Productive infection of primary macrophages with human herpesvirus 7

Here we demonstrate replication of human herpesvirus 7 (HHV-7), a T-lymphotropic virus, in macrophages. Productive replication was lost after 2 weeks, but HHV-7 DNA was detected up to 1 month after infection. Thus, macrophages become infected by HHV-7 and might play an important role as a viral reservoir, as has been demonstrated for human immunodeficiency virus type 1.     

Productive HIV-1 infection of primary CD4+ T cells induces mitochondrial membrane permeabilization leading to a caspase-independent cell death.

We have explored in vitro the mechanism by which human immunodeficiency virus, type 1 (HIV-1) induces cell death of primary CD4+ T cells in conditions of productive infection. Although HIV-1 infection primed phytohemagglutinin-activated CD4+ T cells for death induced by anti-CD95 antibody, T cell death was not prevented by a CD95-Fc decoy receptor, nor by decoy receptors of other members of the TNFR family (TNFR1/R2, TRAILR1/R2/OPG, TRAMP) or by various blocking antibodies, suggesting that triggering of death receptors by their cognate ligands is not involved in HIV-induced CD4 T cell death. HIV-1 induced CD4 T cell shrinkage, cell surface exposure of phosphatidylserine, loss of mitochondrial membrane potential (Deltapsim), and mitochondrial release of cytochrome c and apoptosis-inducing factor. A typical apoptotic phenotype (nuclear chromatin condensation and fragmentation) only occurred in around half of the dying cells. Treatment with benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, a broad spectrum caspase inhibitor, prevented nuclear chromatin condensation and fragmentation in HIV-infected CD4+ T cells and in a cell-free system (in which nuclei were incubated with cytoplasmic extracts from the HIV-infected CD4+ T cells). Nevertheless, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone did not prevent mitochondrial membrane potential loss and cell death, suggesting that caspases are dispensable for HIV-mediated cell death. Our findings suggest a major role of the mitochondria in the process of CD4 T cell death induced by HIV, in which targeting of Bax to the mitochondria may be involved.     

Siglecs facilitate HIV-1 infection of macrophages through adhesion with viral sialic acids

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) infects macrophages effectively, despite relatively low levels of cell surface-expressed CD4. Although HIV-1 infections are defined by viral tropisms according to chemokine receptor usage (R5 and X4), variations in infection are common within both R5- and X4-tropic viruses, indicating additional factors may contribute to viral tropism. METHODOLOGY AND PRINCIPAL FINDINGS: Using both solution and cell surface binding experiments, we showed that R5- and X4-tropic HIV-1 gp120 proteins recognized a family of I-type lectin receptors, the Sialic acid-binding immunoglobulin-like lectins (Siglec). The recognition was through envelope-associated sialic acids that promoted viral adhesion to macrophages. The sialic acid-mediated viral-host interaction facilitated both R5-tropic pseudovirus and HIV-1(BaL) infection of macrophages. The high affinity Siglec-1 contributed the most to HIV-1 infection and the variation in Siglec-1 expression on primary macrophages from different donors was associated statistically with sialic acid-facilitated viral infection. Furthermore, envelope-associated sialoglycan variations on various strains of R5-tropic viruses also affected infection. CONCLUSIONS AND SIGNIFICANCE OF THE FINDINGS: Our study showed that sialic acids on the viral envelope facilitated HIV-1 infection of macrophages through interacting with Siglec receptors, and the expression of Siglec-1 correlated with viral sialic acid-mediated host attachment. This glycan-mediated viral adhesion underscores the importance of viral sialic acids in HIV infection and pathogenesis, and suggests a novel class of antiviral compounds targeting Siglec receptors.     

