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India has the third largest number of HIV-1-infected individuals accounting for approximately 2.1 million people, with a predominance of circulating subtype C strains and a low prevalence of subtype A and A1C and BC recombinant forms, identified over the past two decades. Recovery of near full-length HIV-1 genomes from a plasma source coupled with advances in next generation sequencing (NGS) technologies and development of universal methods for amplifying whole genomes of HIV-1 circulating in a target geography or population provides the opportunity for a detailed analysis of HIV-1 strain identification, evolution and dynamics. Here we describe the development and implementation of approaches for HIV-1 NGS analysis in a southern Indian cohort. Plasma samples (n = 20) were obtained from HIV-1-confirmed individuals living in and around the city of Bengaluru. Near full-length genome recovery was obtained for 9 Indian HIV-1 patients, with recovery of full-length gag and env genes for 10 and 2 additional subjects, respectively. Phylogenetic analyses indicate the majority of sequences to be represented by subtype C viruses branching within a monophyletic clade, comprising viruses from India, Nepal, Myanmar and China and closely related to a southern African cluster, with a low prevalence of the A1C recombinant form also present. Development of algorithms for bespoke recovery and analysis at a local level will further aid clinical management of HIV-1 infected Indian subjects and delineate the progress of the HIV-1 pandemic in this and other geographical regions.

Purpose of Review: To discuss how whey acidic proteins (WAPs), a new class of immunomodulatory soluble mediators, impact innate immunity to HIV infection., Recent Findings: Innate immunity to HIV infection is increasingly being recognized as critical in determining initial virus transmission and dissemination and may, therefore, be exploited in vaccine and microbicide intervention strategies to combat HIV infection. Several important innate immune mediators have recently been shown to regulate HIV infection in vitro and are, thus, implicated in in vivo immunity to the virus. These include soluble mediators, such as type I interferon, the defensins and more recently WAPs. Recent evidence is discussed, which show that WAPs are pleiotropic soluble mediators that may impact the course of HIV infection in two ways: as regulators of HIV replication and as regulators of innate and adaptive immunity., Summary: A better understanding of host factors that regulate HIV transmission is essential in the development of novel therapeutic strategies. This review focuses on recent findings that highlight the HIV regulatory and anti-inflammatory function of WAPs and assesses their potential to be exploited as novel therapeutics.

Background: Elucidating mechanisms that promote HIV-1 transfer between CD4+ T-lymphocytes and their subsequent loss is of importance to HIV-1 pathogenesis. We recently reported that whey acidic protein, ps20, promotes cell-free HIV-1 spread through ICAM-1 modulation. Since ICAM-1 is pivotal in cell conjugation and intercellular HIV-1 transfer, this study examines ps20 effects on HIV-1 spread between T lymphocytes., Results: We demonstrate intrinsic ps20 variability in primary CD4+ T-lymphocyte clonal populations and a significant positive correlation between endogenous ps20 levels and virus transfer involving fusion resulting in a spreading infection that could be reversed by the addition of reverse transcriptase inhibitors. Blocking anti-ps20 antibody or siRNA mediated ps20 knockdown, significantly reduced virus transfer. Conversely, virus transfer was promoted by ectopic ps20 expression or by exogenous addition of recombinant ps20. A higher frequency of virological synapse formation was evident in cocultures of HIV-1 infected donor T-cells with ps20high v ps20low/intermediate targets. Blocking ps20 inhibited T-lymphocyte conjugate formation and ICAM-1 expression, and was as potent as ICAM-1 in inhibiting HIV-1 transfer., Conclusions: Therefore ps20 is a novel marker of CD4+ T-cells rendered vulnerable to HIV-1 infection by regulating the fundamental biologic process of intercellular conjugate formation and consequently of potential importance in HIV-1 pathogenesis.

