Joanne van Harmelen

Characterization and Selection of HIV-1 Subtype C Isolates for Use in Vaccine Development

HIV-1 genetic diversity among circulating strains presents a major challenge for HIV-1 vaccine development, particularly for developing countries where less sequence information is available. To identify representative viruses for inclusion in candidate vaccines targeted for South Africa, we applied an efficient sequence survey strategy to samples from recently and chronically infected persons residing in potential vaccine trial sites. All 111 sequences were subtype C, including 30 partial gag, 26 partial pol, 27 V2-V3 env, and 28 V5-partial gp41 sequences. Of the 10 viruses cultured from recently infected individuals, 9 were R5 and 1 was R5X4. Two isolates, Du151 and Du422, collected within 2 months of infection, were selected as vaccine strains on the basis of their amino acid similarity to a derived South African consensus sequence The selection of recently transmitted R5 isolates for vaccine design may provide an advantage in a subtype C R5-dominant epidemic. The full-length Du422 gag and Du151 pol and env genes were cloned into the Venezuelan equine encephalitis (VEE) replicon particle (VRP) expression system. Du422 Gag protein expressed from the VRP accumulated to a high level and was immunogenic as demonstrated by cytotoxic T lymphocyte responses in mice vaccinated with gag-VRPs. Optimization of codon use for VRP expression in human cells did not enhance expression of the gag gene. The cloned Du151 env gene encoded a functional protein as demonstrated by fusion of VRP-infected cells with cells expressing CD4 and CCR5. Genes identified in this study have been incorporated into the VEE VRP candidate vaccines targeted for clinical trial in South Africa.

Construction, Characterization, and Immunogenicity of a Multigene Modified Vaccinia Ankara (MVA) Vaccine Based on HIV Type 1 Subtype C

Candidate vaccines composed of a DNA construct to prime the immune system, followed by modified vaccinia Ankara (MVA) containing matching genes as a booster vaccination, have produced encouraging immune responses in human volunteers. This study presents the detailed construction and characterization of a recombinant MVA that will be tested in combination with a DNA vaccine in Phase I clinical trials in South Africa and the United States. To match recently transmitted viruses in the southern African region and to maximize epitope coverage, the vaccines were constructed to contain five HIV-1 subtype C genes, namely gag, reverse transcriptase, tat, and nef (grttn), expressed as a polyprotein, and a truncated env (gp150). An initial recombinant MVA construct containing wild-type env was found to be genetically unstable, and thus a human codon-optimized gene was used. Grttn and gp150 were inserted into two different sites in MVA yielding a double recombinant, SAAVI MVA-C. The recombinant MVA was shown to be genetically stable and high level expression of the transgenes was observed. Env retained infectivity in a functional infectivity assay despite a point mutation that arose during virus generation. Mice inoculated with SAAVI MVA-C at various doses developed high levels of Gag, RT, and Env-specific CD8(+) and CD4(+) T cells, and some of these responses could be boosted by a second inoculation. An accompanying paper describes the immunogenicity of SAAVI MVA-C when given in combination with SAAVI DNA-C.

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.

HIV-1 subtype C Pr55gag virus-like particle vaccine efficiently boosts baboons primed with a matched DNA vaccine

