Human herpesvirus 6 variant a infects human term syncytiotrophoblasts in vitro and induces replication of human immunodeficiency virus type 1 in dually infected cells

Human herpesvirus 6 (HHV-6) and human immunodeficiency virus type 1 (HIV-1) may interact during transplacental transmission of HIV-1. The placental syncytiotrophoblast layer serves as the first line of defense of the fetus against viruses. Patterns of replication of HHV-6 variant A (HHV-6A) and HIV-1 were analyzed in singly and dually infected human term syncytiotrophoblast cells cultured in vitro. For this purpose, the GS strain of HHV-6A and the Ba-L and IIIB strains of HIV-1 were used. HHV-6A replication was restricted at the level of early gene products in singly infected syncytiotrophoblasts, whereas no viral protein expression was found in cells infected with HIV-1 alone. Coinfection of syncytiotrophoblast cells with HHV-6A and HIV-1 resulted in production of infectious HIV-1. In contrast, no enhancement of HHV-6A expression was observed in cell cultures infected with both viruses. Uninfected syncytiotrophoblast cells were found to express CXCR4 and CCR3 but not CD4 or CCR5 receptors. Infection of syncytiotrophoblasts with HHV-6A did not induce CD4 expression and had no influence on chemokine receptor expression. Activation of HIV-1 from latency in coinfected cells was mediated by the immediate-early (IE)-A and IE-B gene products of HHV-6A. Open reading frames U86 and U89 of the IE-A region were able to activate HIV-1 replication in a synergistic manner. The data suggest that in vivo double infection of syncytiotrophoblast cells with HHV-6A and HIV-1 could contribute to the transplacental transmission of HIV-1 but not HHV-6A.     

Inhibitory effect of human saliva on HIV-1 infectivity]

Human saliva is known to decrease human immunodeficiency virus type 1 (HIV-1) infectivity in vitro. The purpose of this study was to confirm these findings and to explore the mechanism of action of saliva. Whole saliva from seronegative donors was incubated with HIV-1IIIB chronically infected MOLT 4 cells (MOLT 4/HIV-1IIIB cells) or cell-free HIV-1IIIB or KMT strains. We monitored viral infectivity by using MAGI/CCR5 cells. Whole saliva with Na levels less than 20 mEq/l rapidly damaged MOLT 4/HIV-1IIIB cells, thereby HIV infection to MAGI/CCR5 cells by MOLT 4/HIV-1IIIB cells was nearly abolished. On the contrary, in the cace of whole saliva with Na levels more than 23 mEq/l which damaged few cells, cell-to-cell transmission of HIV-1IIIB was prevented by more than 50%. The infectivity of cell-free HIV-1IIIB to MAGI/CCR5 cells was abolished after incubating and filtering the HIV with whole saliva. Depletion of secretory leukocyte protease inhibitor (SLPI) from whole saliva resulted in a 11-28% decrease in the anti HIV-1KMT activity of saliva. Preincubation of host cells with whole saliva led to an enhancement of the HIV infection rather than inhibition. Whole saliva had no effect on the expression level of the cellular receptors (CD4, CXCR4 and CCR5). These results suggest that the inhibitory effect of whole saliva on HIV-1 infectivity is directly linked to the virus itself rather than on the host cell. Moreover, the physical entrapment of cell-free HIV-1 by whole saliva seems to have major salivaly defence mechanisms against HIV-1 infection through the oral cavity.     

Characterization of a simian human immunodeficiency virus encoding the envelope gene from the CCR5-tropic HIV-1 Ba-L

The tat, rev, vpu, and env genes from the monocytotropic CCR5-dependent HIV-1 Ba-L isolate were substituted for homologous simian immunodeficiency virus (SIV) sequences in the SIV genome. The resultant SHIV (SHIV Ba-L) replicated in CCR5-positive PM-1 cells but not in CCR5-negative CEMX174 cells. Infection of HOS cells expressing different co-receptors showed SHIV Ba-L to be strictly CCR5-dependent. Infection of PM-1 cells and rhesus peripheral blood mononuclear cells (PBMCs) was highly sensitive to RANTES but not to SDF-1. Although SHIV Ba-L infected rhesus and pigtail macaques intravenously or rectally, plasma viremia was controlled after 3 weeks. After serial passage through 4 pigtails by blood and bone marrow transfer, virus from pigtail PBMCs had higher in vitro infectious titers on rhesus PBMCs and was efficiently transmitted vaginally in rhesus and cynomolgus macaques. Plasma viremia generally persisted longer than after infection with unpassaged virus but was eventually controlled with no significant decrease in CD4+ T-cell counts in peripheral blood. The envelope gene of SHIV Ba-L revealed a very little genetic drift during in vivo passage. SHIV Ba-L provides a potentially useful model for R5 HIV-1 infection of humans.     