Understanding why human immunodeficiency virus (HIV) preferentially infects some CD4(+) CD45RO(+) memory T cells has implications for antiviral immunity and pathogenesis. We report that differential expression of a novel secreted factor, ps20, previously implicated in tissue remodeling, may underlie why some CD4 T cells are preferentially targeted. We show that (i) there is a significant positive correlation between endogenous ps20 mRNA in diverse CD4 T-cell populations and in vitro infection, (ii) a ps20(+) permissive cell can be made less permissive by antibody blockade- or small-interference RNA-mediated knockdown of endogenous ps20, and (iii) conversely, a ps20(low) cell can be more permissive by adding ps20 exogenously or engineering stable ps20 expression by retroviral transduction. ps20 expression is normally detectable in CD4 T cells after in vitro activation and interleukin-2 expansion, and such oligoclonal populations comprise ps20(positive) and ps20(low/negative) isogenic clones at an early differentiation stage (CD45RO(+)/CD25(+)/CD28(+)/CD57(-)). This pattern is altered in chronic HIV infection, where ex vivo CD4(+) CD45RO(+) T cells express elevated ps20. ps20 promoted HIV entry via fusion and augmented CD54 integrin expression; both of these effects were reversed by anti-ps20 antibody. We therefore propose ps20 to be a novel signature of HIV-permissive CD4 T cells that promotes infection in an autocrine and paracrine manner and that HIV has coopted a fundamental role of ps20 in promoting cell adhesion for its benefit. Disrupting the ps20 pathway may therefore provide a novel anti-HIV strategy.

Background: The clinical treatment of patients with HIV and adverse drug events may be enhanced by an understanding of the underlying mechanisms. About 4% of patients with HIV receiving the potent antiretroviral drug abacavir develop a hypersensitivity reaction. This idiosyncratic reaction appears to have an immunologic component that has yet to be defined. Given that the T-cell type 2 cytokine IL-4 may be overproduced by patients with allergy or other immunologic dysregulation, an index cytokine profile could help elucidate the character of a drug-specific hypersensitivity reaction., Objective: Quantitation of the production of the type 2 IL-4 and the counterregulatory type 1 cytokine IFN-gamma in patients with abacavir-related hypersensitivity., Methods: Intracellular cytokines were enumerated in blood T cells by flow cytometry. Subjects were grouped for evaluation as patients with a hypersensitive response after abacavir treatment, patients initiating abacavir who also were evaluated again after 1 month on abacavir, patients on abacavir for 6 months without hypersensitivity, and HIV-naive control individuals., Results: There was a significant association between increased IL-4 production by CD4 and CD8 T lymphocytes and hypersensitivity reactions to abacavir. Lymphocytes from hypersensitive subjects expressed CD28 and the anti-HIV chemokine macrophage inflammatory protein 1beta with a frequency comparable with HIV-naive control cells, suggesting the possibility that the activated T cells from patients with hypersensitivity are functional., Conclusion: The expansion of type 0 and type 2 T cells phenotyped by IL-4 production may correlate with abacavir-associated hypersensitivity. The data suggest a cytokine bias that may facilitate B-cell differentiation and downregulate T-cell cytotoxic responses.

