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Background: Cervical tissue-based organ cultures have been used successfully to evaluate microbicides for toxicity and antiviral activity. The antimicrobial peptide retrocyclin RC-101 has been shown to have potent anti-HIV activity in cell culture., Objective: To evaluate RC-101 in organ culture for toxicity and its ability to block HIV-1 transmission across cervical mucosa., Methods: A cervical tissue-based organ culture was used to measure antiviral activity of RC-101. Cytotoxicity in tissues was determined by immunostaining of cellular proteins and by measuring inflammatory cytokines using real-time reverse transcriptase-polymerase chain reaction and Luminex technology., Results: RC-101 blocked transmission of both R5 and X4 HIV-1 across cervical mucosa in this organ culture model. Furthermore, film-formulated RC-101 exhibited potent antiviral activity in organ culture. Such antiviral activity of RC-101 was retained in the presence of semen and vaginal fluid. RC-101 showed no cytotoxicity in cervical tissue. Furthermore, RC-101 did not induce proinflammatory cytokine response in tissues. RC-101 also did not have any effect on natural killer cell activity and proliferation of CD4 and CD8 cells and did not show chemotactic activity., Conclusions: Therefore, because of strong antiviral activity and low cytotoxicity in cervical tissues, RC-101 should be considered as an excellent microbicide candidate against HIV-1.

Background: A potential confounding factor in the treatment of human immunodeficiency virus (HIV) infection in Africa is the frequent occurrence of opportunistic infections (OIs). OI-induced immune activation can interfere with HIV-1 clearance by increasing viral replication and target cell availability., Study Design: Treatment outcomes for patients dually infected with HIV-1 and Mycobacterium tuberculosis or HIV-1 and human herpesvirus (HHV)-8 were assessed by measuring changes in viral load and CD4(+) cell counts and by determining the time taken to reach undetectable HIV-1 RNA levels, assessed by means of Kaplan-Meier survival analysis. Patients with HIV-1 and Kaposi sarcoma (KS) received generic nevirapine, stavudine, and lamivudine (3TC); patients with HIV-1 and tuberculosis (TB) received standard commercial didanosine, 3TC, and efavirenz., Results: Both cohorts exhibited a rapid, near-exponential phase I decline in viral load. Patients with TB and late-stage KS had the steepest decay kinetics. These same patients had the greatest initial increase in CD4(+) cell counts. Phase II clearance was slower and more variable. The proportions of patients reaching undetectable plasma HIV-1 levels at days 7, 14, 28, 60, and 90 were, respectively, 15.8%, 30.0%, 52.6%, 78.9%, and 93.8% (Pearson's chi 2=50.5; P<.001) for patients with TB and 0.0%, 5.0%, 22.2%, 64.7%, and 80.0% (Pearson's chi 2=63.6; P<.001) for patients with KS., Conclusions: Nucleoside reverse-transcriptase inhibitor/nonnucleoside reverse-transcriptase inhibitor-based treatment regimens are highly effective in clearing rapidly replicating (phase I) virus in African patients dually infected with HIV-1 and either TB or KS.

The mononuclear cells and plasma components of semen from HIV-infected subjects have been shown to contain HIV-1. However, there is very little information as to whether distinct HIV-1 population are present in these two seminal compartments or as to their tissue of origin. Phylogenetic analysis of nucleotide sequences of the C2-V5 region of the HIV-1 gp120 from HIV-1 RNA isolated from seminal cells and seminal plasma of five subjects indicates that the HIV-1 population derived from seminal plasma was distinct from that present in seminal cells. Such subcompartmentalization of HIV-1 between seminal cells and seminal plasma was detected as early as 3 months after seroconversion and persisted up to 38 months following seroconversion. Furthermore, comparison of HIV-1 sequences between testis and prostate tissues showed distinct HIV-1 populations in these tissue compartments. In situ real-time (Taqman) PCR analysis of prostate and testis tissues indicated that T lymphocytes were the predominant cells infected with HIV-1 in both of these tissues. Since seminal plasma is derived from prostate and most of the seminal cells originate from the rete testis and epididymis, these results are consistent with the idea that HIV-1 in seminal plasma is derived from the prostate, while HIV-1-infected cells in semen originate mostly from the rete testis and epididymis. These findings provide for the first time evidence of subcompartmentalization of HIV-1 in male genital organs and suggest that intervention strategies such as vasectomy may not prevent sexual transmission.

