Accessory gene components for an HIV-1 subtype C vaccine : functional analysis of mutated Tat, Rev and Nef antigens

[摘要] ENGLISH ABSTRACT:HIV has attained a global distribution and the number of infected people reached anestimated 28.1 million in sub-Saharan Africa at the end of 2001. HIV-1 subtype C isoverwhelmingly prevalent in Botswana and South Africa and to date no interventionshave been successful enough to curb the rapid spread of the virus. A number ofHIV-1 vaccine strategies are being developed, however the breadth and efficacy ofsuch candidate vaccines, many of which are based on the HIV-1 structural genes pol,gag and env, have mostly been found to be inadequate.The HIV-1 accessory genes are attractive components of HIV vaccines due to theirrole in viral pathogenesis, early expression and the high ratio of conserved CTlepitopes. Yet, because of undesirable properties questions regarding their safety asvaccine components are raised. In this study candidate tat, rev and nefmutants wereassessed for efficient expression and inactivation of undesirable functionality./Plasmid constructs that encode the South African HIV-1 subtype C consensus Tat,Rev and Nef proteins were constructed. The coding sequences of the genes werecodon-optimised for optimum protein expression and these synthetic genes wereconstructed using overlapping 50-mer oligonucleotides. Furthermore, the proteinswere mutated at previously described sites by PCR-based site-directed mutagenesisto render them inactive for their respective functions. Corresponding wild-type Tat,Rev and Nef constructs were also made from viral isolates that were least dissimilarto the respective consensus amino acid sequences. tn vitro expression of thedifferent constructs were assessed in 293 cells by Western blotting with polyclonalmouse sera, which were generated by DNA immunisation with one of the Tat, Revand Nef constructs. The transactivation activity of Tat variants and Rev-mediatednuclear export activity of RRE-containing transcripts were studied in cotransfectionexperiments using reporter-gene-based assays while Nef functionality was assessedin a cotransfection assay with subsequent flow cytometric analysis of surface CD4and MHC-I expression on 293 cells.Sequence analysis of the South African HIV-1 subtype C consensus sequences ofTat, Rev and Nef revealed a high degree of similarity with a consensus sequencethat was drawn up from a large number of viruses from southern Africa. Theseconsensus sequences were also closer to individual viral isolate sequences than anyindividual sequences were, indicating that the use of a consensus sequence mayserve to reduce genetic diversity between a vaccine and circulating viruses. Expression levels of the sequence-modified tat and nef gene constructs were notsignificantly higher than the wild-type constructs, however, the codon-optimised revmutant exhibited markedly higher expression than the wild-type rev construct.Immunoreactivity of the protein with the mouse sera demonstrates expression andimmunogenicity of the Tat, Rev and Nef immunogens in mice. In the background ofthe subtype C Tat, a single C22 mutation was insufficient to inactivate l TRdependentCAT expression in 293T and Hela cells. Yet, this activity was significantlyimpaired using the single mutation, C3?, or the double mutation, C22C3? Comparedto the wild-type Rev, the function of the Rev with a double mutation, M5M10, wascompletely abrogated. Similarly, while the wild-type Nef and native, codon-optimisedconsensus Nef proteins mediated CD4 and MHC-I downregulation, CD4downregulation was completely abrogated in one of the mutants, while both Nefmutants were entirely deficient for MHC-I downregulation.These data demonstrate the high expression levels and impaired functionality ofsequence-modified HIV-1 subtype C consensus Tat, Rev and Nef DNA immunogensthat may be used as single-standing vaccine components or form part of a multicomponentHIV-1 vaccine.

[发布机构] Stellenbosch University