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Immunization of cattle with synthetic peptides derived from the Boophilus microplus gut protein (Bm86)
Veterinary Immunology and Immunopathology 88 (2002) 163–172
Immunization of cattle with synthetic peptides derived from the Boophilus microplus gut protein (Bm86) J.H. Patarroyoa,*, R.W. Portelaa, R.O. De Castroa, J. Couto Pimentela, F. Guzmanb, M.E. Patarroyoc, M.I. Vargasa, A.A. Pratesa, M.A. Dias Mendesa a
Laboratory of Biology and Control of Haematozoa, BIOAGRO/Veterinary Department, Federal University of Vic¸osa, 36571-000 Vic¸osa, MG, Brazil b Fundacio´n Instituto de Inmunologia de Colombia (FIDIC), carrera 50, No. 26-00 Santa Fe´ de Bogota´, Colombia c Facultad de Medicina, Universidad Nacional de Colombia, Ciudad Universita´ria, Santa Fe´ de Bogota´, Colombia Received 2 January 2002; received in revised form 1 May 2002; accepted 3 May 2002
Abstract Three synthetic peptides (SBm4912, SBm7462 and SBm19733), derived from the Bm86 glycoprotein from Boophilus microplus gut, were constructed and used to immunize cattle from a tick-free area. The immunized animals received three subcutaneous doses of the peptides, with saponin as adjuvant, at 30-day intervals. The immune response was evaluated by IgG elicited against the peptides by the detection of anti-Bm86 specific antibodies in situ and by Western blotting analysis. After tick challenge, reduction in the number, weight and oviposition capacity of engorged females was observed in the tick population that had fed on immunized animals. The results pointed a high efficacy (81.05%) for the SBm7462 synthetic peptide in relation to the others ðp < 0:01Þ, demonstrating the efficiency of the immune response elicited by synthetic peptides to control the cattle tick B. microplus. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Tick; Boophilus microplus; Cattle; Immunization; Synthetic peptides
1. Introduction The cattle tick Boophilus microplus is one of the most important arthropods in veterinary medicine due to the economic losses and the health problems caused in cattle production in Central and South America and Australia. This tick species causes both direct effects, such as blood sucking, and indirect effects, such as transmission of a wide variety of pathogens, which usually result in lethal infections. *
Corresponding author. Tel.: þ55-31-38992910; fax: þ55-31-38992864. E-mail address: [email protected] (J.H. Patarroyo).
Global economic losses caused by B. microplus ticks have been estimated in US$ 7 per animal per year (Mc Cosker, 1979). Brazil has the fifth largest cattle herd in the world and economic losses around US$ 800 million have been estimated due to direct and indirect effects from B. microplus infestations (Horn and Arteche, 1985). The methods used to control B. microplus ticks are chemical and/or biological. Chemical methods are the most commonly used; however, widespread acaricide resistance to the major classes of chemicals has been reported in many parts of the world (Patarroyo and Costa, 1980; Nolan et al., 1989), in addition to contamination of ecosystems and food by chemical residues. Research on immunological
0165-2427/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 2 ) 0 0 1 5 4 - X
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methods to control B. microplus have been conducted worldwide and constitute an important biological alternative to avoid the disadvantages of chemical products. Usually, the immune response of cattle to natural tick infestations is weak, probably due to competition among antigens. Some antigens, such as saliva constituents, may be irrelevant for induction of resistance but compete with protective antigens. Inoculation of purified antigens associated with an appropriate adjuvant may elicit satisfactory immune responses (Barriga, 1994). Cattle protective immunity against B. microplus has been induced after inoculation of antigens obtained from the midgut of partially engorged females (Johnston et al., 1986; Opdebeeck et al., 1988). Different protocols have been used to isolate and purify these protective antigens. Australian researchers isolated an 89 kDa glycoprotein, named Bm86, some years ago (Willadsen et al., 1989), expressed on midgut cells of B. microplus (Gough and Kemp, 1993). This protein was cloned, expressed in Escherichia coli and used to vaccinate cattle resulting in 77% protection (Rand et al., 1989). This molecule provided 88% protection when expressed in insect cell lines infected with baculovirus (Richardson et al., 1993), but distinct levels of efficiency were obtained when expressed in yeast (Rodriguez et al., 1994; De La Fuente et al., 1995). Furthermore, the use of chemicals in association to immunizations with Bm86 is essential to achieve an efficient control of ticks. Therefore there is still a need to search for other protective antigens, which are able to control populations of B. microplus. The advantages of a synthetic peptide vaccine include: high degree of purity, complete chemical characterization, lack of contaminants, large scale production, easy storage due to high stability as a result of absence of proteolytic enzymes, and low cost of production on industrial scales (Neurath and Kent, 1986). The first tests of synthetic peptides derived from Bm86 were carried out with the purpose of defining epitopes that could elicit protective antibodies. However, only one peptide was inoculated in three animals, but in vivo challenges with ticks were not performed (Sharp et al., 1990). The present paper describes that synthetic oligopeptides derived from different regions of the Bm86
glycoprotein are able to induce a protective immune response against B. microplus when inoculated into cattle.
