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RESEARCH NOTE Phase I and II Open Clinical Trials of a Vaccine Against Leishmania chagasi Infections in Dogs Wilson Mayrink, Odair Genaro/+, Joao Carlos Franca Silva, Roberto Teodoro da Costa, Wagner Luis Tafuri*, Vicente Paulo C Peixoto Toledo**, Alexandre Rotondo da Silva***, Alexandre Barbosa Reis***/++, Paul Williams, Carlos Alberto da Costa** Departamento de Parasitologia *Departamento de Patologia Geral, Instituto de Ciencias Biologicas **Departamento de Analises Clinicas e Toxologicas, Faculdade de Farmacia, Universidade Federal de Minas Gerais, Av. Antonio Carlos 6627, Caixa Postal 486, 31270-901 Belo Horizonte, MG, Brasil ***Departamento de Ciencias Biologicas, Universidade Federal de Ouro Preto, Ouro Preto, MG, Brasil
This work received financial support from Fundacao Banco do
Brasil, FAPEMIG and BIOBRAS. Key words: canine visceral leishmaniasis - Leishmania chagasi - vaccine - dogs
Visceral leishmaniasis occurs in tropical and subtropical parts of the world and is most commonly found in rural areas. In the Americas, more than 90% of the cases have been recorded in Brazil. The disease can be controlled by treatment of all human cases, elimination of infected dogs and application of insecticide to the walls of dwellings and peridomestic buildings (PA Magalhaes et al. 1980 Rev Inst Med Trop Sao Paulo 22: 197-202). After applying these measures, constant vigilance must be exercised. Control measures must be applied again as soon as there is evidence of the reactivation of the transmission cycle. As alternative control measures some authors have emphasized the importance of immunopro-phylaxis for canine visceral leishmaniasis (CVL) (MCA Marzochi et al. 1985 Mem Inst Oswaldo Cruz 80: 349-357, L Monjour et al. 1985 CR Acad Sc Paris 301: 803-806). Observations in Europe, however, have produced contradictory results. The vaccine used by Monjour (loc. cit.) and D Frommel et al. (1988 Infect Immun 56: 843) protected mice against Leishmania mexicana and L. major, and was found to stimulate the production of neutralizing antibodies when given to dogs (S Dunan et al. 1989 Parasite Immunol 11: 397-402). A similar vaccine incorporating L. infantum (semi-purified and lyophilized) was used in a pilot study of domestic dogs in an endemic area of CVL (BV Ogunkolade 1988 Vet Parasitol 28: 33-41). Surprisingly, vaccinated dogs were found to be more susceptible to infection than the controls. In Brazil, W Mayrink et al. (1990 Rev Inst Med Trop Sao Paulo 32: 67-69) found that dogs can be partially protected against cutaneous leishmaniasis by a vaccine prepared from a single stock of L. braziliensis. Presently, this line of study has been developed to explore protection of dogs against infection with L. chagasi. In order to evaluate the safety (phase I) and immunogenicity/efficacy (phase II) of this vaccine against CVL, we carried out experiments in dogs with experimental challenge of promastigotes of L. chagasi (strain MHOM/BR/72/BH46) after immunization. Thirty one 4 month-old laboratory-reared mongrel dogs of both sexes were immunized against parvovirosis, leptospirosis, distemper, parainfluenza and hepatitis and treated with mebendazol for intestinal helminthic infections. The Leishmania vaccine was composed of merthyolated sound-disrupted promastigotes of L. braziliensis, strain MCAN/BR/72/C348 (Mayrink loc. cit.). The promastigotes were cultured in NNN/LIT media (EP Camargo 1964 Rev Inst Med Trop Sao Paulo 6: 43-100). The flagelates were submitted to ultra-sound during 1 min at 40 watts, in an ice bath. The process was repeated three times, at 1 min intervals. Total nitrogen content was then determined and the extracts were diluted in saline mixed with thimerozal (1:10,000), adjusting the final concentration to 240 ug of total N/ml. Bacillus Calmete Guerin (BCG - Fundacao Ataulfo de Paiva, Rio de Janeiro) was added as an adjuvant. The phase I trial was carried out on 12 non-immune dogs, and the aim was to evaluate the kinetics of the inflammatory skin reaction to the vaccine and BCG and to determine localized and systemic side effects. Four groups composed of three dogs each were used. In Group I dogs received an injection of vaccine containing 600 ug protein mixed with 400 ug of BCG. Animals in groups II, III and IV received BCG (400 ug), vaccine or PBS, respectively. All dogs received intradermal injections (300 ul each) in six distinct sites on the back. Skin biopsies were taken from each site of the inocula after 7, 14, 21, 28, 35 and 42 days, fixed in buffered (pH 7.2) formol saline, sectioned (5 um-thick), and stained with haematoxylin and eosin and/or Gomori. The dogs were clinically examined during the experiment and rectal temperature was taken on the days mentioned above. Histologically, all animals from Group I that received BCG combined with vaccine showed an intense, chronic inflammatory reaction that increased progressively in the outer and deeper layers of the dermis. On day 21, the epidermis was replaced by ulcer and the dermis contained an intense exudate of neutrophils, macrophages, lymphocytes and plasma cells, mixed with