Role of beta-chemokines in HIV-1 infection of dendritic cells maturing from CD34+ stem cells.

OBJECTIVES: To study the susceptibility to infection by different strains of HIV-1 viruses and the roles of chemokines (macrophage inflammatory protein-1alpha [MIP-1alpha], MIP-1beta, and regulated-on-activation-T-expressed-and-secreted [RANTES]) in CD34+ stem cells maturing into dendritic cells (DC). DESIGN: It has been controversial whether CD34+ stem cells are susceptible to HIV-1 infection and whether high levels of beta-chemokines are beneficial for suppressing HIV-1 infection during DC maturation. These questions were addressed using different strains of HIV-1 and CD34+ stem cells taken from cord blood and cultured with granulocyte-macrophage colony stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha) to generate mature DC. METHODS: CD34+ stem cells were exposed with M-tropic virus Ba-L or T-tropic viruses IIIB or Rut at day 1. Beta-chemokines were added to some cells before the virus and kept throughout the culture. Virus replication was measured throughout the maturation of these cells into CD1a+ DC and CD1a- CD14+ cells using enzyme-linked immunosorbent assay (ELISA) for p24, nested polymerase chain reaction (PCR) for env and intracellular p24 detection by flow cytometry. RESULTS: First, CD34+ stem cells acquired or were infected by live virus because maturing cells showed infection by both M- and T-tropic viruses. Second, the viruses replicated actively during the maturation of CD34+ stem cells toward CD1a+ DC and CD1a- CD14+ cells. Third, beta-chemokines suppressed infection by M-tropic virus Ba-L. And finally, beta-chemokines enhanced infection by T-tropic viruses IIIB and Rut. CONCLUSIONS: In addition to the initial anti-M-tropic virus effect by beta-chemokines, selective pressure on viruses may also result because of an increase in susceptibility to T-tropic virus. Caution should be taken when evaluating the effect of beta-chemokine receptor agonists in AIDS therapy.     

Transduction efficiency of MLV but not of HIV-1 vectors is pseudotype dependent on human primary T lymphocytes

The success of several gene therapeutic approaches requires efficient transduction of human primary T lymphocytes. For this it is important to enhance the transduction efficiency, and this can be achieved by various means, mainly technical development of transduction procedures and use of different vectors and vector pseudotypes. We analyzed the transduction efficiency of an HIV-1 vector encoding enhanced green fluorescent protein (GFP) as a marker gene and pseudotyped with the envelopes of MLV-A, MLV-10A1, GaLV, RD114, and VSV for human primary T lymphocytes in comparison to an MLV vector pseudotyped with the same envelopes. Pseudotyping of the MLV vector with the envelopes of 10A1 and GaLV resulted in efficient transduction of preactivated human primary T lymphocytes (32.4% and 32.7% CD3⁺/GFP⁺ cells, respectively) while MLV-A (14.0%), RD114 (8.8%), and VSV (1.5%) envelopes were less efficient when using titrated vector stocks equilibrated to a multiplicity of infection of 1. In contrast, the HIV-1 vectors pseudotyped with these envelope proteins transduced preactivated T lymphocytes with similar efficiency (approx. 20% CD3⁺/GFP⁺ cells). Thereby, CD4⁺ and CD8⁺ T lymphocyte subpopulations were transduced at equivalent levels. The similar performance of the different HIV-1 vector pseudotypes may be due in part to the similar half-lives of the vector particles. Independently of the envelope used for pseudotyping neither the MLV nor the HIV-1 vectors yielded any significant transduction in nonactivated T lymphocytes (below 0.55% of GFP⁺ cells)Abbreviations FCS Fetal calf serum - GaLV Gibbon ape leukemia virus - GFP Green fluorescent protein - GFU Green fluorescent colony-forming unit - GP Glycoprotein - HIV Human immunodeficiency virus - IL Interleukin - mAb Monoclonal antibody - MLV Murine leukemia virus - MOI Multiplicity of infection - PBL Peripheral blood lymphocyte - PHA Phytohemagglutinin - VSV-G Vesicular stomatitis virus glycoprotein     

Long-term productive human immunodeficiency virus-1 infection in human infant microglia.