2. Material and methods 2.1. Animals Twenty Jersey-breed Bos Taurus, with ages between 8 and 10 months, with intact spleens, from a tick-free area were used. The animals were individually housed in arthropod-proof isolation pens and were fed on fodder and concentrate (20% protein) receiving water ad libido. The animals were randomly assigned into five groups: immunized groups (groups A, B, C); adjuvant control (group D) and distilled water control (group E). 2.2. Synthetic peptides The inferred sequence of the native Bm86 protein was analyzed by a computer to predict some protein properties, such as antigenic capacity (Hoop and Woods, 1981), a and b potential, beta sheet helix (Chow and Fassman, 1978), and hydrophobic and hydrophilic properties (Kyte and Doolittle, 1982). Three defined sequences were chosen as containing some of the possible immunogenic determinants for Bm86. These sequences were synthesized in the Chemical Synthesis Laboratory of the Immunology Institute of San Juan de Dios Hospital, Bogota´ , Colombia. The synthesis process was carried out according to the Solid Phase Multiple Peptide Synthesis Technique (Merrifield, 1963; Houghten, 1985), using the Good Manufacture Procedure (GMP) system. MBHA resin (0.49 meq/g), t-Boc amino acids, and low–high cleavages were used in the process. The purity of the peptides was evaluated using HPLC in an octadeclysilane (ODS) column and mass spectrometry (MALDITOF). The peptides were named 4822 (a.a. 398–411), 4823 (a.a. 21–35) and 4824 (a.a. 132–145). Each peptide was conjugated to one carrier protein and checked for antigenicity in rabbits. Three different synthetic peptides were constructed: Synthetic B. microplus 4912, 7462 and 19733 (SBm4912, SBm7462 and SBm19733), all with pending patent. The SBm4912 is the continuous synthesis of the three peptides (4822,
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4823 and 4824); the SBm7462 is the continuous synthesis of the three original peptides in a distinct order (4822, 4824, 4823); the SBm19733 is also the continuous synthesis of the three original peptides, but with lysine, glutamic acid and lysine (KEK) between them (4822KEK4823KEK4824). The constructed peptides were quoted with a cysteine added to the N- and the C-terminal to the continuous chain for polymerization. These peptides were submitted to sterility and cytotoxicity tests (Rodriguez et al., 1990). Each constructed peptide was freeze-dried and stored at 20 8C until immediately before its use.
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a 6% methanol solution of hydrogen peroxide (H2O2) for 30 min. The tissues were washed several times in PBS, incubated for 1 h at room temperature with normal rabbit serum diluted at 1:20. Cattle antiserum against the immunogen to be tested, diluted at 1:40, reacted with the sections for 1 h and this was followed by washes. Rabbit IgG anti-bovine IgG horseradish peroxidase-labeled diluted at 1:80 was added. After washes, a chromogen solution (diaminobenzidine (DAB) 20 mg; H2O2 100 ml; H2O dd q.s. 100 ml) was added and reacted for 20 min; this was followed by washes, contrast with Harry’s haematoxylin and examination.
2.3. Cattle immunization and challenge 2.5. ELISA The animals were inoculated subcutaneously in the neck on days 1, 30 and 60. Animals in group A were inoculated with SBm4912, animals in group B with SBm7462 and animals in group C with SBm19733. The formulation used for immunization was composed of 2 mg of synthetic peptide plus 1.5 mg saponin as adjuvant in 4 ml of filtered deionized distilled water (ddw) per dose. Animals in group D received 1.5 mg of adjuvant in 4 ml ddw per dose and animals in group E 4 ml ddw per dose. Twenty-one days after the final inoculation each animal was infested with approximately 1500 larvae (based on egg weight) of B. microplus per day for 3 consecutive days. The strain of B. microplus used in the present experiment (BmUFV1) had been passed through 16 laboratory generations and maintained in a BOD incubator at 28 8C with 80% relative humidity, free from Babesia spp. infections. Collection of naturally detached engorged females was carried out twice a day, always at 9 a.m. and 4 p.m., from all animals throughout the experimental period.
Serum samples were collected weekly for 14 weeks after the first inoculation and tested by ELISA to monitor the antibody response. The plates were coated overnight at 4 8C with synthetic peptides in 0.13 M carbonate buffer, pH 9.6, as follows: 5 mg of synthetic peptide 4824 per well for detection of SBm4912, 2 mg of SBm7462 per well for its own detection, and 0.2 mg of SBm19733 per well for its own detection. Serum samples were diluted from 1:100 up to 1:2400 in incubation buffer containing Tween 20 and tested in triplicate. Bovine anti-peptide antibodies were detected with rabbit IgG anti-bovine IgG (heavy and light specific chains) conjugated with horseradish peroxidase (diluted at 1:4000). A serum sample was considered positive when the reaction showed an optical density (OD) higher than the mean plus two standard deviations of the OD obtained for negative controls. The reading values were adjusted for each plate by check test values (Passos, 1993). 2.6. Western blotting
2.4. Indirect immunoperoxidase of tick gut cells The test was carried out using a modified methodology previously described by Werner et al. (1996). Semi-engorged adult females from nonvaccinated animals were dissected and the midgut removed, fixed, embedded in paraffin wax and sectioned. After removing the paraffin wax, the sections were kept for 30 min in PBS buffer, pH 7.6. The sections were then put in a 0.02% sodium borohydride (NaBH4) solution for 15 min and transferred to
The synthetic peptides, at concentrations of 50 mg, were run on 20% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) following a technique described for separation of proteins ranging from 1 to 100 kDa (Scha¨ gger and Von Jagow, 1987). The bands were transferred to nitrocellulose membranes (pore size 0.45 mm) at 70 V for 1.5 h at 4 8C in 0.13 M carbonate buffer, pH 9.6. The primary antibody was bovine serum (diluted at 1:100) collected 2 weeks after the third inoculation, and bound bovine antibodies were detected
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using rabbit anti-bovine IgG (diluted at 1:1000) conjugated with peroxidase. 2.7. Assessment of tick biological parameters and statistical analyses All naturally detached engorged females were examined for macroscopic lesions, counted and weighed. In order to assess egg laying capacity and fertility, all females were incubated under BOD conditions (28 8C, 80% relative humidity). The effects of immunizations on tick biological parameters were analyzed in a completely randomized design using analysis of variance (ANOVA) and the Tukey multiple comparison test (Gomes, 1990). Significance was indicated by a probability of p < 0:01. The following formulae were used to analyze the results of tick biological parameters (De La Fuente et al., 1995): NTV DT ð%Þ ¼ 100 1 NTC where DT (%) is the percentage reduction of adult females, NTV the number of adult females in the immunized group and NTC the number of adult females in the control group PATV DO ð%Þ ¼ 100 1 PATC where DO (%) is the percentage reduction of mean weight of eggs, PATV the average weight of eggs of the immunized group and PATC the average weight of eggs of the control group PMTV DR ð%Þ ¼ 100 1 PMTC where DR (%) is the percentage reduction of mean weight of adult females, PMTV the mean weight of adult females in the immunized group and PMTC the mean weight of adult females in the control group PPLOV DF ð%Þ ¼ 100 1 PPLOC where DF (%) is the percentage reduction of fertility, PPLOV the mean weight of larvae per gram of eggs in
the immunized group and PPLOC the mean weight of larvae per gram of eggs in the control group E ð%Þ ¼ 100½1 ðCRT CRO CRFÞ where E (%) is the efficacy of immunogens, CRT the reduction in the number of adult females NTV/NTC, CRO the reduction in egg laying capacity PATV/ PATC and CRF the reduction in fertility PPLOV/ PPLOC.