Polymorphism amongst the human leucocyte antigen (HLA) class II genes could influence antigen presentation and the ability to control human immunodeficiency virus (HIV)-1 by modulating the virus specific CD4 immune response. To examine the effect of such polymorphisms on disease progression, we studied a cohort of 46 HIV-1 infected long-term non-progressors (LTNPs), 87 intermediate progressors (IPs) and 26 rapid progressors. Kaplan-Meier survival analysis of all patients in the cohort on time to a CD4 count less than 350 cells/ micro l, showed a trend for a slower rate of CD4 decline in patients with, compared to those without, the DRB1*15-DQB1*06 haplotype (hazard ratio (HR) 0.69, 95% CI 0.46-1.01, P = 0.06). A similar effect was not observed with the DRB1*13-DQB1*06 haplotype (HR 1.18, 95% CI 0.75-1.88, P = 0.46), but was observed when DQB1*06 alleles were considered irrespective of their DR association (HR 0.74, 95% CI 0.52-1.05, P = 0.06). Major HLA-DQ6 alleles encode aspartate (Asp) at position 57 on the DQbeta chain, a phenotype associated with protection from other immune disorders. We therefore examined the frequency of all DQbeta57 Asp+ alleles, but could not detect a significant effect on the rate of CD4 decline. To examine whether the genotype associated with slower CD4 decline was over-represented in patients with a slow rate of disease progression, we conducted a categorical analysis of a subset of patients with an extended follow-up of 14+years. We found a higher proportion of LTNPs at 14+ years possessed the DRB1*15-DQB1*06 haplotype compared to IPs at 14+ years (38.46 versus 18.18%), though this difference did not reach statistical significance. When DQB1*06 alleles irrespective of their DR association were considered, the protective effect was greater (76.9% LTNPs versus 18.18% IPs, P = 0.04). Our results highlight the potential protective effect of HLA DQB1*06 alleles on the course of HIV disease.

Human immunodeficiency virus (HIV) infection of the brain is associated pathologically with neuronal damage and loss. Clinically cognitive impairments can develop, which in some can be improved by highly active antiretroviral therapy (HAART), whereas in others, the infection persists despite treatment. The efficacy of antiretrovirals to treat cognitive impairments may be related to their ability to suppress viral replication in the brain and also to prevent neurodegeneration. To investigate this question, the authors assessed the ability of stavudine (300 nM), zidovudine (2 nM), and abacavir (300 nM) to suppress viral replication in human brain tissue aggregates infected with HIV-1 SF162. Aggregates were cultured for 4 weeks and exposed to nucleoside reverse transcriptase inhibitors (NRTIs) either 24 h prior, simultaneously, or 24 h post infection. Viral replication was assessed by p24 enzyme-linked immunosorbent assay (ELISA) in culture medium. The authors observed a statistically significant reduction in the rate of viral replication for stavudine added 24 h prior to infection univariate analysis of variance ([UANOVA], t = 2.55, df = 17, P =.021). Decreased viral replication observed with zidovudine and abacavir was not statistically significant.

The cellular basis of HIV associated dementia has been correlated with microglial activation and neuronal dysfunction in symptomatic HIV-1 disease. As a cellular model of HIV-1 infection of brain tissue in vitro, we established a stationary human brain aggregate (SHBA) system to compare infection of HIV-1 SF162 (R5 virus) to that of IIIB (X4 virus). Aggregates were analysed by immunohistochemistry, morphometry, flow cytometry and p24 ELISA. SHBAs had a 1 mm(3) size with a mixed cellular composition of 36% neurones, 27% astrocytes, 2% macrophages/microglia and 14% oligodendrocytes. Infection of SHBA's with the R5 HIV-1 SF162 virus led to the expression of HIV-1 p24 antigen in 6% of cells. Infection with this R5 using virus culminated in transient neuronal damage and a decrease in mitotically active progenitor cells within aggregates. Infection with X4 using HIV-1 IIIB was associated with astrocytosis and neurotoxicity. We propose that: (1) the pattern of cellular damage elicited by HIV-1 infection of brain tissue in vitro depends on virus subtype as determined by its preferential use of R5 or X4 chemokine receptors for entry into cells; (2) SHBAs are a reliable and readily established model of the cellular complexity of human brain tissue in vitro.

