Preclinical development of BCG.HIVA2auxo.int, harboring an integrative expression vector, for a HIV-TB Pediatric vaccine. Enhancement of stability and specific HIV-1 T-cell immunity.

One of the critical issues that should be addressed in the development of a BCG-based HIV vaccine is genetic plasmid stability. Therefore, to address this issue we have considered using integrative vectors and the auxotrophic mutant of BCG complemented with a plasmid carrying a wild-type complementing gene. In this study, we have constructed an integrative E. coli-mycobacterial shuttle plasmid, p2auxo.HIVAint, expressing the HIV-1 clade A immunogen HIVA. This shuttle vector uses an antibiotic resistance-free mechanism for plasmid selection and maintenance. It was first transformed into a glycine auxotrophic E. coli strain and subsequently transformed into a lysine auxotrophic Mycobacterium bovis BCG strain to generate the vaccine BCG.HIVA2auxo.int. Presence of the HIVA gene sequence and protein expression was confirmed. We demonstrated that the in vitro stability of the integrative plasmid p2auxo.HIVAint was increased 4-fold, as compared with the BCG strain harboring the episomal plasmid, and was genetically and phenotypically characterized. The BCG.HIVA2auxo.int vaccine in combination with modified vaccinia virus Ankara (MVA).HIVA was found to be safe and induced HIV-1 and Mycobacterium tuberculosis-specific interferon-γ-producing T-cell responses in adult BALB/c mice. We have engineered a more stable and immunogenic BCG-vectored vaccine using the prototype immunogen HIVA. Thus, the use of integrative expression vectors and the antibiotic-free plasmid selection system based on "double" auxotrophic complementation are likely to improve the mycobacterial vaccine stability in vivo and immunogenicity to develop not only recombinant BCG-based vaccines expressing second generation of HIV-1 immunogens but also other major pediatric pathogens to prime protective responses shortly following birth.

Dual neonate vaccine platform against HIV-1 and M. tuberculosis.

Acquired immunodeficiency syndrome and tuberculosis (TB) are two of the world's most devastating diseases. The first vaccine the majority of infants born in Africa receive is Mycobacterium bovis bacillus Calmette-Guérin (BCG) as a prevention against TB. BCG protects against disseminated disease in the first 10 years of life, but provides a variable protection against pulmonary TB and enhancing boost delivered by recombinant modified vaccinia virus Ankara (rMVA) expressing antigen 85A (Ag85A) of M. tuberculosis is currently in phase IIb evaluation in African neonates. If the newborn's mother is positive for human immunodeficiency virus type 1 (HIV-1), the baby is at high risk of acquiring HIV-1 through breastfeeding. We suggested that a vaccination consisting of recombinant BCG expressing HIV-1 immunogen administered at birth followed by a boost with rMVA sharing the same immunogen could serve as a strategy for prevention of mother-to-child transmission of HIV-1 and rMVA expressing an African HIV-1-derived immunogen HIVA is currently in phase I trials in African neonates. Here, we aim to develop a dual neonate vaccine platform against HIV-1 and TB consisting of BCG.HIVA administered at birth followed by a boost with MVA.HIVA.85A. Thus, mMVA.HIVA.85A and sMVA.HIVA.85A vaccines were constructed, in which the transgene transcription is driven by either modified H5 or short synthetic promoters, respectively, and tested for immunogenicity alone and in combination with BCG.HIVA(222). mMVA.HIVA.85A was produced markerless and thus suitable for clinical manufacture. While sMVA.HIVA.85A expressed higher levels of the immunogens, it was less immunogenic than mMVA.HIVA.85A in BALB/c mice. A BCG.HIVA(222)-mMVA.HIVA.85A prime-boost regimen induced robust T cell responses to both HIV-1 and M. tuberculosis. Therefore, proof-of-principle for a dual anti-HIV-1/M. tuberculosis infant vaccine platform is established. Induction of immune responses against these pathogens soon after birth is highly desirable and may provide a basis for lifetime protection maintained by boosts later in life.

Design and pre-clinical evaluation of a universal HIV-1 vaccine.

BACKGROUND: One of the big roadblocks in development of HIV-1/AIDS vaccines is the enormous diversity of HIV-1, which could limit the value of any HIV-1 vaccine candidate currently under test. METHODOLOGY AND FINDINGS: To address the HIV-1 variation, we designed a novel T cell immunogen, designated HIV(CONSV), by assembling the 14 most conserved regions of the HIV-1 proteome into one chimaeric protein. Each segment is a consensus sequence from one of the four major HIV-1 clades A, B, C and D, which alternate to ensure equal clade coverage. The gene coding for the HIV(CONSV) protein was inserted into the three most studied vaccine vectors, plasmid DNA, human adenovirus serotype 5 and modified vaccine virus Ankara (MVA), and induced HIV-1-specific T cell responses in mice. We also demonstrated that these conserved regions prime CD8(+) and CD4(+) T cell to highly conserved epitopes in humans and that these epitopes, although usually subdominant, generate memory T cells in patients during natural HIV-1 infection. SIGNIFICANCE: Therefore, this vaccine approach provides an attractive and testable alternative for overcoming the HIV-1 variability, while focusing T cell responses on regions of the virus that are less likely to mutate and escape. Furthermore, this approach has merit in the simplicity of design and delivery, requiring only a single immunogen to provide extensive coverage of global HIV-1 population diversity.

