Over the last 25 years, numerous studies have been conducted in an attempt to develop effective vaccines against ticks (Guerrero et al., 2012; Willadsen, 2008). Currently, there is no vaccine with protective antigens against infestations of Rhipicephalus sanguineus (Latreille, 1806) in dogs. It has been previously shown that immunization using a 20 amino acid synthetic peptide from the tick ribosomal protein P0 (pP0) chemically conjugated to Keyhole Limpet Hemocyanin of Megathura crenulata (KLH) or Bm86 as carrier protein in an oily formulation containing Montanide^TM ISA 50 (SEPPIC, France) was effective to generate specific antibody titers against the peptide which produced a remarkable diminution in the viability and fertility of R. sanguineus ticks fed on vaccinated animals (Rodríguez-Mallon et al., 2012; Rodríguez-Mallon et al., 2020). These anti-tick effects were demonstrated to be positively correlated with the specific immune response against the pP0. However, the oily formulation of this effective antigen produced some side undesired effects on the inoculation site of immunized dogs (Rodríguez-Mallon et al., 2013, 2020). The objective of this study was to assess the  immunogenicity  of  the  pP0-Bm86  chemical conjugate formulated with the polymeric adjuvant Montanide™ GEL 01 (SEPPIC, France) in dogs and compare the specific antibody response obtained with that of the same antigen in oily formulation using Montanide^TM ISA 50 (SEPPIC, France). General health parameters and in situ changes at the injection site were also evaluated in vaccinated dogs using quantitative and qualitative data.

Immunization schedule

Twenty-two dogs of different breeds and sex ranging between 1 to 5 years old from the Cuban police service were randomly assigned to five experimental groups as shown in Table 1. Dogs were fed with a commercial pellet diet (produced by ALICAN S.A, Argentina) and water ad libitum. The trial was conducted in the Altahabana Veterinary Clinic of the Canine Unit in the Ministry of Interior (MININT, Cuba). Dogs were maintained in separated boxes until the end of the experiment. The sampling exercise and all procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (2011) and were approved by the Ethics Committee at the CIGB.

The P0 synthetic peptide (NH2-AAGGGAAAAKPEESKKEEAK-CONH2) (pP0) was chemically conjugated to the Bm86 protein produced by the fermentation of a recombinant Pichia pastoris yeast clone (Canales et al., 1997) at the CIGB of Camagüey (Gavac^TM active ingredient, Lot: 14.1302-4) (pP0-Bm86). Considering the adjuvant properties described for the Bm86 nanoparticles produced by this yeast (Garcia-Garcia et al., 1998), this protein could be the most suitable and feasible candidate of carrier for the efficient presentation of pP0 to the host immune system, because this protein is itself an immunogen against ticks. The pP0-Bm86 conjugate was obtained by using the maleimide method (Carter 1994). Briefly, the carrier protein was activated using 3-maleimidopropionic acid N-hydroxysuccinimide ester (MPS) as the crosslinking reagent and one Cysteine residue was added to N-terminal end of the P0 peptide in order to be linked to the carrier protein. The immunogen diluted in PBS (NaCl 8 g/L, Na₂HPO₄ 1.15 g/L, KCl 0.2 g/L, KH₂PO₄ 0.2 g/L, pH 7-7.2) was formulated with two different adjuvants: Water-in-Oil (W/O) emulsion (Montanide^TM ISA 50; SEPPIC, France) in a 60/40 proportion of immunogen/adjuvant and a new adjuvant based on the dispersion of a high molecular weight polyacrylic polymer in water (Montanide™ GEL 01) kindly provided by SEPPIC (France) using 3 and 8% of gel in the final preparation. Dogs were subcutaneously immunized with 1mL of pP0-Bm86 in doses of 500 g/ animal on days 0, 21 and 36. Dogs in negative control groups were injected with PBS prepared in Montanide^TM ISA50 or Montanide™ GEL 01. The general behavior of the animals was observed daily and body temperature was measured each two days throughout the test. Body weights were recorded and hematological tests were performed the same days in which dogs were immunized and fifteen days after the last immunization. Inoculation sites were evaluated during 1 week after each immunization and a score was assigned to each dog according to the observed local injuries. Body weights were compared using repeated measures such as one way ANOVA followed by Bonferroni multiple comparisons test; scores of local injuries were compared by ANOVA and Bonferroni multiple comparisons test on the statistical program Prism (version 6.0 for Windows; GraphPad Software, USA).