HIV-1-specific T cell precursors with high proliferative capacity correlate with low viremia and high CD4 counts in untreated individuals

Evidences have recently suggested that the preservation of vaccine-induced memory rather than effector T cells is essential for better outcome and survival following pathogenic SIV challenge in macaques. However, an equivalent demonstration in humans is missing, and the immune correlates of HIV-1 control have been only partially characterized. We focused on the quantification of Ag-specific T cell precursors with high proliferative capacity (PHPC) using a peptide-based cultured IFN-gamma ELISPOT assay (PHPC assay), which has been shown to identify expandable memory T cells. To determine which responses correlate with viral suppression and positive immunologic outcome, PBMC from 32 chronically untreated HIV-1-infected individuals were evaluated in response to peptide pools, representing the complete HIV-1 Gag, Nef, and Rev proteins, by PHPC and IFN-gamma ELISPOT assay, which instead identifies effector T cells with low proliferative capacity. High magnitude of Gag-specific PHPC, but not ELISPOT, responses significantly correlated with low plasma viremia, due to responses directed toward p17 and p15 subunits. Only Gag p17-specific PHPC response significantly correlated with high CD4 counts. Analysis of 20 additional PBMC samples from an independent cohort of chronically untreated HIV-1-infected individuals confirmed the correlation between Gag p17-specific PHPC response and either plasma viremia (inverse correlation) or CD4 counts (direct correlation). Our results indicate that the PHPC assay is quantitatively and qualitatively different from the ELISPOT assay, supporting that different T cell populations are being evaluated. The PHPC assay might be an attractive option for individual patient management and for the design and testing of therapeutic and prophylactic vaccines.     

Morphine and galectin-1 modulate HIV-1 infection of human monocyte-derived macrophages

Morphine is a widely abused, addictive drug that modulates immune function. Macrophages are a primary reservoir of HIV-1; therefore, they play a role in the development of this disease, as well as impact the overall course of disease progression. Galectin-1 is a member of a family of β-galactoside-binding lectins that are soluble adhesion molecules and that mediate direct cell-pathogen interactions during HIV-1 viral adhesion. Because the drug abuse epidemic and the HIV-1 epidemic are closely interrelated, we propose that increased expression of galectin-1 induced by morphine may modulate HIV-1 infection of human monocyte-derived macrophages (MDMs). In this article, we show that galectin-1 gene and protein expression are potentiated by incubation with morphine. Confirming previous studies, morphine alone or galectin-1 alone enhance HIV-1 infection of MDMs. Concomitant incubation with exogenous galectin-1 and morphine potentiated HIV-1 infection of MDMs. We used a nanotechnology approach that uses gold nanorod-galectin-1 small interfering RNA complexes (nanoplexes) to inhibit gene expression for galectin-1. We found that nanoplexes silenced gene expression for galectin-1, and they reversed the effects of morphine on galectin-1 expression. Furthermore, the effects of morphine on HIV-1 infection were reduced in the presence of the nanoplex.     

Reconstitution of virus-specific CD4 proliferative responses in pediatric HIV-1 infection

Gag-specific CD4 proliferative responses correlate inversely with HIV-1 RNA levels in infected adults, and robust responses are characteristic of long-term nonprogressive infection. However, strong responses are seldom detected in adult subjects with progressive infection and are not generally reconstituted on highly active antiretroviral therapy (HAART). To date, the role of HIV-1-specific Th responses in children has not been thoroughly examined. We characterized Gag-specific CD4 responses among 35 perinatally infected subjects, including 2 children who spontaneously control viremia without antiretroviral therapy, 21 children with viral loads (VL) of <400 on HAART, and 12 viremic children. Gag-specific Th activity was assessed by lymphoproliferative assay, and responses were mapped using overlapping Gag peptides in an IFN-gamma ELISPOT. Robust proliferative responses were detected in the children exhibiting spontaneous control of viremia, and mapping of targeted Gag regions in one such subject identified multiple epitopes. Among children >or=5 years old, 14 of 17 subjects with VL of <400 on HAART demonstrated a significant p24 proliferative response (median p24 stimulation index, 20), in contrast with only 1 of 9 viremic children (median p24 stimulation index, 2.0; p = 0.0008). However, no subject younger than 5 years of age possessed a significant response, even when viremia was fully suppressed. When compared with adults with VL of <400 on HAART, Th responses among children with VL of <400 were both more frequent (p = 0.009) and of greater magnitude (p = 0.002). These data suggest that children may have a greater intrinsic capacity to reconstitute HIV-1-specific immunity than adults, and may be excellent candidates for immune-based therapies.     