Design and preclinical evaluation of a multigene human immunodeficiency virus type 1 subtype C DNA vaccine for clinical trial.

In this study, the design and preclinical development of a multigene human immunodeficiency virus type 1 (HIV-1) subtype C DNA vaccine are described, developed as part of the South African AIDS Vaccine Initiative (SAAVI). Genetic variation remains a major obstacle in the development of an HIV-1 vaccine and recent strategies have focused on constructing vaccines based on the subtypes dominant in the developing world, where the epidemic is most severe. The vaccine, SAAVI DNA-C, contains an equimolar mixture of two plasmids, pTHr.grttnC and pTHr.gp150CT, which express a polyprotein derived from Gag, reverse transcriptase (RT), Tat and Nef, and a truncated Env, respectively. Genes included in the vaccine were obtained from individuals within 3 months of infection and selection was based on closeness to a South African subtype C consensus sequence. All genes were codon-optimized for increased expression in humans. The genes have been modified for safety, stability and immunogenicity. Tat was inactivated through shuffling of gene fragments, whilst maintaining all potential epitopes; the active site of RT was mutated; 124 aa were removed from the cytoplasmic tail of gp160; and Nef and Gag myristylation sites were inactivated. Following vaccination of BALB/c mice, high levels of cytotoxic T lymphocytes were induced against multiple epitopes and the vaccine stimulated strong CD8+ gamma interferon responses. In addition, high titres of antibodies to gp120 were induced in guinea pigs. This vaccine is the first component of a prime-boost regimen that is scheduled for clinical trials in humans in the USA and South Africa.

Enhanced immunogenicity using an alphavirus replicon DNA vaccine against human immunodeficiency virus type 1.

With the human immunodeficiency virus type 1 (HIV-1) epidemic expanding at increasing speed, development of a safe and effective vaccine remains a high priority. One of the most central vaccine platforms considered is plasmid DNA. However, high doses of DNA and several immunizations are typically needed to achieve detectable T-cell responses. In this study, a Semliki Forest virus replicon DNA vaccine designed for human clinical trials, DREP.HIVA, encoding an antigen that is currently being used in human trials in the context of a conventional DNA plasmid, pTHr.HIVA, was generated. It was shown that a single immunization of DREP.HIVA stimulated HIV-1-specific T-cell responses in mice, suggesting that the poor immunogenicity of conventional DNA vaccines may be enhanced by using viral replicon-based plasmid systems. The results presented here support the evaluation of Semliki Forest virus replicon DNA vaccines in non-human primates and in clinical studies.

Biodistribution and persistence of an MVA-vectored candidate HIV vaccine in SIV-infected rhesus macaques and SCID mice.

Recombinant modified vaccinia virus Ankara (MVA) is together with a few other attenuated viral vectors on the forefront of human immunodeficiency virus type 1 (HIV-1) vaccine development. As such, MVA-vectored vaccines are likely to be administered into immunocompromized individuals. Here, we demonstrated in a good laboratory practice study safety and biological clearance of candidate HIV-1 vaccine MVA.HIVA in simian immunodeficiency virus (SIV)-infected rhesus macaques and mice with a severe combined immunodeficiency (SCID) following an intradermal vaccine administration. In SIV-infected macaques, MVA.HIVA DNA was undetectable by nested PCR 6 weeks after dosing. In SCID mice, the MVA.HIVA vaccine was well tolerated and a positive PCR signal was only observed at the site of injection 49 days after dosing in four out of six mice, but even these sites were negative by day 81 post-injection. Therefore, the MVA.HIVA vaccine is considered safe for application in phase I clinical trials in HIV-1-infected human subjects. These results also contribute to the confidence of using MVA as a smallpox vaccine.

Construction and characterisation of a candidate HIV-1 subtype C DNA vaccine for South Africa.

A candidate DNA vaccine pTHgagC expressing the immunodeficiency virus-1 (HIV-1) gag gene from South African isolate Du422 was constructed and characterised. The isolate was selected on the basis of being the closest to the South African subtype C consensus sequence. Sequence analysis of cytotoxic T lymphocyte (CTL) epitopes showed that HIV subtype C-infected individuals have CTL responses to a number of epitopes present in the vaccine, but also revealed a more limited presence of subtype A- and any B-derived epitopes. A high level of expression of the immunogen was demonstrated in human cells and a potent, long-lived CTL response to a single inoculation of the DNA vaccine was elicited in BALB/c mice, which could be significantly increased by a boost vaccination at 4 weeks. This is the first candidate HIV-1 DNA vaccine employing the South African subtype C sequences, and constitutes a part of a vaccine scheduled to enter a clinical evaluation in South Africa in 2004.