3. Results As shown in Fig. 1, only sera from immunized animals recognized the whole protein in situ. The reaction was confined to the surface of gut digestive cells, with recognition of the protein inside digestive vacuoles. Anti-peptide antibodies in sera from animals in groups A and B showed a strong reaction, while sera from animals in group C showed a weak reaction. The dynamics of specific anti-synthetic peptides IgG observed in immunized and control animals is shown in Fig. 2. Specific antibodies in sera from immunized animals increased 2 weeks after the first inoculation, while sera from control animals remained negative. Antibody levels increased 1 week after the second inoculation and the greatest values were observed 15 days later in all immunized animals. After the third dose of peptides, antibody levels increased significantly with the highest peak occurring again 15 days after the second inoculation, with titres between 400 and 2400 (data not shown). No significant differences ðp < 0:01Þ were seen, throughout the experimental period, between groups D and E. None of the pre-immunization sera had significant antibody titres against the synthetic peptides. In Western blotting, serum from immunized animals recognized a single band with molecular weight corresponding to each immunogen (Fig. 3). No reaction was observed with sera from control animals. The results indicate effectiveness of immunization with synthetic peptides in the control of B. microplus. Regarding biological parameters of ticks, the number (DT, %) and weight (DR, %) of engorged females, weight of eggs (DO, %), and fertility (DF, %) were significantly lower ð p < 0:01Þ for ticks
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Fig. 1. Microphotography of indirect immunoperoxidase staining of the Bm86 protein on B. microplus gut cells (600). In (A) and (B) cells were probed with serum samples collected from control animals (distilled water and adjuvant, respectively). In (C) cells were probed with sera collected from cattle immunized with peptide SBm4912, in (D) with sera from cattle immunized with peptide SBm7462 (note the strong reaction and the enlargement of digestive cells), and in (E) with serum from cattle immunized with peptide SBm19733. Arrows indicate the digestive cells. DV: digestive vacuoles; N: nuclei; E: erythrocytes. Scale bar, 100 mm.
detached from the immunized animals than for ticks detached from the control animals. When all three immunogens (SBm4912, SBm7462 and SBm19733) were compared to groups D and E (Table 1), peptide
SBm7462 showed the best efficacy (E, %). Some ticks that were feeding on animals immunized with SBm7462 were visibly damaged, showing a dark-red color.
Table 1 Reduction (%) of biological parameters of detached B. microplus ticks from cattle immunized with synthetic peptides in relation to ticks detached from animals in control groups: ddw and adjuvanta Biological parameterb
Immunogen SBm4912 ddw
DT DO DR DF Efficacy (%)c a
45.39 29.78 8.32 28.04 72.40
c c c c c
SBm7462 Adjuvant
ddw
28.11 33.58 10.74 25.48 64.42
60.92 27.53 5.44 33.12 81.05
d d c c d
e e e c e
SBm19733 Adjuvant
ddw
48.56 31.04 7.98 30.80 75.58
14.32 14.78 0.00 12.18 35.87
f f e c f
Adjuvant g g d g
12.78 19.31 0.00 9.13 22.57
g h e h
Values within a line with different letters differ significantly ðp < 0:01Þ. DT: engorged females; DO: egg laying capacity; DR: weight of engorged females; DF: fertility. c Efficacy ð%Þ ¼ 100½1ðCRT CRO CRFÞ. CRT: reduction of engorged females; CRO: reduction in the egg laying capacity; CRF: reduction in fertility. b
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Fig. 2. Kinetics of antibodies detected by indirect ELISA in serum samples from cattle immunized with synthetic peptide SBm4912 (A), SBm7462 (B), and SBm19733 (C). Arrows indicate weeks after immunization and the star indicates the tick challenge.
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Fig. 3. Western blotting analysis of synthetic peptides derived from the gut protein Bm86 (SDS-PAGE, 20%). Lane 2 was probed with sera from group D (distilled water control); lane 3 was probed with sera from group E (adjuvant control); lanes 4 and 5 were probed with sera from group C (SBm19733); lanes 6 and 7 were probed with sera from group B (SBm7462); lanes 8 and 9 were probed with sera from group A (SBm4912).
