Mechanisms of HIV-associated lymphocyte apoptosis: 2010

The inevitable decline of CD4T cells in untreated infection with the Human immunodeficiency virus (HIV) is due in large part to apoptosis, one type of programmed cell death. There is accumulating evidence that the accelerated apoptosis of CD4T cells in HIV infection is multifactorial, with direct viral cytotoxicity, signaling events triggered by viral proteins and aberrant immune activation adding to normal immune defense mechanisms to contribute to this phenomenon. Current antiviral treatment strategies generally lead to reduced apoptosis, but this approach may come at the cost of preserving latent viral reservoirs. It is the purpose of this review to provide an update on the current understanding of the role and mechanisms of accelerated apoptosis of T cells in the immunopathogenesis of HIV infection, and to highlight potential ways in which this seemingly deleterious process could be harnessed to not just control, but treat HIV infection

Expression and purification of nonglycosylated SIV proteins, and their use in induction and detection of SIV-specific immune responses.

Two commercially available expression vectors were modified to generate plasmids pGEXcPk and pQ9cPk. Proteins expressed from pGEXcPk and pQ9cPk had a short oligopeptide tag termed Pk at their carboxy termini and either glutathione S-transferase (GST) or a small histidine (His) tag, respectively, at their N termini. GST fusion proteins can be purified on immobilized glutathione and proteins coupled to the His tag selectively bind to Ni(2+)-NTA columns. The Pk tag is recognized by monoclonal antibody (MAb) SV5-P-k, previously produced in our laboratory. Thus proteins expressed from the pGEXcPk and pQ9cPk vectors can be purified in a two-step procedure, first via the N-terminal tag and second via the C-terminal tag. The combination of two affinity purification steps significantly improves the antigen purity and selects for full-size proteins. Moreover, by using the MAbSV5-P-k in the second purification step, Pk-linked antigens can be assembled directly into solid matrix-antibody-antigen (SMAA) complexes for use as vaccines. The genes for nef, endonuclease, p15, p17, p27, protease, Rev, reverse transcriptase (rt), tat, vif, vpr, and vpx of simian immunodeficiency virus (SIV mac 251) were cloned and expressed as both GST-SIV-Pk and His-SIV-Pk proteins. Multivalent SMAA complexes were made that contained His-p17-Pk, His-p27-Pk, His-rt-Pk, His-vpx-Pk, and His-vpr-Pk. Following two immunizations of mice with this mixture, antibodies could be detected to all five SIV antigens. When compared to single-protein immunizations, the immunogenicity of some of the proteins in this cocktail was either enhanced or decreased. Mice were also immunized with His-p17-Pk or His-p17-Pk-antibody complexes in the presence or absence of alum. The antibody-antigen complexes induced two- to four-fold higher antibody levels than antigen alone but did not appear to be more immunogenic in inducing lymphoproliferative responses. Sera from SIV-infected macaques were tested for the presence of antibodies reacting with the recombinant proteins by Western blot analysis. Antibodies to endonuclease, p15, p17, p27, rt, and vif were readily detected, antibodies against protease and vpx were present at much lower levels, but no antibodies were detected to nef, rev, tat, or vpr. Thus, we have developed a comprehensive range of reagents (available on request) that can be used to examine immune responses to SIV in both mice and monkeys

Expression and purification of nonglycosylated SIV proteins, and their use in induction and detection of SIV-specific immune responses