The course of human immunodeficiency virus 1 (HIV-1) infection in human infant microglia was studied using purified primary cultures of microglia derived from brain autopsy tissue. Previous in vitro studies have used fetal or adult brain tissue. Important differences may exist between brain tissues of different maturational ages with regard to HIV-1 replication and other neuropathogenic effects. Infant microglia were infected with four different strains of HIV-1 (JR-FL, JR-CSF, Ba-L, and IIIB). Productive infection was demonstrated by p24 antigen production, immunocytochemistry, and recovery of replication-competent virus from the supernatants of the infected cultures. Multinucleated giant cells developed in culture mimicking the neuropathological changes seen in the brains of patients with HIV encephalopathy. Productive infection was more readily established by monocyte-tropic strains (JR-FL and Ba-L) of HIV-1 than by a lymphocyte-tropic strain (IIIB). p24 antigen production in this system peaked at 47 to 51 days postinfection. Viral persistence in giant cells was demonstrated by immunocytochemistry for the gp120 and gp41 viral antigens as late as 70 days postinfection. This in vitro culture system, using infant microglia that support viral replication for more than 2 months, may provide a useful model for studying the pathogenesis of progressive HIV encephalopathy.     

Induction of HIV-1 replication in latently infected syncytiotrophoblast cells by contact with placental macrophages: role of interleukin-6 and tumor necrosis factor-alpha.

The syncytiotrophoblast (ST) layer of the human placenta has an important role in limiting transplacental viral spread from mother to fetus. Although certain strains of human immunodeficiency virus type 1 (HIV-1) may enter ST cells, the trophoblast does not exhibit permissiveness for HIV-1. The present study tested the possibility that placental macrophages might induce replication of HIV-1 carried in ST cells and, further, that infected ST cells would be capable of transmitting virus into neighboring macrophages. For this purpose, we investigated HIV-1 replication in ST cells grown alone or cocultured with uninfected placental macrophages. The macrophage-tropic Ba-L strain of HIV-1, capable of entering ST cells, was used throughout our studies. We demonstrated that interactions between ST cells and macrophages activated HIV-1 from latency and induced its replication in ST cells. After having become permissive for viral replication, ST cells delivered HIV-1 to the cocultured macrophages, as evidenced by detection of virus-specific antigens in these cells. The stimulatory effect of coculture on HIV-1 gene expression in ST cells was mediated by marked tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) release from macrophages, an effect caused by contact between the different placental cells. Results of this study suggest an interactive role for the ST layer and placental macrophages in the dissemination of HIV-1 among placental tissue. Data reported here may also explain why macrophage-tropic HIV-1 strains are transmitted preferentially during pregnancy.     

Inhibition of M-tropic HIV-1 infection by the fd phage-gene 3 protein with MIP-1alpha-binding activity

CCR5 is a chemokine receptor with seven transmembrane-domains. It is expressed on T cells and macrophages and functions as the principal co-receptor for macrophage (M)-tropic strains of HIV-1. The anti-CCR5 monoclonal antibody (mAb) 2D7 inhibits the binding and chemotaxis of the three natural beta-chemokine ligands of CCR5, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES, to CCR5(+) cells. The mAb also efficiently blocks the infectivity of several M-tropic and dual-tropic HIV-1 strains in vitro.In this study, we attempted to determine the peptide motif recognized with the 2D7 mAb. We isolated phage clones by panning a phage display library using 2D7 and identified three peptide motifs. One of these phage clones (M23) showed a marked inhibitory activity on HIV-1 infection. The unique sequence of 15 amino acids with an internal disulfide bond was inserted in the g3p of the M23 phage clone (M23-g3p). The M23-g3p was purified by fast-performance liquid chromatography (FPLC). We show here that (1) M23-g3p was specifically recognized with anti-CCR5 mAb; (2) M23-g3p showed inhibitory activity on the infectivity of M-tropic but not T-tropic HIV-1 strains; (3) M23-g3p bound to MIP-1alpha, MIP-1beta, and RANTES but not MCP-1. These results suggested that the M23-g3p might mimic the CCR5-binding domain shared by beta-chemokines, MIP-1alpha, MIP-1beta, and RANTES as well as the HIV-1 infection.     

