Prime-boost vaccination using recombinant Mycobacterium bovis BCG and recombinant vaccinia virus DIs harboring HIV-1 CRF01_AE gag in mice: influence of immunization routes.

We have previously reported that live vector-based HIV-1 gag vaccine candidate using BCG as a vector was achievable in BALB/c mice. Although the gag-specific CTL induced by this live candidate vaccine is significantly high, persistence of CTL remains unclear. Thus, efforts were made to explore the potential of recombinant Vaccinia virus DIs strain harboring the same HIV-1 CRF01_AE gag gene (rVaccinia/ HIV-1gagE) present in the BCG construct, using different immunization routes. After one month following a single subcutaneous (s.c.) injection of rBCG/HIV-1gagE, higher CTL responses were recognized against various peptide epitopes along the whole gag protein compared to that by intradermal (i.d.) route. A prime-boost regimen having only rDIs/HIV-1gagE injected i.d. induced very low CTL levels. However, within two months, by priming with rBCG/HIV-1gagE s.c. and boosting with rVaccinia/HIV-1gagE intravenously (i.v.), CTL levels were greater (20-68% specific cell lysis) than those obtained by priming and boosting both i.d. (18-35%). After seven months, both prime-boost immunization with rBCG/HIV-lgagE s.c. and with rVaccinia/HIV-1gagE either i.v. or i.d. sustained similar CTL levels. Our studies exhibit that the prime-boost vaccination of rBCG/HIV-1gagE following by rVaccinia/HIV-1gagE i.d. could be used to elicit prolonged CTL responses as well as memory T-cells in mice, which might be more practical than using i.v. route.

Specific immune response and pathological findings in BALB/c mice inoculated with recombinant BCG expressing HIV-1 antigen.

Recombinant BCGs (rBCGs) containing extrachromosomal plasmids with different HIV-1 insert sequences: nef, env (V3J1 and E9Q), gag p17 or whole gag p55 were evaluated for their immunogenicity, safety and persistent infection in BALB/c mice. Animal injected with, rBCG-plJKV3J1, rBCG-pSO gag p17 or rBCG-pSO gag p55 could elicit lymphocyte proliferation as tested by specific HIV-1 peptides or protein antigen. Inoculation with various concentration of rBCG-pSO gag p55 generated satisfactory specific lymphocyte proliferation in dose escalation trials. The rBCG-pSO gag p55 recovered from spleen tissues at different time interval post-inoculation could express the HIV protein as determined by ELISA p24 antigen detection kit. This result indicated that the extrachromosomal plasmid was stable and capable to express Gag protein. It was also demonstrated that rBCGs did not cause serious pathological change in the inoculated animals. The present study suggested the role of BCG as a potential vehicle for using in HIV vaccine development.

The role of the Tat gene in the pathogenesis of HIV infection.

The human immunodeficiency virus Tat regulatory protein is essential for virus replication and for the efficient transcription of HIV-1 provirus, and in the pathogenesis of AIDS. The role of the tat gene was investigated in 300 samples. It was found that 71.7% were subtype CRF_01AE, 9.3% were subtype B, while 11.7 and 7.3% of them were cross-reactive and non-typeable, respectively. Moreover the results from peptide ELISA also showed that a low CD4 cell count was related to a low anti-Tat antibody (p < 0.05), which may be due to the progression of HIV-1, which can be found predominantly in AIDS patients. The results of nested PCR showed that the second Tat exon might also play a role in T-cell activation. Reverse transcription polymerase chain reaction (RT-PCR) was used to measure HIV-1 mRNA expression in PBMC. RT-PCR negative results were found mostly in the asymptomatic HIV-seropositive group (88%). HIV-1 mRNA expression was found to correlate with current immunologic status. The differences in Tat protein sequences from DNA sequencing between the patients who had anti-Tat antibody positive and anti-Tat antibody negative, were not significant (p > 0.05). These results suggested that the Tat amino acid sequences were conserved among each group of samples and did not change significantly compared with the consensus sequence in previous studies. Several factors make Tat an attractive target for vaccine design.

Characteristic of HIV-1 in V3 loop region based on seroreactivity and amino acid sequences in Thailand.

The third variable (V3) domain of the envelop (env) protein has been used for determining genetic subtype and phenotypic characteristics of human immunodeficiency virus type 1 (HIV-1) isolates. Based on the seroreactivity of the HIV-1 subtype by V3 peptide binding enzyme immunoassay (EIA) of 351 samples obtained in 1998 from HIV-1 infected individuals and AIDS patients, we found that 283 (80.6%) were subtype E, 20 (5.7%) were subtype B, 28 (8.0%) were cross-reactive between both types and 20 (5.7%) were non-typeable. The degree of seroreactivity of HIV-1 subtype E decreased significantly when the amino acid at the crown of the V3 loop was substituted from a GPGQ motif to GPGR motif. Interestingly, AIDS patients who had V3 sequences of subtype E as GPGR motif had a stronger immunoreactivity to GPGQ motif peptides than to GPGR motif peptides, in contradiction for their proviral sequences. The results suggested that mutations in the V3 loop may lead to a changed immunoreactivity that makes HIV-1 mutants unrecognizable or allow escape from the primary immune response by means of neutralizing sensitivity. In connection with vaccine development, it should be pointed out that the combination of V3 sequencing and peptide EIA could provide a novel approach to obtain a primarily infected virus sequence as a target for a preventive AIDS vaccine.