Sequence variation within the dominant amino terminus epitope affects antibody binding and neutralization of human immunodeficiency virus type 1 Tat protein.

Tat is among the required regulatory genes of human immunodeficiency virus type 1 (HIV-1). Tat functions both within infected cells as a transcription factor and as an extracellular factor that binds and alters bystander cells. Some functions of extracellular Tat can be neutralized by immune serum or monoclonal antibodies. In order to understand the antibody response to Tat, we are defining antibody epitopes and the effects of natural Tat sequence variation on antibody recognition. The dominant Tat epitope in macaque sera is within the first 15 amino acids of the protein amino terminus. Together with a subdominant response to amino acids 57 to 60, these two regions account for most of the macaque response to linear Tat epitopes and both regions are also sites for the binding of neutralizing antibodies. However, the dominant and subdominant epitope sequences differ among virus strains, and this natural variation can preclude antibody binding and Tat neutralization. We also examined serum samples from 31 HIV-positive individuals that contained Tat binding antibodies; 23 of the 31 sera recognized the amino terminus peptide. Similar to binding in macaques, human antibody binding to the amino terminus was affected by variations at positions 7 and 12, sequences that are distinct for clade B compared to other viral clades. Tat-neutralizing antibodies to the dominant amino terminus epitope are affected by HIV clade variation.

Furin cleavage of the HIV-1 Tat protein.

Extracellular human immunodeficiency virus-1 (HIV-1) Tat protein and Tat-derived peptides are biologically active but mechanisms of Tat processing are not known. Within the highly conserved basic region of HIV-1 Tat protein (amino acids, a.a. 48-56), we identified two putative furin cleavage sites and showed that Tat protein was cleaved in vitro at the second site, RQRR\ (a.a. 53-56\). This in vitro cleavage was blocked by a monoclonal antibody that binds near the cleavage site or by the furin inhibitor alpha-1 PDX. Monocytoid cells rich in furin also degraded Tat and this process was slowed by the furin inhibitor or the specific monoclonal antibody. Furin processing did not affect the rates for Tat uptake and nuclear accumulation in HeLa or Jurkat cells, but the transactivation activity was greatly reduced. Furin processing is a likely mechanism for inactivating extracellular HIV-1 Tat protein.

Effects of virus burden and chemokine expression on immunity to SHIV in nonhuman primates.

HIV-1 vaccine candidates are designed to elicit Type 1 immune responses, including cytotoxic T cells and neutralizing antibodies. The type of immune response is influenced by many factors, including the levels of antigen expression and production of cytokines or chemokines; we designed a nonhuman primate study to evaluate the influence of these factors on protective immunity. Recombinant SHIV were engineered to express macrophage inflammatory protein-1 alpha (MIP-1alpha), regulated upon activation, normal T-cell expressed and secreted (RANTES), or Lymphotactin (Ltn) in place of nef in SHIV(89.6) (SHIV(89.6-MIP-1), SHIV(89.6-RANTES), SHIV(89.6-Ltn)). The parental virus SHIV(89.6) was included because it replicates to higher titer while still not causing disease. Control groups included animals that received a recombinant SHIV with a truncated chemokine construct (SHIV(89.6-dLtn)) and unvaccinated macaques. After pathogenic challenge with SHIV(89.6pd), animals from groups that received recombinant (nef-deleted) viruses had peak viremia levels three orders of magnitude lower than unvaccinated controls and increased survival times. Animals that received the original SHIV(89.6) (nef+) were highly resistant to both intrarectal and intravenous challenge with SHIV(89.6PD), and showed no signs of disease. There were no differences in survival times comparing unvaccinated and SHIV(89.6-dLtn) (control) groups, indicating that nef deleted viruses did not provide durable protection in this model. Strongest protection was seen in animals with the highest replicating virus (SHIV(89.6)), and the lower effect on survival after SHIV(89.6) nef-deleted vaccination, likely reflects differences in replication capacity. The protective effect of nef-deleted virus was partly restored by expressing Type 1 chemokines to augment viral immunity.

Tat-neutralizing antibodies in vaccinated macaques.

The human immunodeficiency virus Tat protein is essential for virus replication and is a candidate vaccine antigen. Macaques immunized with Tat or chemically modified Tat toxoid having the same clade B sequence developed strong antibody responses. We compared these antisera for their abilities to recognize diverse Tat sequences. An overlapping peptide array covering three clade B and two clade C Tat sequences was constructed to help identify reactive linear epitopes. Sera from Tat-immunized macaques were broadly cross-reactive with clade B and clade C sequences but recognized a clade B-specific epitope in the basic domain. Sera from Tat toxoid-immunized macaques had a more restricted pattern of recognition, reacting mainly with clade B and with only one clade B basic domain sequence, which included the rare amino acids RPPQ at positions 57 to 60. Monoclonal antibodies against the amino terminus or the domain RPPQ sequence blocked Tat uptake into T cells and neutralized Tat in a cell-based transactivation assay. Macaques immunized with Tat or Tat toxoid proteins varied in their responses to minor epitopes, but all developed a strong response to the amino terminus, and antisera were capable of neutralizing Tat in a transactivation assay.