Inhibition of M-tropic HIV-1 infection by the fd phage-gene 3 protein with MIP-1α-binding activity

CCR5 is a chemokine receptor with seven transmembrane-domains. It is expressed on T cells and macrophages and functions as the principal co-receptor for macrophage (M)-tropic strains of HIV-1. The anti-CCR5 monoclonal antibody (mAb) 2D7 inhibits the binding and chemotaxis of the three natural β-chemokine ligands of CCR5, macrophage inflammatory protein (MIP)-1α, MIP-1β, and RANTES, to CCR5⁺ cells. The mAb also efficiently blocks the infectivity of several M-tropic and dual-tropic HIV-1 strains in vitro.In this study, we attempted to determine the peptide motif recognized with the 2D7 mAb. We isolated phage clones by panning a phage display library using 2D7 and identified three peptide motifs. One of these phage clones (M23) showed a marked inhibitory activity on HIV-1 infection. The unique sequence of 15 amino acids with an internal disulfide bond was inserted in the g3p of the M23 phage clone (M23-g3p). The M23-g3p was purified by fast-performance liquid chromatography (FPLC). We show here that (1) M23-g3p was specifically recognized with anti-CCR5 mAb; (2) M23-g3p showed inhibitory activity on the infectivity of M-tropic but not T-tropic HIV-1 strains; (3) M23-g3p bound to MIP-1α, MIP-1β, and RANTES but not MCP-1. These results suggested that the M23-g3p might mimic the CCR5-binding domain shared by β-chemokines, MIP-1α, MIP-1β, and RANTES as well as the HIV-1 infection.     

Oral epithelial cells are susceptible to cell-free and cell-associated HIV-1 infection in vitro

Epithelial cells lining the oral cavity are exposed to HIV-1 through breast-feeding and oral-genital contact. Genital secretions and breast milk of HIV-1-infected subjects contain both cell-free and cell-associated virus. To determine if oral epithelial cells can be infected with HIV-1 we exposed gingival keratinocytes and adenoid epithelial cells to cell-free virus and HIV-1-infected peripheral blood mononuclear cells and monocytes. Using primary isolates we determined that gingival keratinocytes are susceptible to HIV-1 infection via cell-free CD4-independent infection only. R5 but not X4 viral strains were capable of infecting the keratinocytes. Further, infected cells were able to release infectious virus. In addition, primary epithelial cells isolated from adenoids were also susceptible to infection; both cell-free and cell-associated virus infected these cells. These data have potential implications in the transmission of HIV-1 in the oral cavity.     

Enhanced replication of R5 HIV-1 over X4 HIV-1 in CD4(+)CCR5(+)CXCR4(+) T cells

To enter human cells, HIV-1 usually uses CD4 and 1 of 2 coreceptors: CCR5 and CXCR4. Interestingly, even though CCR5 is expressed on far fewer T cells than is CXCR4, many patients in early- and late-stage HIV disease maintain high levels of CCR5-tropic (R5) viruses. We hypothesized that such high R5 viral loads may be sustained because, relative to CXCR4-tropic (X4) HIV-1 infection, R5 HIV-1 infection of permissive CD4(+)CCR5(+)CXCR4(+) T cells results in the production of significantly more infectious virus particles per target cell. To investigate this possibility, we compared the levels of virus production per target cell after isogenic R5 and X4 HIV-1 infection of 2 in vitro primary human lymphocyte culture systems: T-cell receptor-stimulated blood-derived CD4(+) T cells and tonsil histoculture (which requires no exogenous stimulation for ex vivo infection). We provide evidence that R5 HIV-1 does indeed compensate for a small target cell population by producing, on average, 5 to 10 times more infectious virus per CCR5(+) target cell than X4 HIV-1. This replicative advantage may contribute to the predominance of R5 HIV-1 in vivo.     