Characterization of the immune response by indirect ELISA

Dog serum  samples  were  taken on days 0, 21, 36 and 51 in order to evaluate the specific antibody response against pP0 by an indirect ELISA as previously described (Rodríguez-Mallon et al., 2012). Briefly, 100 ng per well of pP0 chemically conjugated to Keyhole Limpet Hemocyanin of Megathura crenulata (KLH) was used to coat ELISA plates overnight at 4°C because the plate binding of the peptide alone is not efficient and the use of a different chemical conjugate of this peptide will guarantee that only the immune response against pP0 is measured. Sera were serially diluted in base 1:2 in PBS 1X. The plates were incubated with the diluted sera for 1h at 37°C and then incubated with 1:10,000 anti-dog IgG–HRP conjugate (Sigma) for 1h at 37°C. The color reaction was developed with a substrate solution containing o-phenylenediamine, 0.4 mg/ml in 0.1 M citric acid and 0.2 M Na₂HPO₄, pH 5.0 and 0.015% of hydrogen peroxide. The reaction was stopped with 2.5 M H₂SO₄ and the OD490 nm was determined. The antibody titer was established as the reciprocal of the highest dilution, at which the mean OD of the serum in question was three times the mean OD of the negative control serum. The geometric mean of antibody titers in each group was determined from individual values and the data were log base 10 transformed and compared by using ANOVA and Bonferroni multiple comparisons test on the statistical program Prism (version 6.0 for Windows; GraphPad Software, USA).

Specific antibody responses against pP0 were obtained in all animals immunized with the pP0-Bm86 chemical conjugate (Figure 1A). Fifteen days after booster on experimental day 36, the antibody titers against pP0 in vaccinated groups become statistically significant (P<0.01) compared to negative control groups (G1 and G2) reaching an average value above 1000 for all groups vaccinated with the conjugate. There were no statistically significant differences between anti-pP0 antibody titers among groups 3, 4 and 5 vaccinated with the pP0-Bm86 conjugate with different adjuvants. According to previous experiments, this specific antibody response against pP0 will produce an important reduction in the tick reproductive potential with a calculated efficacy around 85% (Rodriguez Mallon et al., 2020).

There were no statistical significant differences among the body weight averages at different times during the experiment  in  each  group  (Figure  1B).  There  was  no change in normal behavior, or any fever in any of the animals during immunization experiment days according to the body temperature reference values reported for dogs (Kahn et al., 2010) (Figure 1C). The averages of all hematological parameters remained within the normal ranges reported for the species during the experiment (Kahn et al. 2010) as shown in Table 2. The assigned scores to local injuries at the inoculation sites showed statistical significant differences (P<0.001) between the groups that received Montanide^TM ISA50 and those that were injected with Montanide™ GEL 01 as adjuvant (Figure 2A). Some dogs in groups 1 and 3 presented large and hard lesions with an evident volume increase. One of them in group 3 developed an access as shown in Figure 2B. This access disappeared after three days of local treatment.

These results demonstrated that the pP0-Bm86 chemical conjugate formulated with both adjuvants, Montanide^TM ISA50 and Montanide™ GEL 01, was able to generate a similar specific immune response against the pP0 in dogs which is effective against ticks as previously demonstrated (Rodríguez-Mallon et al., 2020). However, the evaluation of the local reactions after subcutaneous administration of the vaccine formulations showed significant diminutions in adverse effects when Montanide™ GEL 01 is employed which could suggest the convenience to use this adjuvant when sensitive animals as pets are vaccinated.

As it is very well known, the adjuvant selection during the vaccine design is crucial for the vaccine properties. There is no universal adjuvant, and there is always a tradeoff between safety and efficacy. Therefore, the selected adjuvant will depend on the target species, the antigen, the inoculation route and the desired immune response and its duration. Alum (aluminum salts) is still the most frequently used adjuvant for human vaccines and is also used in many veterinary vaccines, especially for pets.  Nevertheless,  histological  evidence of adverse  effect related to alum in pets has been described (Hendrick et al., 1992; Vascellari et al., 2003; Verdier et al., 2005). On the other hand, oil-based adjuvants as emulsions of water-in-oil, or vice versa are among the most efficient adjuvants known and they are in wide use for veterinary vaccines (Aucouturier et al., 2001). In our experience, water-in-oil (W/O) emulsions induce strong, long-term immunity but can sometimes induce local reactions which could be severe in some cases (Rodríguez-Mallon et al., 2013, 2012, 2020). Generally, water-in-oil emulsions (W/O) are recommended for bovines, small ruminants, poultry and fish when long-term immunity is required. Conversely, vaccines for pets and horses must not induce any local reactions at the injection site.

This study provides direct evidence that Montanide™ GEL 01 can act as a powerful adjuvant in a synthetic peptide based vaccine and constitute an easy-to-use and low-cost veterinary vaccine with a more safety profile in pets  than  oily   Montanide.  In   summary,    the    results obtained in this work show the convenience of using Montanide™ GEL 01 for dogs instead of Montanide^TM ISA50 because the specific antibody titers against the P0 peptide were similar with both adjuvants but the adverse events were significantly lower.

The authors have not declared any conflict of interests.

This work was supported by the Center for Genetic Engineering and Biotechnology, Havana, Cuba and the Altahabana Veterinary Clinic of the Canine Unit in the Ministry of Interior, Cuba. PEEG, YBS, LlGP, MPE and ARM belong to the CYTED network INCOGARR 118RT0541  which  is  devoted to the development of tick immunological control. Permission from SEPPIC has been obtained to use and mention their products in this manuscript.