Characterization of immune responses to immunodominant CD4 epitopes in HIV-1 that are associated with control of HIV infection could be used to strengthen the efficacy of polyepitope HIV vaccines. We measured both the proliferative and the CD4 interferon (IFN)-gamma and interleukin (IL)-2 cytokine responses specific for 11 previously identified HIV-1 T helper epitopes in 10 HIV-infected non-progressors (LTNPs) (infected for a median of 15 years with a stable CD4 count of >500 cells x 10(6)/l), and seven slow progressors (SPs) (infected for a median of 15 years with a CD4 count that had declined to <500 cells x 10(6)/l). Both groups were antiretroviral treatment-naive at the time of evaluation. The median virus load of SP group was higher than that of the LTNP group (P = 0.0002). The CD4 response to a peptide pool representing all potential CD4 Gag epitopes and to Gag p24 protein was also studied. Compared to SPs, LTNPs had higher numbers of Gag-specific IFN-gamma+IL-2+ CD4s (P = 0.0059). The Gag-specific cytokine and proliferative responses correlated inversely with virus load (P = 0.03 and 0.0002, respectively), highlighting the potential importance of this response in immunity to HIV. A direct correlation was noted between proliferation and the Gag-specific IL-2 (P = 0.0053) rather than IFN-gamma response (P = 0.1336), demonstrating that the proliferation assay reflected the IL-2 rather than the IFN-gamma secreting capacity of CD4 cells. Several subjects with diverse class II DRB1 alleles responded, confirming the 11 selected peptides to be both antigenic and conserved. CD4 cytokine responses to one Gag and two conserved Pol peptides correlated negatively with virus load. The cytokine response to two additional Pol peptides correlated positively with virus load. The data indicate that there is not an absolute correlation between the CD4 immune response to conserved and broadly antigenic helper T cell epitopes in HIV non-progression.

Aim: To characterize HIV-1 Gag p24-specific CD4 cell responses in HIV-exposed-seronegative (ES) individuals., Methodology: Twelve ES individuals, of diverse ethnicity and wild type for the CCR5 Delta-32 mutation, were identified. Controls were HIV-negative blood donors. Gag p24-specific and total Vbeta+ CD4 cells that expressed MIP-1beta, IFN-gamma and IL-2 were enumerated by intracytoplasmic cytokine staining. beta-Chemokine expression was correlated with susceptibility to R5 HIV-1 infection, as measured by polymerase chain reaction for integrated HIV-1 and by p24 enzyme-linked immunosorbent assay., Results: Similar numbers of mitogen-stimulated and Vbeta+ MIP-1beta+, IFN-gamma+ and IL-2+ T cells were found in ES and HIV-negative control subjects. However, all ES subjects tested had an HIV Gag p24-specific MIP-1beta+, IFN-gamma+ and IL-2+ CD4 T-cell response that was rare in controls. p24-Specific cells of all ES but no control subjects could be expanded by in-vitro Ag/IL-2 stimulation, and when re-stimulated with an overlapping peptide series showed evidence of a broad CD4 cell memory response directed against multiple regions of Gag p24. Mitogen-stimulated ES CD4 cells were as susceptible to HIV infection as those from control subjects, but p24-specific IFN-gamma+ CD4 cells of six out of seven ES subjects tested were less susceptible to R5 HIV-1 infection than the counterpart fraction depleted of p24-specific IFN-gamma+ cells. The addition of blocking anti-beta-chemokine antibodies did not promote R5 HIV-1 infection of p24-specific IFN-gamma+ cells., Conclusion: Specific CD4 cell immunity, characterized by a broadly directed memory Gag-p24 CD4 cell response and reduced susceptibility of specific CD4 cells to R5 HIV-1 infection, is a likely correlate of non-transmission.