Objective: To determine if low level, persistent, HIV-1 replication within specific immune cells contributes to HIV-1-specific immune responsiveness., Design: We analyzed 59 HIV-1-infected subjects on stable highly active antiretroviral therapy (HAART) therapy (not including zidovudine) with suppressed plasma viremia (< 400 copies/ml) for phenotypic and lymphoproliferative correlates of immune function., Methods: Peripheral blood mononuclear cells were collected for immunophenotyping, lymphoproliferative assays, and simultaneous immunophenotyping/ultrasensitive in situ hybridization. Plasma was collected for plasma viral load as determined by the Ultra Sensitive Roche Amplicor RT-PCR. Descriptive statistics (mean and SD, median, first and third quartiles) were determined for all variables in two groups defined as having persistent viral replication present or absent. The two-sided Wilcoxon test (continuity correction, 0.5) was used to compare lymphocyte phenotypes, lymphoproliferative assay responses, intracellular gag-pol mRNA, lowest CD4 counts and CD4% of these two groups., Results: HIV-1 replication in CD4, CD45RO memory T lymphocytes persists in spite of undetectable plasma viral load. Patients (n = 24) with persistent intracellular expression of HIV-1 mRNA (> 0.3%) showed significant in vitro proliferative responses to HIV-1 p24 (stimulation index > or = 10) compared to patients (n = 35) without persistent intracellular replication. The group with persistent HIV-1 replication in cells showed no significant response to the recall antigen tetanus toxoid but a trend toward higher responses to pathogen antigens. There were no differences between the groups in the prevalence of AIDS or occurrences of opportunistic infections; however, the high viral persistence group was more HAART experienced (P < 0.05)., Conclusions: These results suggest that HIV-1-specific immune responses correlate with evidence of ongoing HIV-1 replication.

Objective: To develop a model to predict transmission of HIV-1 from men to women., Design: HIV-1 in seminal plasma, and endocervical CCR5 receptors were correlated with epidemiological studies of HIV-1 transmission to develop a probabilistic model., Settings: Semen samples were collected from patient subjects in Seattle Washington, Chapel Hill, North Carolina, and St. Gallen, Switzerland. Endocervical biopsy specimens were obtained from women in Chicago, Illinois., Participants: Eighty-six men (not receiving antiretroviral therapy) in whom CD4 cell count and semen volume were available, and 24 women in whom the number of endocervical CCR5 receptors were determined., Main Outcome Measures: Prediction of transmission of HIV-1 from men to women per episode of vaginal intercourse based on the absolute burden of HIV (volume x HIV RNA copies/ml seminal plasma)., Results: The model suggests efficient heterosexual transmission of HIV-1 when semen viral burden is high. When semen contains 100 000 copies of non-syncytium-inducing (NSI) HIV RNA the probability of HIV-1 transmission is 1 per 100 episodes of intercourse; conversely, with 1000 copies NSI HIV RNA in semen, transmission probability is 3 per 10 000 episodes of intercourse., Conclusions: This model links biological and epidemiological data related to heterosexual HIV-1 transmission. The model can be used to estimate transmission of HIV from men with high semen viral burden from inflammation, or reduced burden after antiretroviral therapy. The results offer a biological explanation for the magnitude of the HIV epidemic in places where earlier studies have shown men have high semen viral burden, such as in sub-Saharan Africa. The model can be used to develop and test HIV-1 prevention strategies.

The placenta may play a critical role in inhibiting vertical transmission of HIV-1. Here we demonstrate that leukemia inhibitory factor (LIF) is a potent endogenous HIV-1-suppressive factor produced locally in placentae. In vitro, LIF exerted a potent, gp130-LIFRbeta-dependent, HIV coreceptor-independent inhibition of HIV-1 replication with IC50 values between 0.1 pg/ml and 0.7 pg/ml, depending on the HIV-1 isolate. LIF also inhibited HIV-1 in placenta and thymus tissues grown in ex vivo organ culture. The level of LIF mRNA and the incidence of LIF protein-expressing cells were significantly greater in placentae from HIV-1-infected women who did not transmit HIV-1 to their fetuses compared with women who transmitted the infection, but they were not significantly different from placentae of uninfected mothers. These findings demonstrate a novel pathway for endogenous HIV suppression that may prove to be an effective immune therapy for HIV infection.

The role of placenta in vertical transmission is not yet fully understood. A protective role of the placenta during gestation is suggested by the finding that caesarian sections reduce the risk of transmission of human immunodeficiency virus (HIV)-1 from mother to child three- to fourfold. Here we investigated whether the immunological milieu of the placenta might be important in HIV-1 transmission. In situ imaging of immunohistochemically stained placenta sections and reverse transcriptase-polymerase chain reaction demonstrated a fourfold increase in CCR5:CXCR4 expression ratio in placentae from transmitting women compared to placentae from nontransmitting women. This chemokine receptor repertoire was consistent with an up-regulation of interleukin-4 and interleukin-10 expression in placentae from nontransmitting placentae compared to transmitting placentae. In situ imaging demonstrated that CCR5 and CXCR4 were expressed on placental macrophages and lymphocytes but not in trophoblasts. Simultaneous immunofluorescence/ultrasensitive in situ hybridization for HIV-1 gag-pol mRNA revealed that HIV-1 infects primarily CXCR4-expressing cells in placentae from nontransmitting women whereas predominantly CCR5-expressing cells were infected in placentae from transmitting women. These data are consistent with transmission of a homogeneous population of nonsyncytium-inducing HIV-1 isolates that use CCR5 as co-receptor.

