
P. falciparum.
Figure 3. Functional approach strategy. Flow diagram describing the steps for prioritising in silico selection and laboratory experiments for identifying ideal vaccine component peptides. The strategy involves identifying the entire gene repertoire (using genome, transcriptome and proteome data mining) and their encoding sequence analysis by bioinformatics tools based on in silico characteristics and their intra (from 5 genomes)- and inter-species (phylogenetically related) conservation level
[45]. Peptides encoded by regions under functional constraint are then chemically synthesised and target cell binding activity must be tested by radio-iodination-based peptide-cell interaction and competition assays
[46]. Identified HABPs must be evaluated in in vitro culture and then modified to fit inside MHC-II to evaluate their role as vaccine component in in vivo tests in the pertinent experimental model. The figure shows the required resources for carrying out the functional approach.
43. Conclusions
Anti-
B. bovis vaccine development has been focused on live-attenuated parasites, recombinant proteins and antigenic peptides. However, such traditional strategies have highlighted the variability of these approaches, along with the many limitations related to their success. This has emphasised the need for adopting safe alternative vaccine design approaches, capable of inducing a long-lasting protection-inducing immune response. A functional approach is thus worth considering, taking into account the implicit advantages of safety, efficiency, ease of maintenance and production.