Infectious simian/human immunodeficiency virus with human immunodeficiency virus type 1 subtype C from an African isolate: rhesus macaque model.

Human immunodeficiency virus type 1 (HIV-1) subtype C is responsible for more than 56% of all infections in the HIV and AIDS pandemic. It is the predominant subtype in the rapidly expanding epidemic in southern Africa. To develop a relevant model that would facilitate studies of transmission, pathogenesis, and vaccine development for this subtype, we generated SHIV(MJ4), a simian/human immunodeficiency virus (SHIV) chimera based on HIV-1 subtype C. SHIV(MJ4) contains the majority of env, the entire second exon of tat, and a partial sequence of the second exon of rev, all derived from a CCR5-tropic, primary isolate envelope clone from southern Africa. SHIV(MJ4) replicated efficiently in human, rhesus, and pig-tailed macaque peripheral blood mononuclear cells (PBMCs) in vitro but not in CEMx174 cells. To assess in vivo infectivity, SHIV(MJ4) was intravenously inoculated into four rhesus macaques (Macaca mulatta). All four animals became infected as determined through virus isolation, PCR analysis, and viral loads of 10(7) to 10(8) copies of viral RNA per ml of plasma during the primary infection phase. We have established a CCR5-tropic SHIV(MJ4)/rhesus macaque model that may be useful in the studies of HIV-1 subtype C immunology and biology and may also facilitate the evaluation of vaccines to control the spread of HIV-1 subtype C in southern Africa and elsewhere.

Genetically modified anthrax lethal toxin safely delivers whole HIV protein antigens into the cytosol to induce T cell immunity.

Bacillus anthrax lethal toxin can be engineered to deliver foreign proteins to the cytosol for antigen presentation to CD8 T cells. Vaccination with modified toxins carrying 8-9 amino acid peptide epitopes induces protective immunity in mice. To evaluate whether large protein antigens can be used with this system, recombinant constructs encoding several HIV antigens up to 500 amino acids were produced. These candidate HIV vaccines are safe in animals and induce CD8 T cells in mice. Constructs encoding gag p24 and nef stimulate gag-specific CD4 proliferation and a secondary cytotoxic T lymphocyte response in HIV-infected donor peripheral blood mononuclear cells in vitro. These results lay the foundation for future clinical vaccine studies.

Utility of SHIV for testing HIV-1 vaccine candidates in macaques.

SUMMARY: Intravenous injection of SHIV (simian/human immunodeficiency virus, chimeric virus) into rhesus macaques resulted in a viremia in peripheral blood lymphocytes (PBL) and the generation of anti-HIV-1 (human immunodeficiency virus type 1) envelope immune responses. A challenge stock of a SHIV containing HIV-1 HXBc2 envelope glycoproteins was prepared from infected rhesus monkey peripheral blood mononuclear cells (PBMC). The minimum animal infectious dose of the SHIV stock was determined and used in a challenge experiment to test protection. The vaccination of two rhesus monkeys with whole inactivated HIV-1 plus polydicarboxylatophenoxy phosphazene (PCPP) as the adjuvant protected the animals from becoming infected by a SHIV challenge. This experiment demonstrated for the first time that monkeys immunized with HIV-1 antigens can be protected against an HIV-1 envelope-containing virus. As the challenge virus was prepared from monkey PBMC, human antigens were unlikely to be involved in the protection. Protection of rhesus monkeys from SHIV challenge may help,define protective immune responses stimulated by HIV-1 vaccine candidates.

The NF kappa B independent cis-acting sequences in HIV-1 LTR responsive to T-cell activation.

The rate of transcription initiation directed by the long terminal repeat (LTR) of HIV-1 increases in response to mitogenic stimuli of T cells. Here we show that the response of the HIV-1 LTR may be governed by two independent sequences located 5' to the site of transcription initiation sequences that bind either NFAT-1 or NF kappa B. The rate of LTR-directed gene expression increased in response to treatment with either a phorbol ester or tumor necrosis factor alpha if either the NFAT-1 or NF kappa B binding sites were deleted, but failed to respond to these mitogenic stimuli if both sequences were absent. The HIV-1 mutant virus containing both NF kappa B and NFAT-1 deletion was able to replicate although at a much decreased growth rate, while the deletion of NFAT-1 alone increased the viral growth rate in Jurkat cells. Neither deletion of NF kappa B nor deletion of NFAT-1 decreased activation of viral replication by phorbol ester.

Identification of cis-acting repressive sequences within the negative regulatory element of human immunodeficiency virus type 1.

The negative regulatory element of human immunodeficiency virus type 1 is a 260-nucleotide-long sequence that decreases the rate of RNA transcription initiation specified by the long terminal repeat. This region has the potential to bind several cellular transcription factors. Here it is shown that sequences which recognize the NFAT-1 and USF cellular transcription factors contribute to this negative regulatory effect. The sequences within the negative regulatory element which resemble the AP-1 site and the URS do not negatively regulate human immunodeficiency virus long terminal repeat transcription initiation.