Recent studies of human immunodeficiency virus (HIV)-specific CD8(+) T cells have focused on responses to single, usually HLA-A2-restricted epitopes as surrogate measures of the overall response to HIV. However, the assumption that a response to one epitope is representative of the total response is unconfirmed. Here we assess epitope immunodominance and HIV-specific CD8(+) T-cell response complexity using cytokine flow cytometry to examine CD8(+) T-cell responses in 11 HLA-A2(+) HIV(+) individuals. Initial studies demonstrated that only 4 of 11 patients recognized the putative immunodominant HLA-A2-restricted p17 epitope SLYNTVATL, suggesting that the remaining subjects might lack significant HIV-specific CD8(+) T-cell responses. However, five of six SLYNTVATL nonresponders recognized other HIV epitopes, and two of four SLYNTVATL responders had greater responses to HIV peptides restricted by other class I alleles. In several individuals, no HLA-A2-restricted epitopes were recognized, but CD8(+) T-cell responses were detected to epitopes restricted by other HLA class I alleles. These data indicate that an individual's overall CD8(+) T-cell response to HIV is not adequately represented by the response to a single epitope and that individual major histocompatibility complex class I alleles do not predict an immunodominant response restricted by that allele. Accurate quantification of total HIV-specific CD8(+) T-cell responses will require assessment of the response to all possible epitopes.

High levels of human immunodeficiency virus (HIV) type 1 have been detected in semen at all stages of disease. However, it is not clear whether HIV-1 is shed in semen continuously or intermittently. In a prospective longitudinal study, viral RNA was measured weekly for 10 weeks in semen and blood of HIV-seropositive subjects. Results showed three different patterns of HIV-1 shedding in semen: none (28%), continuous (28%), and intermittent (44%). In contrast, there was no change in blood plasma virus load during the study period. Phylogenetic analysis of the envelope sequences of HIV-1 RNA in semen and blood revealed distinct virus populations in semen and blood of intermittent shedders but similar virus populations in the semen and blood of continuous shedder. These results indicate for the first time that HIV-1 is shed primarily in an intermittent manner and that shedding patterns of HIV-1 in semen are related to compartmentalization of HIV-1 between semen and blood.

It is now recognized that, in addition to drug-mediated therapies against human immunodeficiency virus type 1 (HIV-1), the immune system can exert antiviral effects via CD8(+) T-cell-generated anti-HIV factors. This study demonstrates that (i) supernatants from peripheral blood mononuclear cells (PBMC) stimulated with influenza A virus inhibit replication of CCR5- and CXCR4-tropic HIV-1 isolates prior to reverse transcription; (ii) the HIV-suppressive supernatants can be generated by CD4- or CD8-depleted PBMC; (iii) this anti-HIV activity is partially due to alpha interferon (IFN-alpha), but not to IFN-gamma, IFN-beta, the beta-chemokines MIP-1alpha, MIP-1beta, and RANTES, or interleukin-16; (iv) the anti-HIV activity is generated equally well by PBMC cultured with either infectious or UV-inactivated influenza A virus; and (v) the antiviral activity can be generated by influenza A-stimulated PBMC from HIV-infected individuals. These findings represent a novel mechanism for inhibition of HIV-1 replication that differs from the previously described CD8 anti-HIV factors (MIP-1alpha, MIP-1beta, RANTES, and CD8 antiviral factor).

CD8(+) T cells from human immunodeficiency virus (HIV)-infected individuals can suppress HIV replication in CD4(+) cells by a noncytotoxic mechanism that inhibits the expression of viral RNA. The present study examined whether other step(s) in the virus replicative cycle could be affected by the CD8(+) cells. Culturing HIV-infected CD4(+) T cells with antiviral CD8(+) T cells did not significantly reduce the amounts of (i) early HIV DNA reverse transcripts (detected by LTR-U3/R), (ii) total nuclear HIV gag DNA, or (iii) integrated proviral DNA. However, exposure to the CD8(+) T cells did cause a reduction in the amount of multiply spliced tat and full-length gag mRNA expressed by the infected CD4(+) T cells, confirming previous observations. The levels of glyceraldehyde-3-phosphate dehydrogenase and interleukin-2 receptor-alpha mRNA were not affected. The results support the conclusion that the noncytotoxic anti-HIV response of CD8(+) T cells, demonstrable in vitro, does not affect any of the virus replication steps leading to the integration of proviral HIV, but specifically interrupts the expression of viral RNA.