A DNA vaccine expressing human immunodeficiency virus type 1 (HIV-1) southern African subtype C Gag (pTHGag) and a recombinant baculovirus Pr55gag virus-like particle prepared using a subtype C Pr55gag protein (Gag VLP) was tested in a prime-boost inoculation regimen in Chacma baboons. The response of five baboons to Gag peptides in a gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay after three pTHGag immunizations ranged from 100 to 515 spot-forming units (s.f.u.) per 10(6) peripheral blood mononuclear cells (PBMCs), whilst the response of two baboons to the Gag VLP vaccine ranged from 415 to 465 s.f.u. per 10(6) PBMCs. An increase in the Gag-specific response to a range of 775-3583 s.f.u. per 10(6) PBMCs was achieved by boosting with Gag VLPs the five baboons that were primed with pTHGag. No improvement in Gag responses was achieved in this prime-boost inoculation regimen by increasing the number of pTHGag inoculations to six. IFN-gamma responses were mapped to several peptides, some of which have been reported to be targeted by PBMCs from HIV-1 subtype C-infected individuals. Gag VLPs, given as a single-modality regimen, induced a predominantly CD8+ T-cell IFN-gamma response and interleukin-2 was a major cytokine within a mix of predominantly Th1 cytokines produced by a DNA-VLP prime-boost modality. The prime-boost inoculation regimen induced high serum p24 antibody titres in all baboons, which were several fold above that induced by the individual vaccines. Overall, this study demonstrated that these DNA prime/VLP boost vaccine regimens are highly immunogenic in baboons, inducing high-magnitude and broad multifunctional responses, providing support for the development of these products for clinical trials.

A Multigene HIV Type 1 Subtype C Modified Vaccinia Ankara (MVA) Vaccine Efficiently Boosts Immune Responses to a DNA Vaccine in Mice

Heterologous prime-boost vaccine strategies have generated high frequencies of antigen-specific T cells in preclinical and clinical trials of candidate HIV vaccines. We have developed a DNA (SAAVI DNA-C) and MVA (SAAVI MVA-C) vaccine based on HIV-1 subtype C for testing in clinical trials. Both vaccines contain five subtype C genes: gag, reverse transcriptase, tat, and nef, expressed as a polyprotein, and a truncated env (gp150). The individual vaccines induced CD8(+) and CD4(+) T cells specific for the vaccine-expressed antigens in BALB/c mice. Combining the vaccines in a DNA prime and MVA boost regimen increased the cumulative peptide response compared to the DNA vaccine alone 10-fold, to over 6000 SFU/10(6) splenocytes in the IFN-gamma ELISPOT assay. Th1 cytokine IFN-gamma and TNF-alpha levels from HIV-specific CD8(+) and CD4(+) T cells increased 20- and 8-fold, respectively, with a SAAVI MVA-C boost. Effector and effector memory RT- and Env-specific memory CD8(+) T cell subsets were boosted after MVA immunization, and over time the cells returned to an intermediate memory phenotype similar to that prior to the boost. Immunization of guinea pigs with the DNA-MVA combination induced high titers of antibodies to gp120, although neutralizing activity was weak or absent. The demonstration that these vaccines induce potent cellular immune responses merits their testing in clinical trials.

Restriction fragment length polymorphism analysis for rapid gag subtype determination of human immunodeficiency virus Type 1 in South Africa

A rapid method for identification of human immunodeficiency virus Type 1 (HIV-1) gag subtypes was developed based on restriction fragment length polymorphism (RFLP) analysis of 400 or 650 bp long polymerase chain reaction (PCR) fragments encompassing the start of the p17 (400 bp) and part of the p24 (650bp) regions. The consensus sequences of subtypes A-D, the only subtypes identified in South Africa, were analyzed to detect restriction endonucleases which generate unique patterns for each subtype. Four restriction endonucleases were identified: AluI, AccI, SwaI and XmnI. Digestion of a 400 bp fragment with AluI allowed identification of subtype C. Samples not identified were then reamplified, and a 650 bp fragment digested with AccI to identify subtype B, followed by SwaI and XmnI to distinguish between subtypes A and D. This strategy was applied to 87 samples previously subtyped by either sequence analysis of the gag p17 region (n = 33); or heteroduplex mobility assay (HMA) based on the env gene (n = 75); or both (n = 21). Out of the 87 samples, RFLP identified two samples as subtype A, 28 as subtype B, 56 as subtype C and one as a subtype D virus. No discrepancies were found between RFLP gag subtypes and gag sequence subtypes demonstrating the reliability of this method. There was also no discordance between gag RFLP subtypes and env HMA subtypes, suggesting that there were no recombinant viruses detected relating to the genomic regions analyzed. RFLP is an effective technique for the rapid screening in an HIV epidemic of limited diversity, such as in South Africa.