Selective elimination of HIV-1-infected cells by Env-directed, HIV-1-based virus-like particles

We recently showed that both replicating and resting cells cultivated with ganciclovir (GCV) were killed when challenged with vesicular stomatitis virus G glycoprotein pseudotyped HIV-1-based virus-like particles (VLPs) carrying the Nef7 (i.e., an HIV-1 Nef mutant incorporating in virions at high levels)/herpes simplex virus-1 thymidine kinase (HSV-TK) fusion product. On this basis, a novel anti-HIV therapeutic approach based on Nef7/TK VLPs expressing X4 or R5 HIV cell receptor complexes has been attempted. We here report that (CD4-CXCR4) and (CD4-CCR5) Nef7-based VLPs efficiently enter cells infected by X4- or R5-tropic HIV-1 strains, respectively. Importantly, the delivery of the VLP-associated Nef7/TK led to cell death upon GCV treatment. Of interest, VLPs were effective also against non-replicating, HIV-1-infected primary human monocyte-derived macrophages. HIV-targeted VLPs represent a promising candidate for the treatment of persistently HIV-1-infected cells that are part of virus reservoirs resistant to HAART therapies.     

Glutathione and growth inhibition of Mycobacterium tuberculosis in healthy and HIV infected subjects

Background

The high rate of HIV-1 mutation and increasing resistance to currently available antiretroviral (ART) therapies highlight the need for new antiviral agents. Products derived from natural sources have been shown to inhibit HIV-1 replication during various stages of the virus life cycle, and therefore represent a potential source of novel therapeutic agents. To expand our arsenal of therapeutics against HIV-1 infection, we investigated aqueous extract from Sargassum fusiforme (S. fusiforme) for ability to inhibit HIV-1 infection in the periphery, in T cells and human macrophages, and for ability to inhibit in the central nervous system (CNS), in microglia and astrocytes.

Results

S. fusiforme extract blocked HIV-1 infection and replication by over 90% in T cells, human macrophages and microglia, and it also inhibited pseudotyped HIV-1 (VSV/NL4-3) infection in human astrocytes by over 70%. Inhibition was mediated against both CXCR4 (X4) and CCR5 (R5)-tropic HIV-1, was dose dependant and long lasting, did not inhibit cell growth or viability, was not toxic to cells, and was comparable to inhibition by the nucleoside analogue 2', 3'-didoxycytidine (ddC). S. fusiforme treatment blocked direct cell-to-cell infection spread. To investigate at which point of the virus life cycle this inhibition occurs, we infected T cells and CD4-negative primary human astrocytes with HIV-1 pseudotyped with envelope glycoprotein of vesicular stomatitis virus (VSV), which bypasses the HIV receptor requirements. Infection by pseudotyped HIV-1 (VSV/NL4-3) was also inhibited in a dose dependant manner, although up to 57% less, as compared to inhibition of native NL4-3, indicating post-entry interferences.

Conclusion

This is the first report demonstrating S. fusiforme to be a potent inhibitor of highly productive HIV-1 infection and replication in T cells, in primary human macrophages, microglia, and astrocytes. Results with VSV/NL4-3 infection, suggest inhibition of both entry and post-entry events of the virus life cycle. Absence of cytotoxicity and high viability of treated cells also suggest that S. fusiforme is a potential source of novel naturally occurring antiretroviral compounds that inhibit HIV-1 infection and replication at more than one site of the virus life cycle.     