4. Discussion As shown in Fig. 1, antibodies against peptides SBm7462 and SBm4912 showed a strong reaction on the surface of gut epithelial cells of ticks, with intense reactivity inside the digestive vacuoles (Fig. 1D). This strong reaction may be due to the fact that the serum sample used in this reaction had been collected 2 weeks after the third inoculation, when antibody levels were high, or due to a better affinity of SBm7462 and SBm4912 antibodies to the whole protein than the antibodies elicited by SBm19733. Previous studies using bovine anti-synthetic peptide antibodies for in situ recognition of tick gut proteins were made with construction corresponding to the Bm86 ‘‘B-loop’’. A weak reaction was detected by immunofluorescence (Sharp et al., 1990), possibly due to distinct aspects of antigenicity involved in the construction of synthetic peptides. The reaction inside the digestive vacuoles described in the present work probably corresponds to the Bm86 protein. In fact,
according to Willadsen (1997), the Bm86 protein could be involved in a digestive process such as endocytosis. The results confirmed that the construction and the structure of the peptides used in the present study stimulated the immune system of inoculated animals, allowing recognition of the whole protein in situ by the elicited antibodies. This is not a unique case in the literature. IgG anti-synthetic peptide (SPf 66), used as a vaccine against Plasmodium falciparum, also recognized the proteins from which the amino acid sequence had been originated (Salcedo et al., 1991). The ELISA plates used for detection of antibodies against SBm4912 were coated with synthetic peptide 4824 based on B epitope mapping. The 4824 antigen discriminated more efficiently positive and negative sera and it was more efficient in recognizing sera from cattle inoculated with the SBm4912 peptide than the other fractions. This may be explained by the spatial conformation of the hybridized peptide. Algorithms were used to predict the hydrophobicity of the synthetic
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immunogen, and the fraction corresponding to 4824 was highly hydrophilic, which nowadays is considered a representation of a more exposed region of the protein, possibly acting as an antibody inducer. The anti-peptide antibodies have clearly shown the boosting effect after the third inoculation of animals in groups A and B. Animals in group C not showed a similar kinetics of antibodies, possibly due to the structure of peptide SBm19733. Specific antibodies against the immunogens were not detected in preimmunization sera. Levels of specific IgG in serum samples from immunized animals showed decay with the time, common to all the IgG immune responses regardless the antigen used (Fig. 2). The Western blotting analysis reveals that the synthetic peptides used in the present study elicited specific IgG in immunized animals, while sera from animals in groups D and E did not recognize the peptides. Immunization of animals with synthetic peptides SBm4912 and SBm7462 resulted in greater reduction of the reproductive potential of ticks, represented by the number and weight of adult females, and their oviposition capacity than the SBm19733 antigen. Furthermore, the percentages of efficacy of peptides were statistically different, with the SBm7462 antigen showing the highest efficacy (Table 1). The reduction of the different parameters calculated with respect to saponin group (group D) has shown a minor efficacy, statistically different to the results obtained when compared with the group not immunized (Table 1). Other researchers have previously described the use of saponin as an adjuvant for immunization of cattle against ticks, but its interaction with the bovine immune system has not yet been completely explained (Jackson and Opdebeeck, 1994). Our results suggest that saponin may play a role as a non-specific immune stimulator against ticks, and is very interesting to note that saponin did not elicit specific antibodies against the immunogens used (Figs. 1–3). The level of antibodies against the SBm19733 antigen was higher than those elicited by the other peptides. In contrast, its efficiency was lower than those obtained with SBm4912 or SBm7462 antigens (Fig. 2; Table 1). This may indicate that the construction of the peptide with KEK between the original sequences may have caused a structural alteration that interfered with the epitope presentation to the immune
system, perhaps eliciting mainly a humoral response. In accordance to results reported by Tellam et al. (1992), our results suggest that not only specific antibodies produced against one synthetic peptide, but also other components of the host immune system play an important role in the development of protection of cattle against B. microplus ticks. A comparison between the results of the present study and previous pen trials carried out in Brazil with rBm86 originated from Cuba (Massard et al., 1995) showed that animals immunized with SBm4912 and SBm7462 had better performances, as efficacy of rBM86 was 51%, while efficacy of SBm4912 and SBm7462 were 72.4% and 81.05%, respectively. The same formulas were used to evaluate tick biological parameters in both studies. Although controlled pen trials with a small number of animals present some limitations, regarding the analysis of results, those are indicative of the efficient control that could be obtained under field conditions. The data obtained with synthetic peptides SBm4912 and Sbm7462 suggest that the sequences used in their construction contained antigenic and immunogenic epitopes from the Bm86 protein and that this could be sufficient for eliciting an immunological response to control B. microplus ticks. It is not expected that a vaccine against ticks would be able to suppress completely the tick population in a single generation, but rather to progressively control successive generations. The most relevant effect of a vaccine against B. microplus would be the reduction of reproductive performance of ticks rather than the direct effect on the number of individuals. Under this prospective, the aim of a vaccination program is to reduce pasture contamination and the number of parasites in future generations (Cobon et al., 1995). In addition, it should be emphasized that the immunity produced by using a tick vaccine results in herd immunity, and not only individual bovine immunity. Finally, synthetic vaccines against B. microplus may replace the existing vaccines because, among other advantages, they are cheaper, do not need culture fermentation and are not contaminated with biological sub-products. More information is required regarding protection of different cattle breeds, different geographic populations of ticks, and distinct strains, particularly those resistant to acaricides. There is also
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a need to perform experiments under field conditions and optimize methods of immunogen presentation and formulation, to achieve consistent protection. The data obtained with the use of SBm4912 and SBm7462 reinforces the use of chemically defined immunogens as an effective approach to control the cattle tick B. microplus.
Acknowledgements The authors thank FAPEMIG (Minas Gerais State Research Foundation Brazil) for providing financial support for the project and CNPq (Brazilian National Research Council) for different scholarships.