Objectives: Dendritic cells (DC) are potential first target cells in sexually transmitted HIV-1 infection. They are also considered to be central in the activation of naive T cells, which thereupon can become permissive for HIV-1. In addition, activated DC express effector molecules, which likely contribute to the direction of T helper (Th1/Th2)-specific immune responses., Methods: The capacity of cytokine and chemokine production in in vitro DC infected and uninfected with HIV-1 was assessed by enzyme-linked immunosorbent assay (ELISA) and by in situ immunocytochemical detection at the single cell level. Fluorescent in situ 5'-nuclease assay (FISNA) was used for quantitative evaluation of HIV-1 gag-positive cells., Results: Macrophage-tropic HIV-1 effectively infected 20-40% of in vitro cultured DC. However, this activity alone did not induce detectable cytokine or chemokine protein expression in DC. In contrast, lipopolysaccharide (LPS) stimulation of these HIV-1-infected DC resulted in a significantly increased level of cells producing tumour necrosis factor alpha (TNF-alpha) and interleukin (IL) 1beta but reduced frequencies of cells producing IL-1 receptor antagonist (IL-1ra) compared with the LPS-stimulated but uninfected DC cultures (P < 0.05). Furthermore, an extensive production of the beta-chemokines [RANTES, macrophage inflammatory proteins (MIP) 1alpha and 1beta] was detected in DC in response to both LPS and HIV-1 plus LPS., Conclusions: These findings indicate that HIV-1 infected DC may have an increased proinflammatory activity. Elevated production of cytokines such as TNF-alpha and IL-1beta and reduced IL-1ra may contribute to enhanced replication of HIV-1 in bystander T cells. Gram-negative bacterial infection and gut-associated bacterial translocation in HIV-1-infected individuals may also result in endotoxin-mediated reactivation of HIV-1 in bystander CD4 CD45RO T cells caused by the increased production of proinflammatory cytokines in DC.

Objective: We examined the effect of an HIV-1-specific immune-based therapy on cell-associated HIV-1 DNA and RNA., Design: Five HIV-1-infected subjects receiving HIV-1 immunogen plus HAART were compared with three HIV-1-infected subjects who received incomplete Freund's adjuvant (IFA) plus HAART., Methods: Cell-associated HIV-1 RNA or DNA in lymphocytes and monocytes was determined using a dual immunophenotyping/in situ hybridization assay with or without in situ PCR amplification., Results: Cell-associated HIV-1 RNA in CD4 cells correlated with plasma RNA overall. CD4, HIV-1 gag-pol messenger (m)RNA+ cells decreased in the immunogen plus HAART group compared with the IFA plus HAART group. Decreases in HIV-1 DNA+ CD4 cells were observed in the immunogen plus HAART compared with the IFA plus HAART group. Decreases in HIV-1 gag-pol mRNA+ monocytes were observed in the immunogen plus HAART group compared with the IFA plus HAART group. Consistent with the findings in CD4 cells, decreases in HIV-1 DNA+ monocytes were observed in the immunogen plus HAART group compared with the IFA plus HAART group., Conclusions: These preliminary observations support the rationale for examining the combination of immune-based therapies and antiretroviral drugs for effective HIV-1 control.

Infection of HIV-1-transgenic mice with Mycobacterium avium, a common opportunistic pathogen in AIDS patients, was shown to result in increased tissue expression of viral specific transcripts. Moreover, by coculturing splenocytes from the transgenic animals with human T cells it was possible to demonstrate that the elevation in HIV-1 mRNA triggered by M. avium infection reflects increased production of infectious virions. Viral immune activation was also shown to correlate with a marked elevation of p24 in supernatants of ex vivo-cultured tissues and, more importantly, in systemic increases in the HIV-1 protein in plasma. Interestingly, these tissue and systemic p24 responses were found to be differentially regulated. Thus, while in vitro p24 production by cultured splenocytes increased concurrently with bacterial loads during the first 6 wk of infection, levels of the Ag in plasma actually decreased. In situ localization experiments together with FACS analysis of HIV-1-expressing splenocytes indicated that virus production is restricted largely to cells of the monocyte/macrophage lineage. Indeed, in vitro p24 expression by cells from noninfected transgenic mice was up-regulated by polyclonal stimulation of macrophages but not T cells. Together these results underscore the importance of the macrophage reservoir in persistent virus expression and establish a convenient and relevant animal model for studying the factors responsible for immune activation of HIV-1 induced by mycobacterial as well as other common coinfections encountered by AIDS patients.