An anti-CCR5 monoclonal antibody and small molecule CCR5 antagonists synergize by inhibiting different stages of human immunodeficiency virus type 1 entry

HIV-1 coreceptors are attractive targets for novel antivirals. Here, inhibition of entry by two classes of CCR5 antagonists was investigated. We confirmed previous findings that HIV-1 isolates vary greatly in their sensitivity to small molecule inhibitors of CCR5-mediated entry, SCH-C and TAK-779. In contrast, an anti-CCR5 monoclonal antibody (PA14) similarly inhibited entry of diverse viral isolates. Sensitivity to small molecules was V3 loop-dependent and inversely proportional to the level of gp120 binding to CCR5. Moreover, combinations of the MAb and small molecules were highly synergistic in blocking HIV-1 entry, suggesting different mechanisms of action. This was confirmed by time course of inhibition experiments wherein the PA14 MAb and small molecules were shown to inhibit temporally distinct stages of CCR5 usage. We propose that small molecules inhibit V3 binding to the second extracellular loop of CCR5, whereas PA14 preferentially inhibits subsequent events such as CCR5 recruitment into the fusion complex or conformational changes in the gp120-CCR5 complex that trigger fusion. Importantly, our findings suggest that combinations of CCR5 inhibitors with different mechanisms of action will be central to controlling HIV-1 infection and slowing the emergence of resistant strains.     

CCR5 as target for HIV-1 gene therapy

Acquired immune deficiency syndrome (AIDS) is caused by a lentivirus, human immunodeficiency virus type-1 (HIV-1). Viral entry is mediated by specific interaction of the viral envelope (Env) glycoprotein with a cell surface molecule CD4 which serves as the primary receptor and a chemokine (C-C or C-X-C motif) receptor CCR5 or CXCR4 which serves as a co-receptor. The viral Env, the cellular CD4 receptor, or the CCR5/CXCR4 co-receptors may be the targets of therapeutic interventions. Compared to the high variability of the viral Env protein, lack of variability in the CD4 receptor and the CCR5 or CXCR4 co-receptor makes them better targets to prevent viral entry. Downregulation of CD4 or CXCR4 is likely to have harmful consequences for the immune function or cellular maturation and homing. In contrast, individuals who lack functional CCR5 have no apparent immune defects, and show decreased susceptibility to HIV-1 infection and delayed progression to AIDS. CCR5 is essential for HIV-1 infection through all routes of transmission. Therefore, its downregulation may not only prevent disease progression, but also the spread of HIV-1 transmission. To block CCR5 function, a number of molecules were developed, including low molecular weight compounds, chemokines, N-terminally-modified chemokine analogues, chemokine-derived molecules, chemokine-based synthetic peptides, and anti-CCR5 monoclonal antibodies. Gene therapy strategies were developed using intrakines and intrabodies to prevent cell surface expression of CCR5 and zinc finger-nucleases, or using small interfering RNAs, antisense RNAs, or ribozymes to decrease co-receptor synthesis. This review describes the importance of targeting CCR5 and summarizes the status of various anti-CCR5 gene therapy strategies.     

Uncoupling coreceptor usage of human immunodeficiency virus type 1 (HIV-1) from macrophage tropism reveals biological properties of CCR5-restricted HIV-1 isolates from patients with acquired immunodeficiency syndrome

The mechanisms underlying the pathogenicity of CCR5-restricted (R5) human immunodeficiency virus type-1 (HIV-1) strains are incompletely understood. Acquisition or enhancement of macrophage (M)-tropism by R5 viruses contributes to R5 HIV-1 pathogenesis. In this study, we show that M-tropic R5 viruses isolated from individuals with acquired immunodeficiency syndrome (late R5 viruses) require lower levels of CD4/CCR5 expression for entry, have decreased sensitivity to inhibition by the entry inhibitors TAK-779 and T-20, and have increased sensitivity to neutralization by the Env MAb IgG1b12 compared with non-M-tropic R5 viruses isolated from asymptomatic, immunocompetent individuals (early R5 viruses). Augmenting CCR5 expression levels on monocyte-derived macrophages via retroviral transduction led to a complete or marginal restoration of M-tropism by early R5 viruses, depending on the viral strain. Thus, reduced CD4/CCR5 dependence is a phenotype of R5 HIV-1 associated with M-tropism and late stage infection, which may affect the efficacy of HIV-1 entry inhibitors.     