Two commercially available expression vectors were modified to generate plasmids pGEXcPk and pQ9cPk. Proteins expressed from pGEXcPk and pQ9cPk had a short oligopeptide tag termed Pk at their carboxy termini and either glutathione S-transferase (GST) or a small histidine (His) tag, respectively, at their N termini. GST fusion proteins can be purified on immobilized glutathione and proteins coupled to the His tag selectively bind to Ni2+-NTA columns. The Pk tag is recognized by monoclonal antibody (MAb) SV5-P-k, previously produced in our laboratory. Thus proteins expressed from the pGEXcPk and pQ9cPk vectors can be purified in a two-step procedure, first via the N-terminal tag and second via the C-terminal tag. The combination of two affinity purification steps significantly improves the antigen purity and selects for full-size proteins. Moreover, by using the MAb SV5-P-k in the second purification step, Pk-linked antigens can be assembled directly into solid matrix-antibody-antigen (SMAA) complexes for use as vaccines. The genes for nef, endonuclease, p15, p17, p27, protease, Rev, reverse transcriptase (rt), tat, vif, vpr, and vpx of simian immunodeficiency virus (SIVmac 251) were cloned and expressed as both GST-SIV-Pk and His-SIV-Pk proteins. Multivalent SMAA complexes were made that contained His-p17-Pk, His-p27-Pk, His-rt-Pk, His-vpx-Pk, and His-vpr-Pk. Following two immunizations of mice with this mixture, antibodies could be detected to all five SIV antigens. When compared to single-protein immunizations, the immunogenicity of some of the proteins in this cocktail was either enhanced or decreased. Mice were also immunized with His-p17-Pk or His-p17-Pk-antibody complexes in the presence or absence of alum. The antibody-antigen complexes induced two- to four-fold higher antibody levels than antigen alone but did not appear to be more immunogenic in inducing lymphoproliferative responses. Sera from SIV-infected macaques were tested for the presence of antibodies reacting with the recombinant proteins by Western blot analysis. Antibodies to endonuclease, p15, p17, p27, rt, and vif were readily detected, antibodies against protease and vpx were present at much lower levels, but no antibodies were detected to nef, rev, tat, or vpr. Thus, we have developed a comprehensive range of reagents (available on request) that can be used to examine immune responses to SIV in both mice and monkeys.</p

VEINCTR-N, an immunogenic epitope of Fas (CD95/Apo-I), and soluble Fas enhance T-cell apoptosis in vitro. II. Functional analysis and possible implications in HIV-1 disease.

BACKGROUND: Recent studies indicate that soluble Fas (sFas) may modulate T-cell apoptosis, since it inhibits Fas-ligand (Fas-L)-mediated cytotoxicity in vitro. Here, we explored whether the soluble receptor and its major immunogenic domain, namely VEINCTR-N, interfered with apoptosis of T cells from human immunodeficiency virus-type 1 (HIV-1)+ subjects showing serum elevations of both the soluble receptor and anti-Fas antibodies, and with that of several T-cell lines. MATERIALS AND METHODS: Both proliferation and apoptosis extent of T cells from 16 HIV-1+ patients showing serum anti-VEINCTR-N immunoglobulin G (IgG) and 15 controls were tested after incubation with sFas and three 8-mer peptides of its first consensus sequence that included VEINCTR-N. Several cell lines were also investigated by flow cytometry for their expression of Ki-67, the APO2.7-related mitochondrial protein, and the annexin-V. In addition, we evaluated the expression of Fas-L and caspases FLICE, CPP32 and ICE either by flow cytometry, immunoblotting, and/or reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Cell proliferation in cultures from both patients and controls was affected significantly by sFas and VEINCTR-N. However, a prevalent increase of the subdiploid DNA-containing cell population occurred within these cultures. Similarly, Jurkat, CEM cells, and a mouse WR19L transformant overexpressing native human Fas underwent prompt apoptosis, which was detected as enlargement of APO2.7-reactive and annexin-V-positive populations. By exploring the Fas pathway in Jurkat cells, we found that both apoptosis inducers acted through Fas, since Fas-L, as well as CPP32 and FLICE were activated. By contrast, ICE was up-regulated only in control cells treated with tumor necrosis factor alpha (TNFalpha). CONCLUSIONS: These data suggest that the soluble molecular forms of Fas prime cell death in Fas-positive cells. Therefore, the shedding of high amounts of sFas in HIV- 1 disease is possibly entrusted with amplification of the death execution program by cells functionally exhausted and committed to die. It is conceivable that the appearance of anti-Fas antibodies reflects an attempt by the immune system to neutralize these effective forms of the receptor and its structurally degraded domains, such as VEINCTR-N