Background: Residual HIV-1-infected cells are poorly eliminated from lymphoid tissue (LT) reservoirs by effector cytotoxic T lymphocytes (eCTL) despite antiretroviral therapy. Perforin and granzyme A (grA) constitute major effector molecules within eCTL granules that induce apoptosis and lysis of virally infected cells., Objective: Expression of perforin and grA was studied at the single cell level in LT and blood from 16 patients infected with HIV-1 (stage A1-C) who were not taking antiretroviral therapy., Method: Immunohistochemical analysis by in situ imaging of cells from blood and LT., Results: Quantitative in situ imaging showed that perforin-expressing CD8 T cells comprised 0.3-1.5% of total cells within the LT from recent HIV-1 seroconverters, while grA was found in 2.1-7.2% of total cells. However, despite high-level grA upregulation (1.5-4.5% of total cells) compared with that in non-infected individuals (0.4-0.9%), perforin expression remained low (< 0.1% of total cells) (P < 0.02) in LT from patients with chronic HIV-1 infection (stage A2-C). This contrasted with findings in peripheral blood mononuclear cells (PBMC) from the same HIV-1 infected cohort where perforin was detected in 13-31% of all PBMC, which was 10- to 100-fold higher than in lymphoid tissue (P < 0.001); grA was found in 14-32% of total PBMC. Two-colour staining showed that granular expression of perforin and grA was restricted to CD8 T cells in over 90% of total cells in both LT and blood., Conclusions: These findings indicate that cytotoxic perforin expression is impaired at local sites of HIV replication within lymphoid tissue. Since perforin is required together with grA for granule-mediated cytolysis, the low perforin expression in the LT may limit the ability of eCTL to eliminate HIV-1 infected cells in lymphoid tissue.

HIV-1 enters target cells mainly via binding to CD4 and its coreceptors. The presence of HIV-1 in CD4- cells suggests, however, that there exist other mechanisms for viral entry. Here it is reported that HIV-1 DNA may be transferred from one cell to another by uptake of apoptotic bodies in a CD4-independent way. This was investigated by coculturing CD4-, chemokine receptor CCR5- and CXCR4- human fetal fibroblasts with apoptotic HIV-1-infected HuT78 cells or apoptotic PBMC isolated from HIV-1-infected patients. After 2 wk of coculture, fibroblasts contained HIV-1 DNA and expressed HIV-1 proteins p24 and gp120. Transfer of HIV-1 DNA was verified by coculturing fibroblasts with apoptotic bodies derived from cells infected with a defective HIV-1 virus. These cells contain one integrated copy of a reverse transcriptase (RT)-negative HIV-1 strain (8E5/LAV RT- cells) and consequently cannot produce free virus. Intracellular HIV-1 gag DNA was detected in both fibroblasts and dendritic cells after coculture with apoptotic 8E5/LAV RT- cells. Transfer of viral DNA after uptake of apoptotic bodies may explain HIV-1 infection of CD4- cells in vivo and furthermore may be relevant for Ag presentation.

HIV-1 transmission and disease progression is, in general, characterized by initial predominance of macrophage tropic, non-syncytium-inducing strains followed by a switch to T-cell tropic, syncytium-inducing strains. Using sensitive, quantitative kinetic RT-PCR, we examined cytokine regulation of tropism-specific HIV-1 coreceptor expression in PBMCs from HIV-1-seronegative individuals. Proinflammatory (TNF-alpha and IL-12) and type 1 cytokines (IFN-gamma and IL-2) significantly upregulated CCR5 (wt allele) mRNA expression in CCR5 homozygous wild-type (wt/wt) and heterozygous individuals (wt/del) (P < 0.02). CCR5 (wt) mRNA expression in unstimulated PBMCs was significantly increased in wt/wt individuals compared to that of wt/del individuals (P < 0.01). In wt/del individuals, del CCR5 mRNA was expressed at 10-fold greater levels than wt CCR5 mRNA in unstimulated PBMCs from the same individual. Flow cytometry confirmed that upregulated CCR5 mRNA following type 1 cytokine stimulation leads to increased cell surface expression of CCR5 protein. The type 2 cytokine IL-10 downregulated both CCR5 mRNA and protein expression in wt/wt and wt/del individuals. Proinflammatory, type 1, and type 2 cytokines significantly increased CXCR4 mRNA expression in wt/wt, wt/del, and del/del CCR5 genotypes (P < 0.02). These results suggest that changes in the cytokine milieu influence chemokine receptor expression and may explain emergence of tropism-specific strains facilitating HIV transmission and disease progression.

The persistence of HIV replication in infected individuals may reflect an inadequate host HIV-specific CD8+ cytotoxic T lymphocyte (CTL) response. The functional activity of HIV-specific CTLs and the ability of these effector cells to migrate in vivo to sites of infection was directly assessed by expanding autologous HIV-1 Gag-specific CD8+ CTL clones in vitro and adoptively transferring these CTLs to HIV-infected individuals. The transferred CTLs retained lytic function in vivo, accumulated adjacent to HIV-infected cells in lymph nodes and transiently reduced the levels of circulating productively infected CD4+ T cells. These results provide direct evidence that HIV-specific CTLs target sites of HIV replication and mediate antiviral activity, and indicate that the development of immunotherapeutic approaches to sustain a strong CTL response to HIV may be a useful adjunct to treatment of HIV infection.