HIV-1-specific B cell activation. A major constituent of spontaneous B cell activation during HIV-1 infection

B cell activation is a well known consequence of HIV-1 infection, and seropositive subjects show high numbers of spontaneously activated Ig-secreting cells in circulation. To better define the importance of the HIV-1-specific response in this phenomenon, we first studied whether in vitro spontaneous anti-HIV-1 antibody production was accompanied by reactivation of memory B lymphocytes. Unstimulated PBL from HIV-1-infected individuals with prior history of hepatitis B and/or EBV infection did not consistently show spontaneous in vitro synthesis of anti-hepatitis B core Ag or anti-EBV antibodies; in addition, PWM-induced synthesis of anti-hepatitis B virus and anti-EBV antibodies was decreased compared to HIV-1-seronegative subjects. Moreover, in comparing the frequencies of activated HIV-1-specific B cell precursors and activated Ig-secreting precursors in limiting dilution experiments, a sizable fraction (20 to 40%) of circulating cells spontaneously secreting Ig produced antibody against HIV-1 determinants. The ratio between the two frequencies fitted in very well with the amount of Ig removed from unstimulated culture supernatants after HIV-1-specific antibody absorption with solid-phase HIV-1. These findings indicate that B cell activation during HIV-1 infection is mainly oriented toward a specific response to HIV-1 determinants; the possible relevance of this phenomenon to lymphomagenesis in AIDS patients is discussed.     

B cell responses to HIV-1 infection and vaccination: pathways to preventing infection

The B cell arm of the immune response becomes activated soon after HIV-1 transmission, yet the initial antibody response does not control HIV-1 replication, and it takes months for neutralizing antibodies to develop against the autologous virus. Antibodies that can be broadly protective are made only in a minority of subjects and take years to develop--too late to affect the course of disease. New studies of the earliest stages of HIV-1 infection, new techniques to probe the human B cell repertoire, the modest degree of efficacy in a vaccine trial and new studies of human monoclonal antibodies that represent the types of immune responses an HIV-1 vaccine should induce are collectively illuminating paths that a successful HIV-1 vaccine might take.     

NK cell function in HIV-1 infection

NK cells are critical effector cells of the innate immune response to malignancy and infection. These cells have a wide array of direct antiviral activities and have been critically implicated in the regulation and induction of an effective adaptive immune response. Although the pivotal role of this cell subset in the context of a number of viral infections is well established, the role of NK cells in HIV-1 infection is less well understood. Recent data has demonstrated the association between an NK cell receptor, KIR3DS1, and it's ligand, HLA-Bw4 with an isoleucine at position 80, and slower disease progression. This data suggests that NK cells may play an essential role in the control of HIV-1 disease, and has provided the impetus to begin to better understand the role of this cell subset in the context of HIV-1 infection, replication, and pathogenesis. Here we present a review of the literature pertaining to both the effect of HIV-1 infection on NK cell activity and the potential role that this subset of cells may play in controlling HIV-1 disease.     

HIV-1 replication increases HIV-specific CD4+ T cell frequencies but limits proliferative capacity in chronically infected children

This study investigated the relationship between HIV-1 replication and virus (HIV-1; CMV)-specific CD4(+) T cell frequency and function in HIV-1-infected children. HIV-1 gag p55-specific CD4(+) T cell IFN-gamma responses were detected in the majority of children studied. p55-specific responses were detected less commonly and at lower frequencies in children with <50 copies/ml plasma HIV-1 RNA than in children with active HIV-1 replication. In children with <50 copies/ml plasma HIV-1, p55-specific responses were detected only in children with evidence of ongoing HIV-1 replication, indicating a direct relationship between HIV-1 replication and HIV-specific CD4(+) T cell frequencies. In contrast, p55-specific proliferative responses were detected more frequently in children with <50 copies/ml plasma HIV-1. CMV-specific CD4(+) responses were more commonly detected and at higher frequencies in CMV-coinfected children with suppressed HIV-1 replication. The lack of HIV-specific CD4(+) proliferative responses, along with the preservation of CMV-specific CD4(+) responses in children with controlled HIV-1 replication, suggests that viral replication may have deleterious effects on HIV-1 and other virus-specific CD4(+) responses. Vaccination to stimulate HIV-specific CD4(+) T cell responses in these children may synergize with antiretroviral therapy to improve the long-term control of viral replication, and may perhaps allow the eventual discontinuation of antiretroviral therapy.     