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Immunization of cattle with synthetic peptides derived from the Boophilus microplus gut protein (Bm86) J.H. Patarroyoa,*, R.W. Portelaa, R.O. De Castroa, J. Couto Pimentela, F. Guzmanb, M.E. Patarroyoc, M.I. Vargasa, A.A. Pratesa, M.A. Dias Mendesa a
Laboratory of Biology and Control of Haematozoa, BIOAGRO/Veterinary Department, Federal University of Vic¸osa, 36571-000 Vic¸osa, MG, Brazil b Fundacio´n Instituto de Inmunologia de Colombia (FIDIC), carrera 50, No. 26-00 Santa Fe´ de Bogota´, Colombia c Facultad de Medicina, Universidad Nacional de Colombia, Ciudad Universita´ria, Santa Fe´ de Bogota´, Colombia Received 2 January 2002; received in revised form 1 May 2002; accepted 3 May 2002
Abstract Three synthetic peptides (SBm4912, SBm7462 and SBm19733), derived from the Bm86 glycoprotein from Boophilus microplus gut, were constructed and used to immunize cattle from a tick-free area. The immunized animals received three subcutaneous doses of the peptides, with saponin as adjuvant, at 30-day intervals. The immune response was evaluated by IgG elicited against the peptides by the detection of anti-Bm86 specific antibodies in situ and by Western blotting analysis. After tick challenge, reduction in the number, weight and oviposition capacity of engorged females was observed in the tick population that had fed on immunized animals. The results pointed a high efficacy (81.05%) for the SBm7462 synthetic peptide in relation to the others ðp < 0:01Þ, demonstrating the efficiency of the immune response elicited by synthetic peptides to control the cattle tick B. microplus. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Tick; Boophilus microplus; Cattle; Immunization; Synthetic peptides
1. Introduction The cattle tick Boophilus microplus is one of the most important arthropods in veterinary medicine due to the economic losses and the health problems caused in cattle production in Central and South America and Australia. This tick species causes both direct effects, such as blood sucking, and indirect effects, such as transmission of a wide variety of pathogens, which usually result in lethal infections. *
Corresponding author. Tel.: þ55-31-38992910; fax: þ55-31-38992864. E-mail address: [email protected] (J.H. Patarroyo).
Global economic losses caused by B. microplus ticks have been estimated in US$ 7 per animal per year (Mc Cosker, 1979). Brazil has the fifth largest cattle herd in the world and economic losses around US$ 800 million have been estimated due to direct and indirect effects from B. microplus infestations (Horn and Arteche, 1985). The methods used to control B. microplus ticks are chemical and/or biological. Chemical methods are the most commonly used; however, widespread acaricide resistance to the major classes of chemicals has been reported in many parts of the world (Patarroyo and Costa, 1980; Nolan et al., 1989), in addition to contamination of ecosystems and food by chemical residues. Research on immunological
0165-2427/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 2 ) 0 0 1 5 4 - X
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methods to control B. microplus have been conducted worldwide and constitute an important biological alternative to avoid the disadvantages of chemical products. Usually, the immune response of cattle to natural tick infestations is weak, probably due to competition among antigens. Some antigens, such as saliva constituents, may be irrelevant for induction of resistance but compete with protective antigens. Inoculation of purified antigens associated with an appropriate adjuvant may elicit satisfactory immune responses (Barriga, 1994). Cattle protective immunity against B. microplus has been induced after inoculation of antigens obtained from the midgut of partially engorged females (Johnston et al., 1986; Opdebeeck et al., 1988). Different protocols have been used to isolate and purify these protective antigens. Australian researchers isolated an 89 kDa glycoprotein, named Bm86, some years ago (Willadsen et al., 1989), expressed on midgut cells of B. microplus (Gough and Kemp, 1993). This protein was cloned, expressed in Escherichia coli and used to vaccinate cattle resulting in 77% protection (Rand et al., 1989). This molecule provided 88% protection when expressed in insect cell lines infected with baculovirus (Richardson et al., 1993), but distinct levels of efficiency were obtained when expressed in yeast (Rodriguez et al., 1994; De La Fuente et al., 1995). Furthermore, the use of chemicals in association to immunizations with Bm86 is essential to achieve an efficient control of ticks. Therefore there is still a need to search for other protective antigens, which are able to control populations of B. microplus. The advantages of a synthetic peptide vaccine include: high degree of purity, complete chemical characterization, lack of contaminants, large scale production, easy storage due to high stability as a result of absence of proteolytic enzymes, and low cost of production on industrial scales (Neurath and Kent, 1986). The first tests of synthetic peptides derived from Bm86 were carried out with the purpose of defining epitopes that could elicit protective antibodies. However, only one peptide was inoculated in three animals, but in vivo challenges with ticks were not performed (Sharp et al., 1990). The present paper describes that synthetic oligopeptides derived from different regions of the Bm86
glycoprotein are able to induce a protective immune response against B. microplus when inoculated into cattle.