HIV immunity is likely CD4 T cell dependent. HIV-specific CD4 T cell proliferative responses are reported to correlate inversely with virus load and directly with specific CD8 responses. However, the phenotype and cytokine profile of specific CD4 T cells that correlate with disease is unknown. We compared the number/function of Gag p24-specific CD4 T cells in 17 HIV-infected long-term nonprogressors (LTNPs) infected for a median of 14.6 years with those of 16 slow progressors (SPs), also HIV infected for a median of 14 years but whose CD4 count had declined to <500 cells/ micro l. Compared with SPs, LTNPs had higher numbers of specific CD4s that were double positive for IFN-gamma and IL-2 as well as CD28 and IL-2. However, CD4 T cells that produced IL-2 alone (IL-2(+)IFN-gamma(-)) or IFN-gamma alone (IFN-gamma(+)IL-2(-)) did not differ between LTNPs and SPs. The decrease in p24-specific CD28(+)IL-2(+) cells with a concomitant increase of p24-specific CD28(-)IL-2(+) cells occurred before those specific for a non-HIV Ag, CMV. p24-specific CD28(-)IL-2(+) cells were evident in LTNPs and SPs, whereas the CMV-specific CD28(-)IL-2(+) response was confined to SPs. The difference between LTNPs and SPs in the Gag p24 IFN-gamma(+)IL-2(+) response was maintained when responses to total Gag (p17 plus p24) were measured. The percentage and absolute number of Gag-specific IFN-gamma(+)IL-2(+) but not of IFN-gamma(+)IL-2(-) CD4s correlated inversely with virus load. The Gag-specific IFN-gamma(+)IL-2(+) CD4 response also correlated positively with the percentage of Gag-specific IFN-gamma(+) CD8 T cells in these subjects. Accumulation of specific CD28(-)IL-2(+) helpers and loss of IFN-gamma(+)IL-2(+) CD4 T cells may compromise specific CD8 responses and, in turn, immunity to HIV.

The mechanisms underlying non-progression in HIV-1 infection are not well understood; however, this state has been associated previously with strong HIV-1-specific CD8+ T cell responses and the preservation of proliferative CD4+ T cell responses to HIV-1 antigens. Using a combination of interferon-gamma (IFN-gamma) ELISpot assays and tetramer staining, the HIV-1-specific CD8+ T cell populations were quantified and characterized in untreated long-term HIV-1-infected non-progressors and individuals with slowly progressive disease, both in relation to CD4+ T cell responses, and in comparison with responses to cytomegalovirus (CMV) antigens. High levels of CD8+ T cell responses specific for HIV-1 or CMV were observed, but neither their frequency nor their phenotype seemed to differ between the two patient groups. Moreover, while CMV-specific CD4+ T cell responses were preserved in these donors, IFN-gamma release by HIV-1-specific CD4+ T cells was generally low. These data raise questions with regard to the role played by CD8+ T cells in the establishment and maintenance of long-term non-progression.

Background: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is elevated in cerebrospinal fluid in HIV- associated dementia; in addition, therapeutic GM-CSF elevates plasma viral load., Objective: To assess the effect of GM-CSF on viral replication and the potential ameliorative effect of antiretroviral therapy., Design: A primary human brain aggregate system is used as a model of the in vivo situation., Method: Cultured aggregates were infected with the macrophage tropic strain HIV-1SF162 and then exposed to varying GM-CSF concentrations and 0.3 micromol/l stavudine. Viral replication was assessed by p24 expression in the supernatant and aggregates. Immunohistochemistry identified neurons, astrocytes, microglia and oligodendrocytes., Results: A GM-CSF concentration of 1 ng/ml resulted in a fivefold increase in microglial cells, the main HIV cellular reservoir (P = 0.0001). Prior GM-CSF exposure before infection of the aggregates resulted in sixfold increase in p24 levels compared with non-GM-CSF-exposed infected aggregates. Infected aggregates with or without GM-CSF had significant neuronal loss of 50% and 45%, respectively, and astrocytosis. Addition of stavudine to the infected aggregates, even in the presence of GM-CSF, reduced p24 levels to zero and prevented neuronal loss and astrocytosis., Conclusions: This study demonstrates that GM-CSF enhances viral replication while addition of stavudine prevents this potentially detrimental process.

