Oral vaccination with a recombinant Salmonella vaccine vector provokes systemic HIV-1 subtype C Gag-specific CD4+ Th1 and Th2 cell immune responses in mice.

BACKGROUND: Recombinant Salmonella vaccine vectors may potentially be used to induce specific CD4+ T cell responses against foreign viral antigens. Such immune responses are required features of vaccines against pathogens such as human immunodeficiency virus type 1 (HIV-1). The aim of this study was to investigate the induction of systemic HIV-1-specific CD4+ T helper (Th) responses in mice after oral immunization with a live attenuated Salmonella vaccine vector that expressed HIV-1 subtype C Gag. Groups of BALB/c mice were vaccinated orally three times (4 weeks apart) with this recombinant Salmonella. At sacrifice, 28 days after the last immunization, systemic CD4+ Th1 and Th2 cytokine responses were evaluated by enzyme-linked immunospot assay and cytometric bead array. HIV-1 Gag-specific IgG1 and IgG2a humoral responses in the serum were determined by enzyme-linked immunosorbent assay. RESULTS: Mice vaccinated with the recombinant Salmonella elicited both HIV-1-specific Th1 (interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha)) and Th2 (interleukin-4 (IL-4) and interleukin-5 (IL-5)) cytokine responses. The vaccine induced 70 (IFN-gamma) spot-forming units (SFUs)/10e(6) splenocytes and 238 IL-4 SFUs/10e(6) splenocytes. Splenocytes from vaccinated mice also produced high levels of Th1 and Th2 cytokines upon stimulation with a Gag CD4 peptide. The levels of IFN-gamma, TNF-alpha, IL-4 and IL-5 were 7.5-, 29.1-, 26.2- and 89.3-fold above the background, respectively. Both HIV-1 Gag-specific IgG1 and IgG2a antibodies were detected in the sera of vaccinated mice. CONCLUSION: The study highlights the potential of orally-delivered attenuated Salmonella as mucosal vaccine vectors for HIV-1 Subtype C Gag to induce Gag-specific CD4+ Th1 and Th2 cellular immune responses and antibodies which may be important characteristics required for protection against HIV-1 infection.

Creation and characterisation of a high-copy-number version of the pAL5000 mycobacterial replicon.

The majority of mycobacterial plasmid vectors are derived from the pAL5000 replicon and maintained at approximately five copies per cell. We have devised a method that directly selects for high-copy-number plasmids. This involves enriching for high copy number plasmids by repeatedly isolating and retransforming plasmids from a mutant library. Using this method we have selected a copy-up version of the pAL5000 replicon. In Mycobacterium smegmatis the copy-number was shown to have increased 7-fold to between 32 and 64 copies/cell, and the plasmid remained relatively stable after 100 generations in the absence of antibiotic selection. The plasmid also has a high-copy-number phenotype in M. bovis BCG and can be used to increase expression of cloned genes, as we have demonstrated with the green fluorescent protein. The mutation was found to be the deletion of an alanine residue in the C-terminal end of the RepA replication protein. We argue that the mutation exerts its effect through altered RNA folding, thereby affecting the translationally coupled RepA-RepB expression.

The use of directed evolution to create a stable and immunogenic recombinant BCG expressing a modified HIV-1 Gag antigen.

Numerous features make Mycobacterium bovis BCG an attractive vaccine vector for HIV. It has a good safety profile, it elicits long-lasting cellular immune responses and in addition manufacturing costs are affordable. Despite these advantages it is often difficult to express viral antigens in BCG, which results in genetic instability and low immunogenicity. The aim of this study was to generate stable recombinant BCG (rBCG) that express high levels of HIV antigens, by modification of the HIV genes. A directed evolution process was applied to recombinant mycobacteria that expressed HIV-1 Gag fused to the green fluorescent protein (GFP). Higher growth rates and increased GFP expression were selected for. Through this process a modified Gag antigen was selected. Recombinant BCG that expressed the modified Gag (BCG[pWB106] and BCG[pWB206]) were more stable, produced higher levels of antigen and grew faster than those that expressed the unmodified Gag (BCG[pWB105]). The recombinant BCG that expressed the modified HIV-1 Gag induced 2 to 3 fold higher levels of Gag-specific CD4 T cells than those expressing the unmodified Gag (BCG[pWB105]). Mice primed with 10(7) CFU BCG[pWB206] and then boosted with MVA-Gag developed Gag-specific CD8 T cells with a frequency of 1343±17 SFU/10(6) splenocytes, 16 fold greater than the response induced with MVA-Gag alone. Levels of Gag-specific CD4 T cells were approximately 5 fold higher in mice primed with BCG[pWB206] and boosted with MVA-Gag than in those receiving the MVA-Gag boost alone. In addition mice vaccinated with BCG[pWB206] were protected from a surrogate vaccinia virus challenge.

An oral recombinant Salmonella enterica serovar Typhimurium mutant elicits systemic antigen-specific CD8+ T cell cytokine responses in mice.

BACKGROUND: The induction of antigen-specific CD8+ T cell cytokine responses against an attenuated, oral recombinant Salmonella enterica serovar Typhimurium vaccine expressing a green fluorescent protein (GFP) model antigen was investigated. A GFP expression plasmid was constructed in which the gfp gene was fused in-frame with the 5' domain of the Escherichia coli beta-galactosidase alpha-gene fragment with expression under the lac promoter. Groups of mice were orally immunized three times with the bacteria and systemic CD8+ T cell cytokine responses were evaluated. RESULTS: High level of the GFP model antigen was expressed by the recombinant Salmonella vaccine vector. Systemic GFP-specific CD8+ T cell cytokine (IFN-gamma and IL-4) immune responses were detected after mice were orally vaccinated with the bacteria. It was shown that 226 net IFN-gamma and 132 net IL-4 GFP-specific SFUs/10e6 splenocytes were formed in an ELISPOT assay. The level of IFN-gamma produced by GFP peptide-stimulated cells was 65.2-fold above background (p < 0.05). The level of IL-4 produced by the cells was 10.4-fold above background (p < 0.05). CONCLUSION: These results suggested that a high expressing recombinant Salmonella vaccine given orally to mice would elicit antigen-specific CD8+ T cell responses in the spleen. Salmonella bacteria may, therefore, be used as potential mucosal vaccine vectors.