2. Material and methods 2.1. Animals Twenty Jersey-breed Bos Taurus, with ages between 8 and 10 months, with intact spleens, from a tick-free area were used. The animals were individually housed in arthropod-proof isolation pens and were fed on fodder and concentrate (20% protein) receiving water ad libido. The animals were randomly assigned into five groups: immunized groups (groups A, B, C); adjuvant control (group D) and distilled water control (group E). 2.2. Synthetic peptides The inferred sequence of the native Bm86 protein was analyzed by a computer to predict some protein properties, such as antigenic capacity (Hoop and Woods, 1981), a and b potential, beta sheet helix (Chow and Fassman, 1978), and hydrophobic and hydrophilic properties (Kyte and Doolittle, 1982). Three defined sequences were chosen as containing some of the possible immunogenic determinants for Bm86. These sequences were synthesized in the Chemical Synthesis Laboratory of the Immunology Institute of San Juan de Dios Hospital, Bogota´ , Colombia. The synthesis process was carried out according to the Solid Phase Multiple Peptide Synthesis Technique (Merrifield, 1963; Houghten, 1985), using the Good Manufacture Procedure (GMP) system. MBHA resin (0.49 meq/g), t-Boc amino acids, and low–high cleavages were used in the process. The purity of the peptides was evaluated using HPLC in an octadeclysilane (ODS) column and mass spectrometry (MALDITOF). The peptides were named 4822 (a.a. 398–411), 4823 (a.a. 21–35) and 4824 (a.a. 132–145). Each peptide was conjugated to one carrier protein and checked for antigenicity in rabbits. Three different synthetic peptides were constructed: Synthetic B. microplus 4912, 7462 and 19733 (SBm4912, SBm7462 and SBm19733), all with pending patent. The SBm4912 is the continuous synthesis of the three peptides (4822,
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4823 and 4824); the SBm7462 is the continuous synthesis of the three original peptides in a distinct order (4822, 4824, 4823); the SBm19733 is also the continuous synthesis of the three original peptides, but with lysine, glutamic acid and lysine (KEK) between them (4822KEK4823KEK4824). The constructed peptides were quoted with a cysteine added to the N- and the C-terminal to the continuous chain for polymerization. These peptides were submitted to sterility and cytotoxicity tests (Rodriguez et al., 1990). Each constructed peptide was freeze-dried and stored at 20 8C until immediately before its use.
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a 6% methanol solution of hydrogen peroxide (H2O2) for 30 min. The tissues were washed several times in PBS, incubated for 1 h at room temperature with normal rabbit serum diluted at 1:20. Cattle antiserum against the immunogen to be tested, diluted at 1:40, reacted with the sections for 1 h and this was followed by washes. Rabbit IgG anti-bovine IgG horseradish peroxidase-labeled diluted at 1:80 was added. After washes, a chromogen solution (diaminobenzidine (DAB) 20 mg; H2O2 100 ml; H2O dd q.s. 100 ml) was added and reacted for 20 min; this was followed by washes, contrast with Harry’s haematoxylin and examination.
2.3. Cattle immunization and challenge 2.5. ELISA The animals were inoculated subcutaneously in the neck on days 1, 30 and 60. Animals in group A were inoculated with SBm4912, animals in group B with SBm7462 and animals in group C with SBm19733. The formulation used for immunization was composed of 2 mg of synthetic peptide plus 1.5 mg saponin as adjuvant in 4 ml of filtered deionized distilled water (ddw) per dose. Animals in group D received 1.5 mg of adjuvant in 4 ml ddw per dose and animals in group E 4 ml ddw per dose. Twenty-one days after the final inoculation each animal was infested with approximately 1500 larvae (based on egg weight) of B. microplus per day for 3 consecutive days. The strain of B. microplus used in the present experiment (BmUFV1) had been passed through 16 laboratory generations and maintained in a BOD incubator at 28 8C with 80% relative humidity, free from Babesia spp. infections. Collection of naturally detached engorged females was carried out twice a day, always at 9 a.m. and 4 p.m., from all animals throughout the experimental period.
Serum samples were collected weekly for 14 weeks after the first inoculation and tested by ELISA to monitor the antibody response. The plates were coated overnight at 4 8C with synthetic peptides in 0.13 M carbonate buffer, pH 9.6, as follows: 5 mg of synthetic peptide 4824 per well for detection of SBm4912, 2 mg of SBm7462 per well for its own detection, and 0.2 mg of SBm19733 per well for its own detection. Serum samples were diluted from 1:100 up to 1:2400 in incubation buffer containing Tween 20 and tested in triplicate. Bovine anti-peptide antibodies were detected with rabbit IgG anti-bovine IgG (heavy and light specific chains) conjugated with horseradish peroxidase (diluted at 1:4000). A serum sample was considered positive when the reaction showed an optical density (OD) higher than the mean plus two standard deviations of the OD obtained for negative controls. The reading values were adjusted for each plate by check test values (Passos, 1993). 2.6. Western blotting
2.4. Indirect immunoperoxidase of tick gut cells The test was carried out using a modified methodology previously described by Werner et al. (1996). Semi-engorged adult females from nonvaccinated animals were dissected and the midgut removed, fixed, embedded in paraffin wax and sectioned. After removing the paraffin wax, the sections were kept for 30 min in PBS buffer, pH 7.6. The sections were then put in a 0.02% sodium borohydride (NaBH4) solution for 15 min and transferred to
The synthetic peptides, at concentrations of 50 mg, were run on 20% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) following a technique described for separation of proteins ranging from 1 to 100 kDa (Scha¨ gger and Von Jagow, 1987). The bands were transferred to nitrocellulose membranes (pore size 0.45 mm) at 70 V for 1.5 h at 4 8C in 0.13 M carbonate buffer, pH 9.6. The primary antibody was bovine serum (diluted at 1:100) collected 2 weeks after the third inoculation, and bound bovine antibodies were detected
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using rabbit anti-bovine IgG (diluted at 1:1000) conjugated with peroxidase. 2.7. Assessment of tick biological parameters and statistical analyses All naturally detached engorged females were examined for macroscopic lesions, counted and weighed. In order to assess egg laying capacity and fertility, all females were incubated under BOD conditions (28 8C, 80% relative humidity). The effects of immunizations on tick biological parameters were analyzed in a completely randomized design using analysis of variance (ANOVA) and the Tukey multiple comparison test (Gomes, 1990). Significance was indicated by a probability of p < 0:01. The following formulae were used to analyze the results of tick biological parameters (De La Fuente et al., 1995): NTV DT ð%Þ ¼ 100 1 NTC where DT (%) is the percentage reduction of adult females, NTV the number of adult females in the immunized group and NTC the number of adult females in the control group PATV DO ð%Þ ¼ 100 1 PATC where DO (%) is the percentage reduction of mean weight of eggs, PATV the average weight of eggs of the immunized group and PATC the average weight of eggs of the control group PMTV DR ð%Þ ¼ 100 1 PMTC where DR (%) is the percentage reduction of mean weight of adult females, PMTV the mean weight of adult females in the immunized group and PMTC the mean weight of adult females in the control group PPLOV DF ð%Þ ¼ 100 1 PPLOC where DF (%) is the percentage reduction of fertility, PPLOV the mean weight of larvae per gram of eggs in
the immunized group and PPLOC the mean weight of larvae per gram of eggs in the control group E ð%Þ ¼ 100½1 ðCRT CRO CRFÞ where E (%) is the efficacy of immunogens, CRT the reduction in the number of adult females NTV/NTC, CRO the reduction in egg laying capacity PATV/ PATC and CRF the reduction in fertility PPLOV/ PPLOC.