Background: HIV-1 strains R5 and X4 can infect CD4 memory T cells in vivo. Anti-CD3/28 stimulation induces beta-chemokines and CCR5 down-regulation and renders these cells resistant to R5 HIV-1 infection. Here we describe an additional cellular mechanism that blocks productive R5 HIV-1 infection of CD4 memory T cells., Methods: Blood-derived CD4 memory T cells and CD4 T-cell clones were infected with primary R5 and X4 HIV-1 strains. Virus replication was correlated with CCR5 expression and beta-chemokine production. Virus entry and infectivity were measured by PCR for early and late products of HIV reverse transcription respectively., Results: R5 strains were up to 1000-fold less infectious than X4 viruses for CD4 memory T cells. This resistance was independent of CCR5 levels and of the Delta-32 mutation and the CCR2-V64I/CCR5-59653T linked mutations. Blocking endogenous beta-chemokines relieved minimally this restriction. At the single cell level, CD4 memory cells were either permissive or non-permissive for R5 HIV-1 infection. R5 HIV titre was up to 10-fold lower than X4 virus titre even in a permissive clone. However, R5 viruses replicated as efficiently as X4 viruses in the permissive clone when neutralizing anti-beta chemokine antibodies were added. Non-permissive cells blocked a post-entry step of the virus life-cycle and expressed early but not late HIV transcripts. Neutralizing anti-beta chemokine antibodies promoted R5 virus replication marginally in the non-permissive clone., Conclusion: Some blood memory CD4 T cells retard R5 HIV-1 replication via endogenous beta-chemokines whereas others block productive R5 HIV-1 infection by an additional mechanism that interferes with a post-entry step of the virus life cycle. These natural barriers might contribute to lower pathogenicity of R5 HIV-1 strains for CD4 memory T cells than X4 viruses that emerge late in disease.

Enrichment of a subset of CD4(+)CD45R0(+)CD7(-) T cells has been observed in HIV-infected individuals. We have investigated the ability of CD7(+) and CD7(-) T cells to support replication of HIV and show that virus replicates preferentially in CD7(+) cells. Several possible mechanisms that may underlie such differences in susceptibility to HIV were studied. Our data demonstrate that mitogen stimulation induces poor expression of CD25 and IL-2 in CD7(-) compared with CD7(+) cells. We also show that uninfected CD7(-) cells are more resistant to mitogen-induced apoptosis than CD7(+) cells. Our data support the view that the CD7(-) subset is inherently resistant to HIV replication and that this is due in part to reduced CD25 expression and IL-2 production.

Objective: To test the T-helper (TH)1/TH2 cytokine paradigm in HIV infection., Design and Methods: Cytokine profiles in two separate studies of HIV patients and controls are presented: (i) a longitudinal study of HIV patients with CD4 counts > 500 x 10(6)/l tested at three timepoints compared with controls; (ii) a blinded cross-sectional study of controls and patients with high (> 500 x 10(6)/l) and low (< 500 x 10(6)/l) CD4 counts. Peripheral blood mononuclear cells (PBMC) from patients and controls were tested for the production of two type 1 [interleukin (IL)-2, interferon (IFN)-gamma] and two type 2 (IL-4, IL-10) cytokines by enzyme-linked immunosorbent assay. Both spontaneous and mitogen-induced cytokine production was measured., Results: HIV infection was noted to have the following effects on cytokine production: (i) it led to the in vivo activation of type 2 cytokines in a small group of individuals with high CD4 numbers characterized by the spontaneous release of IL-4 and IL-10. Longitudinal data showed high spontaneous IL-4 and IL-10 to be a consistent feature of the patient group (at each timepoint some patients were high producers) but to be variable in a given individual; (ii) HIV infection impaired the ability of PBMC to respond to stimuli (selected for their ability to optimally induce each cytokine) in terms of IL-2, IL-4 and IL-10 production in patients with both high and low CD4 cell counts; and (iii) conversely, HIV infection led to an overproduction of IFN-gamma in patients with high CD4 counts; patients with low CD4 produced normal levels of IFN-gamma., Conclusions: Our observations did not suggest polarization of the type 1/type 2 cytokine profile in HIV patients. Instead, the data suggested more complex changes to type 1/type 2 cytokine patterns in HIV infection than originally proposed by the TH1/TH2 dichotomy.

