Superior induction of T cell responses to conserved HIV-1 regions by electroporated alphavirus replicon DNA compared to that with conventional plasmid DNA vaccine.

Vaccination using "naked" DNA is a highly attractive strategy for induction of pathogen-specific immune responses; however, it has been only weakly immunogenic in humans. Previously, we constructed DNA-launched Semliki Forest virus replicons (DREP), which stimulate pattern recognition receptors and induce augmented immune responses. Also, in vivo electroporation was shown to enhance immune responses induced by conventional DNA vaccines. Here, we combine these two approaches and show that in vivo electroporation increases CD8(+) T cell responses induced by DREP and consequently decreases the DNA dose required to induce a response. The vaccines used in this study encode the multiclade HIV-1 T cell immunogen HIVconsv, which is currently being evaluated in clinical trials. Using intradermal delivery followed by electroporation, the DREP.HIVconsv DNA dose could be reduced to as low as 3.2 ng to elicit frequencies of HIV-1-specific CD8(+) T cells comparable to those induced by 1 µg of a conventional pTH.HIVconsv DNA vaccine, representing a 625-fold molar reduction in dose. Responses induced by both DREP.HIVconsv and pTH.HIVconsv were further increased by heterologous vaccine boosts employing modified vaccinia virus Ankara MVA.HIVconsv and attenuated chimpanzee adenovirus ChAdV63.HIVconsv. Using the same HIVconsv vaccines, the mouse observations were supported by an at least 20-fold-lower dose of DNA vaccine in rhesus macaques. These data point toward a strategy for overcoming the low immunogenicity of DNA vaccines in humans and strongly support further development of the DREP vaccine platform for clinical evaluation.

Optimizing HIV-1-specific CD8+ T-cell induction by recombinant BCG in prime-boost regimens with heterologous viral vectors.

The desire to induce HIV-1-specific responses soon after birth to prevent breast milk transmission of HIV-1 led us to propose a vaccine regimen which primes HIV-1-specific T cells using a recombinant Mycobacterium bovis bacillus Calmette-Guérin (rBCG) vaccine. Because attenuated live bacterial vaccines are typically not sufficiently immunogenic as stand-alone vaccines, rBCG-primed T cells will likely require boost immunization(s). Here, we compared modified Danish (AERAS-401) and Pasteur lysine auxotroph (222) strains of BCG expressing the immunogen HIVA for their potency to prime HIV-1-specific responses in adult BALB/c mice and examined four heterologous boosting HIVA vaccines for their immunogenic synergy. We found that both BCG.HIVA(401) and BCG.HIVA(222) primed HIV-1-specific CD8(+) T-cell-mediated responses. The strongest boosts were delivered by human adenovirus-vectored HAdV5.HIVA and sheep atadenovirus-vectored OAdV7.HIVA vaccines, followed by poxvirus MVA.HIVA; the weakest was plasmid pTH.HIVA DNA. The prime-boost regimens induced T cells capable of efficient in vivo killing of sensitized target cells. We also observed that the BCG.HIVA(401) and BCG.HIVA(222) vaccines have broadly similar immunologic properties, but display a number of differences mainly detected through distinct profiles of soluble intercellular signaling molecules produced by immune splenocytes in response to both HIV-1- and BCG-specific stimuli. These results encourage further development of the rBCG prime-boost regimen.

Pre-clinical development of BCG.HIVA(CAT), an antibiotic-free selection strain, for HIV-TB pediatric vaccine vectored by lysine auxotroph of BCG.

In the past, we proposed to develop a heterologous recombinant BCG prime-recombinant modified vaccinia virus Ankara (MVA) boost dual pediatric vaccine platform against transmission of breast milk HIV-1 and Mycobacterium tuberculosis (Mtb). In this study, we assembled an E. coli-mycobacterial shuttle plasmid pJH222.HIVA(CAT) expressing HIV-1 clade A immunogen HIVA. This shuttle vector employs an antibiotic resistance-free mechanism based on Operator-Repressor Titration (ORT) system for plasmid selection and maintenance in E. coli and lysine complementation in mycobacteria. This shuttle plasmid was electroporated into parental lysine auxotroph (safer) strain of BCG to generate vaccine BCG.HIVA(CAT). All procedures complied with Good Laboratory Practices (GLPs). We demonstrated that the episomal plasmid pJH222.HIVA(CAT) was stable in vivo over a 20-week period, and genetically and phenotypically characterized the BCG.HIVA(CAT) vaccine strain. The BCG.HIVA(CAT) vaccine in combination with MVA.HIVA induced HIV-1- and Mtb-specific interferon γ-producing T-cell responses in newborn and adult BALB/c mice. On the other hand, when adult mice were primed with BCG.HIVA(CAT) and boosted with MVA.HIVA.85A, HIV-1-specific CD8(+) T-cells producing IFN-γ, TNF-α, IL-2 and CD107a were induced. To assess the biosafety profile of BCG.HIVA(CAT)-MVA.HIVA regimen, body mass loss of newborn mice was monitored regularly throughout the vaccination experiment and no difference was observed between the vaccinated and naïve groups of animals. Thus, we demonstrated T-cell immunogenicity of a novel, safer, GLP-compatible BCG-vectored vaccine using prototype immunogen HIVA. Second generation immunogens derived from HIV-1 as well as other major pediatric pathogens can be constructed in a similar fashion to prime protective responses soon after birth.

Prime-boost regimens with adjuvanted synthetic long peptides elicit T cells and antibodies to conserved regions of HIV-1 in macaques.

OBJECTIVES: Administration of synthetic long peptides (SLPs) derived from human papillomavirus to cervical cancer patients resulted in clinical benefit correlated with expansions of tumour-specific T cells. Because vaginal mucosa is an important port of entry for HIV-1, we have explored SLP for HIV-1 vaccination. Using immunogen HIVconsv derived from the conserved regions of HIV-1, we previously showed in rhesus macaques that SLP.HIVconsv delivered as a boost increased the breath of T-cell specificities elicited by single-gene vaccines. Here, we compared and characterized the use of electroporated pSG2.HIVconsv DNA (D) and imiquimod/montanide-adjuvanted SLP.HIVconsv (S) as priming vaccines for boosting with attenuated chimpanzee adenovirus ChAdV63.HIVconsv (C) and modified vaccinia virus Ankara MVA.HIVconsv (M). DESIGN: Prime-boost regimens of DDDCMS, DSSCMS and SSSCMS in rhesus macaques. METHODS: Animals' blood was analysed regularly throughout the vaccination for HIV-1-specific T-cell and antibody responses. RESULTS: We found that electroporation spares DNA dose, both SLP.HIVconsv and pSG2.HIVconsv DNA primed weakly HIVconsv-specific T cells, regimen DDDCM induced the highest frequencies of oligofunctional, proliferating CD4(+) and CD8(+) T cells, and a subsequent SLP.HIVconsv boost expanded primarily CD4(+) cells. DSS was the most efficient regimen inducing antibodies binding to regions of trimeric HIV-1 Env, which are highly conserved among the four major global clades, although no unequivocal neutralizing activity was detected. CONCLUSION: The present results encourage evaluation of the SLP.HIVconsv vaccine modality in human volunteers along the currently trialled pSG2.HIVconsv DNA, ChAdV63.HIVconsv and MVA.HIVconsv vaccines. These results are discussed in the context of the RV144 trial outcome.