3. Results As shown in Fig. 1, only sera from immunized animals recognized the whole protein in situ. The reaction was confined to the surface of gut digestive cells, with recognition of the protein inside digestive vacuoles. Anti-peptide antibodies in sera from animals in groups A and B showed a strong reaction, while sera from animals in group C showed a weak reaction. The dynamics of specific anti-synthetic peptides IgG observed in immunized and control animals is shown in Fig. 2. Specific antibodies in sera from immunized animals increased 2 weeks after the first inoculation, while sera from control animals remained negative. Antibody levels increased 1 week after the second inoculation and the greatest values were observed 15 days later in all immunized animals. After the third dose of peptides, antibody levels increased significantly with the highest peak occurring again 15 days after the second inoculation, with titres between 400 and 2400 (data not shown). No significant differences ðp < 0:01Þ were seen, throughout the experimental period, between groups D and E. None of the pre-immunization sera had significant antibody titres against the synthetic peptides. In Western blotting, serum from immunized animals recognized a single band with molecular weight corresponding to each immunogen (Fig. 3). No reaction was observed with sera from control animals. The results indicate effectiveness of immunization with synthetic peptides in the control of B. microplus. Regarding biological parameters of ticks, the number (DT, %) and weight (DR, %) of engorged females, weight of eggs (DO, %), and fertility (DF, %) were significantly lower ð p < 0:01Þ for ticks
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Fig. 1. Microphotography of indirect immunoperoxidase staining of the Bm86 protein on B. microplus gut cells (600). In (A) and (B) cells were probed with serum samples collected from control animals (distilled water and adjuvant, respectively). In (C) cells were probed with sera collected from cattle immunized with peptide SBm4912, in (D) with sera from cattle immunized with peptide SBm7462 (note the strong reaction and the enlargement of digestive cells), and in (E) with serum from cattle immunized with peptide SBm19733. Arrows indicate the digestive cells. DV: digestive vacuoles; N: nuclei; E: erythrocytes. Scale bar, 100 mm.
detached from the immunized animals than for ticks detached from the control animals. When all three immunogens (SBm4912, SBm7462 and SBm19733) were compared to groups D and E (Table 1), peptide
SBm7462 showed the best efficacy (E, %). Some ticks that were feeding on animals immunized with SBm7462 were visibly damaged, showing a dark-red color.
Table 1 Reduction (%) of biological parameters of detached B. microplus ticks from cattle immunized with synthetic peptides in relation to ticks detached from animals in control groups: ddw and adjuvanta Biological parameterb
Immunogen SBm4912 ddw
DT DO DR DF Efficacy (%)c a
45.39 29.78 8.32 28.04 72.40
c c c c c
SBm7462 Adjuvant
ddw
28.11 33.58 10.74 25.48 64.42
60.92 27.53 5.44 33.12 81.05
d d c c d
e e e c e
SBm19733 Adjuvant
ddw
48.56 31.04 7.98 30.80 75.58
14.32 14.78 0.00 12.18 35.87
f f e c f
Adjuvant g g d g
12.78 19.31 0.00 9.13 22.57
g h e h
Values within a line with different letters differ significantly ðp < 0:01Þ. DT: engorged females; DO: egg laying capacity; DR: weight of engorged females; DF: fertility. c Efficacy ð%Þ ¼ 100½1ðCRT CRO CRFÞ. CRT: reduction of engorged females; CRO: reduction in the egg laying capacity; CRF: reduction in fertility. b
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Fig. 2. Kinetics of antibodies detected by indirect ELISA in serum samples from cattle immunized with synthetic peptide SBm4912 (A), SBm7462 (B), and SBm19733 (C). Arrows indicate weeks after immunization and the star indicates the tick challenge.
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Fig. 3. Western blotting analysis of synthetic peptides derived from the gut protein Bm86 (SDS-PAGE, 20%). Lane 2 was probed with sera from group D (distilled water control); lane 3 was probed with sera from group E (adjuvant control); lanes 4 and 5 were probed with sera from group C (SBm19733); lanes 6 and 7 were probed with sera from group B (SBm7462); lanes 8 and 9 were probed with sera from group A (SBm4912).