This report provides three lines of evidence to suggest that T-helper type 1 (Th1) and type 0 (Th0) cells could play an opposing role in acquired immune deficiency syndrome (AIDS). Using a panel of Th1 and Th0 clones specific for human immunodeficiency virus-1 (HIV-1) gag p24, derived from seronegative volunteers immunized with gag p24: Ty virus-like particles, a Th1 clone specific for tuberculin (PPD), and a Th0 clone derived by random activation from the same volunteer, we have demonstrated the following differences in the capacity of these clones to regulate the in vitro replication of HIV. (1) Th1 clones were less efficient than Th0 clones in supporting HIV replication, both in their resting state (by 10-1000-fold) and after antigen activation (by five to 100-fold). Furthermore, the infectious titre of HIV recovered from the Th0 population was more than 1000-fold higher than virus from the Th1 population, and the number of HIV-infected Th0 cells was five to 16 times higher than the number of infected Th1 cells. (2) Antigen- or mitogen-activated Th1, but not Th0 clones, inhibited HIV in bystander CEM-4 cells. Th1 cells also inhibited HIV in autologous and allogeneic Th0 cells. The level of inhibition in these experiments ranged from 50% to 100% and was three to 10-fold higher and more sustained in the presence of p24-specific clones compared to the PPD-specific Th1 clone. The capacity of Th1 cells to inhibit HIV in neighbouring cells was also reflected in the reduced replication of HIV in the clones immediately after antigen activation compared to unstimulated cells. Kinetic studies of virus production, cytokine release and proliferation showed that inhibition of HIV was associated with peak cytokine release and preceeded proliferation. (3) The Th1 clones had higher cytolytic potential than the Th0 clones. Therefore, the HIV inhibitory activity of Th1 cells could be partly due to cell to cell killing. These data demonstrate the opposing effects of Th1 and Th0 cells on the in vitro replication of HIV, and suggest that Th1 cells might be important in immunity whereas Th0/Th2 cells might lay a role in promoting disease.

The H9 and CEM CD4+ T cell lines were infected with HIV-1 (NY5/LAV-1 isolate) and monitored for losses in cell viability, syncytium formation, and internucleosomal DNA cleavage (a marker for apoptosis). H9 cells were found to undergo cell death via apoptosis as a result of HIVNY5 infection, but this effect was not apparent in CEM cell cultures. The differential effects of HIV-1NY5 in terms of its apoptosis-inducing properties correlated with the relative abilities of H9 and CEM cells in supporting replication of this HIV-1 isolate, since infected CEM cell cultures produced 10-fold lower levels of HIV-1 p24 protein, and very few of these cells stained positive for cell-associated p24 by comparison with H9 cell cultures infected at the same multiplicity of infection. Furthermore, a different HIV-1 isolate (RF), which replicated equally efficiently in both H9 and CEM cells, produced similar levels of apoptosis in these cultures. HIV-1NY5 was also found to be capable of inducing apoptosis in purified peripheral blood CD4+ T cells as well as inhibiting anti-CD3-driven proliferation of these cells. In contrast, incubation of purified CD8+ T cells with HIV-1NY5 under similar conditions produced no cytopathic effects. Substantial levels of apoptosis were also recorded in HIV-1NY5-infected PHA blasts cell cultures. Soluble rHIV-1IIIB type CHO-derived gp120 was found to mimic the effects of HIV in terms of inhibition of anti-CD3/TCR mAb-induced proliferation of T cells, but apoptosis was not detected in gp120-treated T cell cultures whether cross-linked or used in conjunction with anti-CD3 mAb or not. We conclude therefore that both HIV-1NY5 and HIV-1RF isolates have the capacity to directly trigger apoptotic cell death in CD4+ T cells and that this appears to be at least partly associated with the efficiency of virus replication in these cells.

