IMMUNOGENICITY OF DNAHSP65 VACCINE AGAINST TUBERCULOSIS IN LEWIS RAT

ZORZELLA S. F. G.(1), SEGER J.(1), PELIZON A. C.(1), MARTINS D. R.(1), CARVALHO R.(2), MASSON A. P.(3), SILVA C. L.(3), SARTORI A.(1)

(1)Department of Microbiology and Immunology, IB, UNESP, Botucatu, SP, Brazil; (2)Department of Morphology, IB, UNESP, Botucatu, SP, Brazil; (3)Department of Biochemistry and Immunology, FMRP, USP, Ribeirão Preto, SP, Brazil

A DNA vaccine containing the gene of heat shock protein (pVAXhsp65) showed high immunogenicity and protective efficacy against tuberculosis in BALB/c mice. As this mycobacterial protein is highly homologous to the correspondent mammalian protein, an anti-hsp65 immunity could trigger or worsen an autoimmune disease. The purpose of this work was to evaluate the immunogenicity of DNAhsp65 in Lewis rat by using protocols already tested in mice. This analysis will allow a further study of the autoimmune potential of this vaccine in experimental autoimmune encephalomyelitis induced by myelin immunization in these animals. To test DNAhsp65 transcription, RNA was extracted from different tissues (muscle, bone marrow, thymus, spleen and lymph nodes) 3 and 7 days after immunization and assayed by RT-PCR. mRNA for hsp65 was not detected in any of the investigated samples. To test immunogenicity, the animals were immunized with pVAXhsp65 (3 doses/100mg each/intramuscular route) preceded or not by a BCG dose (100mg) by subcutaneous route (prime-boost protocol). Non-immunized rats or rats that received only vector (pVAX) were used as control groups. IgG1 and IgG2b anti-hsp65 were quantified in seric samples and cytokine levels (IFN-g and IL-4) in supernatants from splenic cell cultures stimulated with recombinant hsp65 (rhsp65) or ConA. pVAXhsp65 did not induce significant amounts of antibodies nor increased the low levels of anti-hsp65 antibodies induced by BCG immunization. Levels of IFN-g  induced by rhsp65 were significantly higher in rats that received only pVAXhsp65 but significantly lower in rats submitted to the prime-boost protocol comparing to non-immunized animals. ConA stimulation induced similar IFN-g levels in the different groups. No IL-4 was detected in culture supernatants. These results do not allow a definitive conclusion but they could de interpreted as low immunogenicity of these protocols in the rat or induction of a predominant cellular immune response.

KEY WORDS: tuberculosis, DNA vaccine, Lewis rat, hsp65, RT-PCR

FINANCIAL SUPPORT: FAPESP, CNPq.

CORRESPONDENCE TO:

Alexandrina Sartori, Departamento de Microbiologia e Imunologia do Instituto de Biociências de Botucatu, UNESP, Caixa Postal, 510, CEP 18618-000, Botucatu, SP. Email: sartori@ibb.unesp.br