CD8+ T lymphocytes confer significant but ultimately insufficient protection against HIV infection. Here we report that activated neonatal CD8+ T cells can be productively infected in vitro by macrophage-tropic (M-tropic) HIV-1 isolates, which are responsible for disease transmission, whereas they are resistant to T cell-tropic (T-tropic) HIV strains. Physiological activation of CD8-alpha/beta+ CD4- T cell receptor-alpha/beta+ neonatal T cells, including activation by allogeneic dendritic cells, induces the accumulation of CD4 messenger RNA and the expression of CD4 Ag on the cell surface. The large majority of anti-CD3/B7.1-activated cord blood CD8+ T cells coexpress CD4, the primary HIV receptor, as well as CCR5 and CXCR4, the coreceptors used by M- and T-tropic HIV-1 strains, respectively, to enter target cells. These findings are relevant to the rapid progression of neonatal HIV infection. Infection of primary HIV-specific CD8+ T cells may compromise their survival and thus significantly contribute to the failure of the immune system to control the infection. Furthermore, these results indicate a previously unsuspected level of plasticity in the neonatal immune system in the regulation of CD4 expression by costimulation.

Determinations of plasma HIV viral RNA copy numbers help to define the kinetics of HIV-1 infection in vivo and to monitor antiretroviral therapy. However, questions remain regarding the identity of various infected cell types contributing to this free virus pool and to the in vivo lifecycle of HIV during disease progression. Characterization of a novel fluorescence in situ hybridization (FISH) assay employing a pool of labeled oligonucleotide probes directed against HIV RNA was done followed by coupling of the FISH assay with simultaneous surface immunophenotyping to address these questions. In vitro characterizations of this assay using tumor necrosis factor-alpha stimulated and unstimulated ACH-2 cells demonstrated the ability to detect < 5% HIV RNA positive cells with a sensitivity of < 30 RNA copies per cell. Peripheral blood mononuclear cells from 39 HIV-seropositive patients on no, single, combination, or triple drug therapy and 8 HIV-seronegative patients were examined. The majority of HIV-positive patients (24/39) harbored monocytes positive for HIV RNA and a significantly higher fraction of patients with high plasma viral load carried positive monocytes (13/16) than did patients in the low plasma viral load group (11/23). These results demonstrate the effectiveness of a novel FISH assay for identifying and monitoring HIV-infected cell populations in the peripheral blood of HIV-positive patients. In addition, monocytes are a major source of cellular HIV virus in the peripheral blood of HIV patients, even with progression of disease.

Most human immunodeficiency virus (HIV)-infected individuals show evidence of infection by only one strain of the virus despite possible frequent contact with multiple strains. The reason(s) for the emergence of a dominant strain of virus in HIV-infected people and the mechanism(s) which prevent other strains from establishing an infection is not known. In the present study, we demonstrate that peripheral blood mononuclear cells (PBMC) of asymptomatic HIV-infected individuals can resist productive infection by HIV-1 and HIV-2 strains. Although the PBMC of these individuals are resistant to superinfection, their CD4+ cells are susceptible to infection. Moreover, two weeks after infection of their PBMC in culture, the superinfecting virus can be recovered from isolated CD4+ cells. When CD8+ cells from asymptomatic individuals are added to the superinfected CD4+ cells, replication of the exogenously introduced virus is inhibited. In contrast, PBMC from individuals who have progressed to disease (Progressors) do not resist superinfection and their CD8+ cells do not showthe antiviral activity which controls productive HIV infection. These findings suggest that CD8+ cells suppressing HIV replication in infected individuals may be critical in preventing the establishment of infection by other strains of HIV by blocking virus replication.

The critical aspects of successful in situ amplification include fixation, permeabilization, amplification and detection. We address these aspects and present a novel detection scheme that eliminates hybridization following amplification. We use the 5'-nuclease activity of Taq polymerase to cleave in situ a 5'-reporter dye from an oligonucleotide probe which hybridizes to the target amplicon during amplification. The 5'-reporter dye is disassociated from the 3'-quenching dye and remains localized by charge interactions. In addition, we describe probe design constraints for 5'-nuclease assays both in solution and in situ. Using this technique, we show the sensitive and specific detection of HIV-1 DNA in cells lines and tissue from HIV-1-infected individuals.