Objective: To evaluate the immune response to HIV-1 p24 generated in vivo by p17/p24:Ty virus-like particles (p17/p24:Ty-VLP) by examining the lymphoproliferative and antibody (Ab) responses to HIV-1 p24, as well as Gag-specific cytotoxic T lymphocytes (CTL), in HIV-seronegative volunteers immunized with hybrid p17/p24:Ty-VLP., Design and Methods: Sixteen HIV-seronegative volunteers were immunized with p17/p24:Ty-VLP at two dose levels (100 or 500 micrograms) and monitored for the following 48 weeks for production of anti-p24 and anti-p17 Ab, in vitro lymphoproliferative responses to HIV-1 p24 and p17, and in vitro CTL responses to HIV-1 Gag., Results: Twelve out of the 16 volunteers had significant p24-specific proliferative responses, with volunteers on the higher dose schedule exhibiting earlier proliferative responses than those on the lower dose schedule. Proliferative responses in both volunteer groups were similar in overall magnitude but appeared at different times during the immunization schedule. Anti-p24 Ab were detected in six out of the nine individuals in the lower dose group and in five out of the seven in the higher dose group. There was a good correlation between the presence of p24-specific Ab and the detection of lymphoproliferative responses to the p24 protein in peripheral blood mononuclear cells isolated from the same individuals. Anti-p17 Ab were detected in five volunteers. No Gag-specific CTL responses were detected., Conclusion: We conclude that hybrid HIV-1 p17/p24:Ty-VLP are capable of inducing both cellular and humoral immunity to HIV-1 Gag p17 and p24 components and are worthy of further study as a potential HIV immunotherapeutic.

We demonstrate that peptides (16 amino acids long) covering the sequence of the HIV-1 core protein p24 induce significant proliferation in peripheral blood mononuclear cells (PBMC) of several (greater than 50%) healthy seronegative volunteers as well as seronegative homosexual men. The nature of this response was characterized and compared with those of HIV-infected patients. Several peptides induced responses; however, the most frequent responses in both seropositive and seronegative individuals were noted to the following peptides: 1 and 2 (aa 133-157); 6 and 7 (aa 183-207); 15 (aa 273-287); and 17 and 18 (aa 293-317). The response pattern was related to the disease stage of the patients; seronegative individuals as well as asymptomatic seropositive individuals (CDC II/III) responded to low concentrations of several peptides, but symptomatic patients (CDC IV) only responded to high concentrations of a few peptides. Cell separation studies of PBMC from healthy volunteers showed that the responding cells were CD4+ and expressed the CD45RO differentiation antigen. Furthermore, cord-blood mononuclear cells with less than 5% of CD45RO T cells did not proliferative to any of the peptides. Finally, CD4+ T cell lines specific for both peptides and p24 protein were successfully established from the PBMC of seronegative individuals confirming the data obtained with freshly isolated cells. These studies therefore suggest that the CD4+ cell response to p24 is not strictly disease related, instead, the response may be due to priming of the host with cross-reactive antigens.

Cytokines such as tumour necrosis factor (TNF) can induce HIV-1 production in T-cell tumour lines. However, it is not known if the same occurs in freshly isolated mononuclear cells, nor is it known if the virus can itself regulate cellular cytokine production. In this paper we report that HIV-1 induces peripheral blood mononuclear cells (PBMC) and CD4+ T lymphocytes to secrete TNF alpha, TNF beta and interferon-gamma (IFN gamma), three cytokines having multifunctional activities and complex physiological roles. We also show that separate addition of exogenous recombinant (r) TNF alpha or rTNF beta or rIFN gamma increases HIV-1-induced syncytium formation in both PBMC and CD4+ cells by up to 10,000-fold, with TNF alpha being most potent in this regard. Finally, we show that syncytium formation induced by diverse HIV-1 isolates and LAV-2 is inhibited without the addition of exogenous r-cytokines by the respective anti-cytokine antibodies. Our study therefore demonstrates that efficient HIV replication in primary mononuclear cells is associated with the ability of the virus to induce TNF and IFN gamma secretion.