4. Discussion As shown in Fig. 1, antibodies against peptides SBm7462 and SBm4912 showed a strong reaction on the surface of gut epithelial cells of ticks, with intense reactivity inside the digestive vacuoles (Fig. 1D). This strong reaction may be due to the fact that the serum sample used in this reaction had been collected 2 weeks after the third inoculation, when antibody levels were high, or due to a better affinity of SBm7462 and SBm4912 antibodies to the whole protein than the antibodies elicited by SBm19733. Previous studies using bovine anti-synthetic peptide antibodies for in situ recognition of tick gut proteins were made with construction corresponding to the Bm86 ‘‘B-loop’’. A weak reaction was detected by immunofluorescence (Sharp et al., 1990), possibly due to distinct aspects of antigenicity involved in the construction of synthetic peptides. The reaction inside the digestive vacuoles described in the present work probably corresponds to the Bm86 protein. In fact,
according to Willadsen (1997), the Bm86 protein could be involved in a digestive process such as endocytosis. The results confirmed that the construction and the structure of the peptides used in the present study stimulated the immune system of inoculated animals, allowing recognition of the whole protein in situ by the elicited antibodies. This is not a unique case in the literature. IgG anti-synthetic peptide (SPf 66), used as a vaccine against Plasmodium falciparum, also recognized the proteins from which the amino acid sequence had been originated (Salcedo et al., 1991). The ELISA plates used for detection of antibodies against SBm4912 were coated with synthetic peptide 4824 based on B epitope mapping. The 4824 antigen discriminated more efficiently positive and negative sera and it was more efficient in recognizing sera from cattle inoculated with the SBm4912 peptide than the other fractions. This may be explained by the spatial conformation of the hybridized peptide. Algorithms were used to predict the hydrophobicity of the synthetic
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immunogen, and the fraction corresponding to 4824 was highly hydrophilic, which nowadays is considered a representation of a more exposed region of the protein, possibly acting as an antibody inducer. The anti-peptide antibodies have clearly shown the boosting effect after the third inoculation of animals in groups A and B. Animals in group C not showed a similar kinetics of antibodies, possibly due to the structure of peptide SBm19733. Specific antibodies against the immunogens were not detected in preimmunization sera. Levels of specific IgG in serum samples from immunized animals showed decay with the time, common to all the IgG immune responses regardless the antigen used (Fig. 2). The Western blotting analysis reveals that the synthetic peptides used in the present study elicited specific IgG in immunized animals, while sera from animals in groups D and E did not recognize the peptides. Immunization of animals with synthetic peptides SBm4912 and SBm7462 resulted in greater reduction of the reproductive potential of ticks, represented by the number and weight of adult females, and their oviposition capacity than the SBm19733 antigen. Furthermore, the percentages of efficacy of peptides were statistically different, with the SBm7462 antigen showing the highest efficacy (Table 1). The reduction of the different parameters calculated with respect to saponin group (group D) has shown a minor efficacy, statistically different to the results obtained when compared with the group not immunized (Table 1). Other researchers have previously described the use of saponin as an adjuvant for immunization of cattle against ticks, but its interaction with the bovine immune system has not yet been completely explained (Jackson and Opdebeeck, 1994). Our results suggest that saponin may play a role as a non-specific immune stimulator against ticks, and is very interesting to note that saponin did not elicit specific antibodies against the immunogens used (Figs. 1–3). The level of antibodies against the SBm19733 antigen was higher than those elicited by the other peptides. In contrast, its efficiency was lower than those obtained with SBm4912 or SBm7462 antigens (Fig. 2; Table 1). This may indicate that the construction of the peptide with KEK between the original sequences may have caused a structural alteration that interfered with the epitope presentation to the immune
system, perhaps eliciting mainly a humoral response. In accordance to results reported by Tellam et al. (1992), our results suggest that not only specific antibodies produced against one synthetic peptide, but also other components of the host immune system play an important role in the development of protection of cattle against B. microplus ticks. A comparison between the results of the present study and previous pen trials carried out in Brazil with rBm86 originated from Cuba (Massard et al., 1995) showed that animals immunized with SBm4912 and SBm7462 had better performances, as efficacy of rBM86 was 51%, while efficacy of SBm4912 and SBm7462 were 72.4% and 81.05%, respectively. The same formulas were used to evaluate tick biological parameters in both studies. Although controlled pen trials with a small number of animals present some limitations, regarding the analysis of results, those are indicative of the efficient control that could be obtained under field conditions. The data obtained with synthetic peptides SBm4912 and Sbm7462 suggest that the sequences used in their construction contained antigenic and immunogenic epitopes from the Bm86 protein and that this could be sufficient for eliciting an immunological response to control B. microplus ticks. It is not expected that a vaccine against ticks would be able to suppress completely the tick population in a single generation, but rather to progressively control successive generations. The most relevant effect of a vaccine against B. microplus would be the reduction of reproductive performance of ticks rather than the direct effect on the number of individuals. Under this prospective, the aim of a vaccination program is to reduce pasture contamination and the number of parasites in future generations (Cobon et al., 1995). In addition, it should be emphasized that the immunity produced by using a tick vaccine results in herd immunity, and not only individual bovine immunity. Finally, synthetic vaccines against B. microplus may replace the existing vaccines because, among other advantages, they are cheaper, do not need culture fermentation and are not contaminated with biological sub-products. More information is required regarding protection of different cattle breeds, different geographic populations of ticks, and distinct strains, particularly those resistant to acaricides. There is also
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a need to perform experiments under field conditions and optimize methods of immunogen presentation and formulation, to achieve consistent protection. The data obtained with the use of SBm4912 and SBm7462 reinforces the use of chemically defined immunogens as an effective approach to control the cattle tick B. microplus.
Acknowledgements The authors thank FAPEMIG (Minas Gerais State Research Foundation Brazil) for providing financial support for the project and CNPq (Brazilian National Research Council) for different scholarships.
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