We previously reported that pigtailed macaques (Macaca nemestrina) became infected after intravenous inoculation with the LAI strain of human immunodeficiency virus type 1 (HIV-1), an isolate that replicates in both M. nemestrina lymphocytes and blood-derived monocyte/macrophages. In the current study we investigated the presence of HIV-1 and pathology in the postmortem brains of four of these macaques. Histopathological findings revealed focal lesions in white matter in the frontal and occipital lobes of one macaque, with myelin loss, nerve fibre loss, and gliosis within these lesions. Semi-quantitative, solution-based PCR revealed HIV-1 DNA in the brains of two of the other macaques. Using slide-based PCR-driven in situ hybridization, we studied these two macaques further and detected intranuclear, circular HIV-1 DNA in vascular endothelia and other non-neuronal brain cells. These findings indicate that M. nemestrina brain can be infected with HIV-1 in vivo and may provide a useful animal model for understanding early HIV-1 brain infection in humans.

Previous studies have suggested that resting CD4+ lymphocytes can be infected by human immunodeficiency virus (HIV), but viral production is inhibited. If these cells are activated, progeny virions are released. The present data indicate that CD4+ lymphocytes in the G0/G1 stage of the cell cycle which have been highly purified to remove macrophages and activated HLA-DR+ cells can also be infected by HIV. However, our findings differ from those of earlier reports since in this study, infected quiescent CD4+ cells cannot be activated to produce virus after virus inoculation. PCR analyses indicate that reverse transcription in these CD4+ cells is arrested at a very early step in proviral DNA formation (U3-R region). They do not show any evidence of longer DNA transcripts (e.g., U3-gag). When these quiescent infected CD4+ lymphocytes are activated by exposure to mitogens and macrophages and then reinoculated with HIV, the replication of virus takes place. Resting CD4+ lymphocytes are also resistant to infection when they are cocultured with HIV-infected macrophages. Only activated CD4+ cells are susceptible to cell-to-cell transmission. These observations suggest that in vivo tissue macrophages, susceptible to HIV replication, are the major cells initially productively infected by the virus. Then these cells can transfer HIV to activated CD4+ lymphocytes with resultant virus production. The presence of arrested reverse transcription in quiescent cells raises questions about the cellular factors required to permit production of longer HIV proviral DNA copies. Because they can be reinfected once they have been activated, these infected quiescent cells could be a source of recombinant viruses in the host.

Human immunodeficiency virus type 1 (HIV-1) infection of T cells and cells of the monocyte/macrophage lineage requires a specific interaction between the CD4 antigen expressed on the cell surface and the HIV-1 external envelope glycoprotein (gp120). To study the association between HIV-1 infection and modulation of cell surface expression of the CD4 molecule in vivo, we examined the CD4+ T cells harboring proviral DNA obtained from HIV-1-infected individuals who had received no antiretroviral therapy for at least 90 days. Simultaneous immunophenotyping of CD4 cell surface expression and PCR-driven in situ hybridization for HIV-1 DNA were used to resolve the CD4+ T cells into distinct populations predicted upon the presence or absence of proviral DNA. Among the HIV-1-infected study subjects, the percentage of CD4+ T cells harboring proviral DNA ranged from 17.3 to 55.5%, with a mean of 40.5%. Cell surface fluorescent staining with anti-CD4 antibody directed against a non-gp120 binding site-related epitope (L120) or a conformation-dependent epitope of the gp120 binding site (Leu 3A) demonstrated either an equivalent or a 1.5- to 3-fold-lower cell surface staining intensity for the HIV-1 DNA-positive subpopulation relative to the HIV-1 DNA-negative subpopulation, respectively. These data suggest that masking or alteration of specific epitopes on the CD4 molecule occurs after viral infection.

The third variable domain (V3 domain) of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is an immunodominant region. Anti-V3 domain antibodies neutralize both HIV-1 infection and syncytium formation. The V3 domain has a high density of positive charge which is a potential binding site for anti-HIV-1 sulfated polysaccharides. To investigate the inhibitory effect of sulfated polysaccharides on the binding of anti-V3 domain antibody, fluorescence-activating cell sorting analysis was performed using two kinds of antibodies, NEA9284 (purified, 0.25 micrograms/ml) and 0.5 beta (ascite, 2.0 mg/ml), and HIV-1-infected CEM cells. When the binding assay with a 1:100 dilution of each antibody was performed in the presence of dextran sulfate, heparin, and inositol hexasulfate at concentrations which are antiviral, the compounds did not inhibit the binding of either antibody. As the antibody concentration was decreased with higher dilution, dextran sulfate was able to reduce antibody binding by 50-60%. Thus, antagonism of anti-V3 domain antibody binding by sulfated polysaccharides is not as extensive as reported previously by several groups.