Infection of macrophages and dendritic cells with primary R5-tropic human immunodeficiency virus type 1 inhibited by natural polyreactive anti-CCR5 antibodies purified from cervicovaginal secretions

Heterosexual contact is the primary mode of human immunodeficiency virus (HIV) type 1 (HIV-1) transmission worldwide. The chemokine receptor CCR5 is the major coreceptor that is associated with the mucosal transmission of R5-tropic HIV-1 during sexual intercourse. The CCR5 molecule is thus a target for antibody-based therapeutic strategies aimed at blocking HIV-1 entry into cells. We have previously demonstrated that polyreactive natural antibodies (NAbs) from therapeutic preparations of immunoglobulin G and from human breast milk contain NAbs directed against CCR5. Such antibodies inhibit the infection of human macrophages and T lymphocytes by R5-tropic isolates of HIV in vitro. In the present study, we demonstrate that human immunoglobulins from the cervicovaginal secretions of HIV-seronegative or HIV-seropositive women contain NAbs directed against the HIV-1 coreceptor CCR5. Natural affinity-purified anti-CCR5 antibodies bound to CCR5 expressed on macrophages and dendritic cells and further inhibited the infection of macrophages and dendritic cells with primary and laboratory-adapted R5-tropic HIV but not with X4-tropic HIV. Natural anti-CCR5 antibodies moderately inhibited R5-tropic HIV transfer from monocyte-derived dendritic cells to autologous T cells. Our results suggest that mucosal anti-CCR5 antibodies from healthy immunocompetent donors may hamper the penetration of HIV and may be suitable for use in the development of novel passive immunotherapy regimens in specific clinical settings of HIV infection.     

Neuraminidase from a bacterial source enhances both HIV-1-mediated syncytium formation and the virus binding/entry process

Neuraminidases, also termed sialidases, which catalyze the removal of sialic acid residues from various glycoconjugates, have been previously reported to modulate HIV-1 replication. Given that some of the known opportunistic microbes found in patients infected with HIV-1 harbor neuraminidase (NA) activity, we speculated that pathogen-derived NA might be envisaged as an important factor in the pathogenesis of this retroviral infection. In the present study, we have monitored the putative modulation of HIV-1-mediated syncytium formation and virus replication by highly purified bacterial-derived NA from Arthrobacter ureafaciens. Taking advantage of a luciferase-based syncytium quantitative assay, we demonstrate here that the level of HIV-1-mediated syncytium formation is enhanced in the presence of NA and that it necessitates interaction between gp120 and CD4/chemokine coreceptor. By using pseudotyped recombinant luciferase-encoding HIV-1 particles, we found that NA treatment of human CD4-positive target cells (i.e., T lymphoid, monocytoid, and peripheral blood mononuclear cells) significantly augmented single-round infection by T- and macrophage-tropic isolates of HIV-1. The observed increase in HIV-1 infection was linked with an enhancement in the initial steps of the virus replicative cycle as monitored by viral binding and entry assays. Interestingly, NA treatment also enhances infectivity of HIV-1 pseudotypes with envelope glycoprotein from the amphotropic murine leukemia virus or the vesicular stomatitis virus. Taken together, our results provide useful information regarding the possible contribution of microbial agents carrying NA activity to HIV-1 pathogenesis.     

In vitro exposure to highly cytopathic HIV-1 X4 strains increases expression of mucosa-associated integrins on CD4(+) T cells

To determine whether infection with HIV-1 strains of different tropisms would influence expression of the mucosa-associated integrins alpha 4 beta 7 and alpha E beta 7 or the lymph node homing receptor L-selectin on peripheral T lymphocytes, cells were infected with the CXCR4-tropic (X4)/syncytium-inducing (SI) HIV-1(IIIB) strain or with X4/SI or CCR5-tropic (R5)/non-SI (NSI) primary human isolates. Flow cytometric analyses of CD4(+) T cells from cultures infected with HIV-1(IIIB) and one X4/SI primary HIV-1 isolate revealed a significant increase in surface expression of alpha 4 beta 7 and alpha E beta 7 12 days after infection. L-selectin expression was not significantly affected on CD4(+) T cells. However, infection with another X4/SI and two R5/NSI primary HIV-1 isolates did not significantly alter homing receptor expression on CD4(+) T cells. Since a higher degree of CD4 cytopathicity occurred in those cultures having increased integrin expression, these data suggest that significantly altered mucosal homing receptor expression on CD4(+) T cells may result as a "bystander" effect after infection with some cytopathic isolates of HIV-1.