necrotic tissue on the bed of the ulcer. On days 28 and 35, the inflammatory reaction became chronic, productive, and granulomatous. After 35 days, there was a tendency for fibrosis, with fibroblastic proliferation and local deposition of collagen. Dogs receiving only BCG (Group II) presented a chronic and diffuse inflammatory reaction that was qualitatively similar to the vaccine plus BCG group, but quantitatively less intense, mainly at days 21 and 28. Animals from Group III, receiving only vaccine, displayed a moderate inflammatory reaction observed only on day 21, characterized by an exudate composed by mononuclear cells, plasma cells and a few neutrophils. In the animals from Group IV that received only PBS no inflammatory reaction was observed. This study showed an absence of adverse reactions in dogs inoculated with the vaccine and/or BCG. The local reaction with granuloma and ulcer formation was circumscribed at the site of injection. No fever or satellite adenitis were observed. Phase II was carried out on 19 dogs randomly divided in two groups: vaccinated group (10 dogs vaccinated with vaccine plus BCG) and control group (9 unvaccinated controls). Three doses of vaccine (600 ug protein/dose) mixed with BCG (400 ug/dose) were given intradermally at 21-day intervals. Sixty days after the third dose, all dogs received an intravenous challenge with 2.3x106 infective promastigotes of the parasites. All dogs were followed up at two month intervals with aspirative biopsies of bone marrow (Giemsa-stained smears and by culture in NNN/LIT medium) together with blood collections for anti-Leishmania immunofluorescence antibody test (IFAT) to detect IgG. Cell-mediated immune response was assessed by lymphocyte proliferation assay, employing peripheral mononuclear blood cells (E Nascimento et al. 1990 Infect Immun 58: 2198-2203). Table shows the final results after 26-months of follow up (necropsy was performed in all dogs to detect parasites). At this time, 1/10 dogs in the vaccinated group showed patent L. chagasi infection whereas in the unvaccinated control dogs all animals developed infection. On the subsequent days after the immunization, the vaccinated dogs did not produce specific anti-Leishmania antibodies detectable by IFAT. These were detected in some animals at different levels three months after the inocula of the promastigotes, indicating infection by Leishmania and not antibody production due to immunization. Nine of ten vaccinated dogs showed positive stimulation index responses 15 days after the third immunization dose and during the follow-up, but the control dogs were unresponsive. A proliferation response was considered positive when calculated lymphocyte proliferation rate in face of Leishmania antigens was +/- 2.5 (SC Mendonca et al. 1986 Clin Exp Immunol 64: 269-276). These results indicate that the combined vaccine/BCG tested is safe and did not give rise to adverse side effects in inoculated dogs. The immunogenic effect of the combined vaccine/BCG was demonstrated by the induction of cellular immunity and the partial protection of dogs when challenged. Use of a strain of L. braziliensis combined with BCG as a vaccine against L. chagasi infection could be effective against the disease in dogs, since Ogunkolade (loc. cit.) has already shown that a vaccine prepared with L. infantum increases the susceptibility of dogs to the parasite. It is possible that antigens of L. chagasi have an immunossupressive effect. The use of the BCG as adjuvant together with the first generation vaccines composed of killed parasite for clinical trial has been recommended by the Leishmaniasis Vaccine Steering Committee of the WHO/TDR Programme. In view of these results, we have begun a third phase consisting of a randomized, double blind clinical trial of the vaccine combined with BCG. This is being carried out in Montes Claros, in the north of the State of Minas Gerais, Brazil, where the disease is endemic and 34 autochthonous human cases were diagnosed in 1994. TABLE: Parasitological and immunological observations on 19 dogs challenged with promastigotes of Leishmania chagasi after immunization with a vaccine against visceral leishmaniasis
Group Dog Parasite Stimulation Reciprocal Conclusion
isolation^a index of of IFAT (infection)
lymphocyte titres^c
proliferation
assay^b
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1 Negative 14.3 Negative No
2 Negative 13.0 Negative No
3 Negative 5.6 Negative No
4 Negative 3.3 Negative No
5 Negative 5.6 Negative No
Vaccinated 6 Negative 3.3 Negative No
7 Negative 6.6 Negative No
8 Positive 1.9 1:160 Yes
9 Negative 3.9 Negative No
10 Negative 4.7 Negative No
11 Negative 1.0 1:360 Yes
12 Negative 1.7 1:640 Yes
13 Negative 2.1 1:360 Yes
14 Positive 2.1 1:1280 Yes
Control 15 Positive 1.7 1:160 Yes
16 Positive 1.3 1:2560 Yes
17 Negative 0.8 1:640 Yes
18 Positive 0.7 1:1280 Yes
19 Positive 0.8 1:1280 Yes
^a: final results obtained on the day of the animals'
necropsies; ^b: results obtained after the third dose of the
vaccine; ^c: IFAT= immunofluorescent antibody test.Copyright 1996 Fundacao Oswaldo Cruz
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