Human immunodeficiency virus type 1 (HIV-1) sequences were generated from blood and from brain tissue obtained by stereotactic biopsy from six patients undergoing a diagnostic neurosurgical procedure. Proviral DNA was directly amplified by nested PCR, and 8 to 36 clones from each sample were sequenced. Phylogenetic analysis of intrapatient envelope V3-V5 region HIV-1 DNA sequence sets revealed that brain viral sequences were clustered relative to the blood viral sequences, suggestive of tissue-specific compartmentalization of the virus in four of the six cases. In the other two cases, the blood and brain virus sequences were intermingled in the phylogenetic analyses, suggesting trafficking of virus between the two tissues. Slide-based PCR-driven in situ hybridization of two of the patients' brain biopsy samples confirmed our interpretation of the intrapatient phylogenetic analyses. Interpatient V3 region brain-derived sequence distances were significantly less than blood-derived sequence distances. Relative to the tip of the loop, the set of brain-derived viral sequences had a tendency towards negative or neutral charge compared with the set of blood-derived viral sequences. Entropy calculations were used as a measure of the variability at each position in alignments of blood and brain viral sequences. A relatively conserved set of positions were found, with a significantly lower entropy in the brain-than in the blood-derived viral sequences. These sites constitute a brain "signature pattern," or a noncontiguous set of amino acids in the V3 region conserved in viral sequences derived from brain tissue. This brain-derived signature pattern was also well preserved among isolates previously characterized in vitro as macrophage tropic. Macrophage-monocyte tropism may be the biological constraint that results in the conservation of the viral brain signature pattern.

The third variable domain (V3 domain) of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is a major determinant of phenotypic variability. The V3 domain of HIV-1 has many basic amino acid residues. Lymphocytotropic HIV-1 tends to have a V3 domain with a higher density of positive charge than does monocytotropic HIV-1. The importance of basic residues in the V3 domain for the HIV-1 infectivity, however, has not been well investigated. Here we show that mutation of basic amino acid residues at positions 303, 306, 309, 313, and 325 in the V3 domain of the lymphocytotropic isolate NL4-3 results in a dramatic elimination of both virus infectivity and syncytium-inducing ability. Three basic amino acid substitutions (at position 306, 309, and 313) induced a decrease in the binding ability of two kinds of neutralizing antibodies (NEA9284 and 0.5 beta) that recognize a different site in the V3 domain. This suggests that the basic residues play an important role in maintaining the tertiary structure of the V3 domain. Monocytotropism was not simply dependent on either decreased positive charge in the V3 domain of NL4-3 or on mutation of lysine to glutamate at position 320, which is a characteristic amino acid of monocytotropic HIV-1. These findings contribute to our understanding of the significance of basic residues on the function of envelope glycoprotein.

Human immunodeficiency virus type-1 (HIV-1) DNA and messenger RNA sequences in both cell lines and blood obtained directly from HIV-1-infected patients were amplified by polymerase chain reaction and hybridized to fluorescein-labeled probes in situ, and the individually labeled cells were analyzed by flow cytometry. After flow cytometric analysis, heterogeneous cell populations were reproducibly resolved into HIV-1-positive and -negative distributions. Fluorescence microscopy showed that the cellular morphology was preserved and intracellular localization of amplified product DNA was maintained. Retention of nonspecific probe was not observed. Analysis of proviral DNA and viral messenger RNA in cells in the blood of HIV-1-infected patients showed that the HIV-1 genome persists in a large reservoir of latently infected cells. With the use of this technique it is now possible to detect single-copy DNA or low-abundance messenger RNA rapidly and reproducibly in a minor subpopulation of cells in suspension at single-cell resolution and to sort those cells for further characterization.

Aurothioglucose and aurothiomalate have anti-HIV-1 activity in vitro. Antiviral activity requires the formation of a reactive intermediate with a molar equivalent amount of a thiol ligand. This activates gold(I) ligand exchange between the reactive species bis(thiolato)gold(I) and acidic thiol groups exposed on the surface of proteins. Bis(thioglucose)gold(I) (bisAuTG) which is formed by the reaction of molar equivalent amounts of aurothioglucose and 1-thio-beta-D-glucose completely protected MT-4 and CEM cells against HIV-1NL4-3-induced cytopathogenicity. Although bisAuTG is an inhibitor of human immunodeficiency virus-1 (HIV-1) reverse transcriptase in a cell-free assay, its antiviral effect is due to modification of a surface component of the virion. The HIV-1 strain NL4-3 is 200-fold more sensitive to inhibition of infectivity by bisAuTG than are the strains MN, RF, and SF-2. HIV-1NL4-3 has a unique cysteine residue close to the amino terminus of its gp41 envelope glycoprotein (residue 532 of gp160) which we hypothesize is the target of bisAuTG binding. Mutation of that residue alters HIV-1NL4-3 infectivity and dominantly suppresses virus assembly when coexpressed with the wild-type NL4-3 genome. We show that bisAuTG treatment releases gp120 from the surface of cells expressing wild-type HIV-1NL4-3 envelope glycoprotein, but it does not release gp120 if Cys532 is mutationally altered to Ala. Thus, the antiviral effect of bisAuTG on HIV-1NL4-3 is due to an effect on the association of gp120 with gp41.