Peptide mapping of H-2d restricted T-cell epitope against six antigens of HIV-1 subtype B'/C by ELISPOT assay / 病毒学报

The purpose is to screen and identify the specific H-2d restricted T-cell epitopes. These epitopes are used to investigate the cellular immune response of BALB/c (H-2d) mice immunized with a HIV-1 vaccine which expresses six antigens of gp160, gag, pol, rev, tat and nef of HIV subtype B'/C. A replicating DNA vaccine and a non-replicating recombinant vaccinia virus vector, both expressing the six antigens mentioned above, were used to immune BALB/c (H-2d) mice in a prime-boost regiment. The six peptide libraries of HIV B'/C corresponding respectively to the six complete antigens were pooled according to a designed matrix format and used to test for IFN-gamma production from splenocytes of immunized mice by an enzyme-linked immunospot (IFN-gamma ELISPOT) assay. The ELISPOT data indicated that two of seven Gag-specific T-cell epitope peptides were identified to be the novel epitopes. One of three Pol-specific T-cell epitope is unreported. One novel epitope was confirmed in two gp160-specific T-cell epitope peptides. One Nef-specific T-cell epitope was identified. Three Tat-specific T-cell epitope peptides were continuous sequences in Tat peptide library and all contained either complete or partial sequence reported. Rev-specific T-cell epitope was not be found. The specific T-cell epitopes (H-2d restricted) were identified by IFN-7 ELISPOT assay, which could be used to detect the cellular immune response of BALB/c mice immunized with the HIV-1 vaccine expressing six antigens of gp160, gag, pol, rev, tat and nef of HIV subtype B'/C.

The non-replicating recombinant vaccinia virus expressing six genes of HIV-1 can be passaged stably in CEF / 病毒学报

To investigate the genetic stability (including the vector of vaccinia virus and six foreign genes: gp160, gag, pol, rev, tat and nef) of the HIV-1 non-replicating recombinant vaccinia virus (rNTV-C). rNTV-C was serially passaged to passage 25 (P25) in primary chicken embryo fibroblast (CEF). P9, P12, P15 and P25 were selected to study the genetic stability in four aspects, including the genetic stability of viral vector, the genetic stability of six foreign genes, the expressing stability of foreign genes and the genetic loss of foreign genes. The results showed that the viral vector was non-replicated vaccinia virus of Tiantan strain and was passaged stably; foreign gene sequences matched with designed sequences, the insert sites were right, and the nucleotide mutation rate was less than one over ten thousands within different passages of rNTV-C; the target proteins could be expressed effectively, and the expression level was stable within different passages of rNTV-C; the genetic loss of gag and nef was less than 5% within different passages of rNTV-C. The above results provided important data for the vaccine production.

Expression and immunity of multi-HIV B'/C subype genes in replicating DNA vaccines / 病毒学报

To understand the effect of various gene structures of HIV B'/C subtype on the gene expression and immunity in DNA vaccine, replicating DNA vector pSCK2 was used to construct seven DNA vaccines carrying one or more of HIV B'/C subtype genes: gagpol, gp160 and rtn (rev, tat and nef fusion gene). Immunofluorescence staining indicated that Gag, Gp160, Rev, Tat and Nef could be expressed from the seven DNA vaccines. Stronger expression was observed with the gene in single-gene expression plasmid or with the gene located at upper-IRES in double- or multi-gene expression plasmid. ELISA test showed that Gag induced higher antibody response, but the antibody titers stimulated by Gp160, Pol, or RTN were very low. Both Gag single-gene expression plasmid and Gag-RTN double-gene expression plasmid separately inoculating induced stronger antibody response against Gag than Gag-Gp160 double-gene expression plasmid and Gagpol-Gp160-RTN multi-gene expression plasmid or combined inoculation of Gag and Gp160 single-gene expression plasmids did. ELISPOT detection showed that all the seven DNA vaccines could stimulate cellular immune response against Gag, Pol, Gp160, Tat, and Nef, respectively. Gagpol or Gp160 single-gene expression plasmid separately inoculating stimulated the strongest cellular immune response. Tat and Nef expressed in all the plasmids induced similar immune response. These results indicated that HIV B'/C subtype genes gagpol, gp160 and rtn could be efficiently expressed in the replicating DNA vaccine vector, single-gene expression plasmid had the higher gene expression level and induced stronger immune response; combined immunization of Gagpol and Gp160 had dramatically lower immunity than Gagpol or Gp160 separated immunization did. Immunity of RTN had no difference between combined and separated immunizations. Therefore, in case of immunization with DNA vaccines containing different HIV genes, it is necessary to optimize the combined immunization procedure, especially for the combination of Gag and Gp160-containing vaccines.

Vaccination with three HIV-1 cross neutralizing epitopes fused to HBV S antigen could induce robust antibody immune response in mice / 病毒学报

To enhance immunogenicity of HIV-1 cross neutralizing epitopes , three HIV-1 cross neutralizing epitopes (ELDKWA, NWFDIT, GPGRAFY) were fused to 3' end of HBV S gene by PCR cloning technology, respectively. Three vaccinia virus (Tiantan strain) recombinants expressing separately the three fusion genes were subsequently constructed, named as RVJ1175S-2F5 (ELDKWA), RVJ1175S-4E10 (NWFDIT) and RVJ1175S-447-52D (GPGRAFY), respectively. From the supernatants of CEF cells infected by these vaccinia recombinants, three subunit vaccines (PS-2F5, PS-4E10 and PS-447-52D) were prepared after purification. Biology and immunology characteristics of these fusion antigens in vaccinia recombinants and subunit vaccines were comparatively studied. It was confirmed by PCR and sequencing that the fusion genes were inserted into the TK locus of vaccinia virus (Tiantan strain) correctly. The Fusion proteins were expressed efficiently and secreted into supernatant of the infected cells, which was demonstrated by HBsAg ELISA test. Two typical HBsAg bands of 23kD and 27kD were detected in all the purified samples by SDS-PAGE. These two bands were reacted well to HBsAb and corresponding HIV-1 monoclonal antibodies 2F5, 4E10 and 447-52D. BALB/c mice were immunized with subunit and vaccinia recombinant vaccines by intraperitoneal injection. High levels of HBsAb and anti-HIV-1 cross neutralizing epitope antibody in peripheral blood of immunized mice were tested by ELISA, and all the antibody titers induced by three subunit vaccines were higher than that induced by correlated vaccinia recombinants in mice. This work provides a basis for future study on neutralizing activity of these immunized sera and enhancing immune effect through the combined immunization with different type of vaccines.