Fumagillin suppresses HIV-1 infection of macrophages through the inhibition of Vpr activity

HIV-1 viral protein R (Vpr) is one of the human immunodeficiency virus type 1 encoded proteins that have important roles in viral pathogenesis. However, no clinical drug for AIDS therapy that targets Vpr has been developed. Here, we have established a screening system to isolate Vpr inhibitors using budding yeast cells. We purified a Vpr inhibitory compound from fungal metabolites and identified it as fumagillin, a chemical already known to be a potent inhibitor of angiogenesis. Fumagillin not only reversed the growth inhibitory activity of Vpr in yeast and human cells, but also inhibited Vpr-dependent viral gene expression upon the infection of human macrophages.     

Preservation of T cell proliferation restricted by protective HLA alleles is critical for immune control of HIV-1 infection

HIV-1-infected persons with HLA-B27 and -B57 alleles commonly remain healthy for decades without antiretroviral therapy. Properties of CD8+ T cells restricted by these alleles considered to confer disease protection in these individuals are elusive but important to understand and potentially elicit by vaccination. To address this, we compared CD8+ T cell function induced by HIV-1 immunogens and natural infection using polychromatic flow cytometry. HIV-1-specific CD8+ T cells from all four uninfected immunized and 21 infected subjects secreted IFN-gamma and TNF-alpha. However, CD8+ T cells induced by vaccination and primary infection, but not chronic infection, proliferated to their cognate epitopes. Notably, B27- and B57-restricted CD8+ T cells from nonprogressors exhibited greater expansion than those restricted by other alleles. Hence, CD8+ T cells restricted by certain protective alleles can resist replicative defects, which permits expansion and antiviral effector activities. Our findings suggest that the capacity to maintain CD8+ T cell proliferation, regardless of MHC-restriction, may serve as an important correlate of disease protection in the event of infection following vaccination.     

Purinergic receptors are required for HIV-1 infection of primary human macrophages

Macrophages play a significant role in HIV infection, viral rebound, and the development of AIDS. However, the function of host proteins in viral replication is incompletely characterized in macrophages. Purinergic receptors P2X and P2Y are major components of the macrophage immune response to pathogens, inflammation, and cellular damage. We demonstrate that these receptors are necessary for HIV infection of primary human macrophages. Inhibition of purinergic receptors results in a significant reduction in HIV replication in macrophages. This inhibition is independent of viral strain and is dose dependent. We also identify that P2X(1), P2X(7), and P2Y(1) receptors are involved in viral replication. We show that P2X(1), but not P2X(7) or P2Y(1), is necessary for HIV entry into macrophages. We demonstrate that interaction of the HIV surface protein gp120 with macrophages stimulates an increase in ATP release. Thus, we propose that HIV's binding to macrophages triggers a local release of ATP that stimulates purinergic receptors and facilitates HIV entry and subsequent stages of viral replication. Our data implicate a novel role for a family of host proteins in HIV replication in macrophages and suggest new therapeutic targets to reduce the devastating consequences of HIV infection and AIDS.     

Effect of mannosylated derivatives on HIV-1 infection of macrophages and lymphocytes

We previously demonstrated that gpl20/160 (Env) of HIV-1 interactin a carbohydrate-specific manner with mannosyI/N-acetylglucosaminylderivatives and that HTV-1_LAI infection of monocytic U937 andlymphoid CEM cells was inhibited by CD4-free Concanavalin A-reactiveglyco-peptides from U937 cells. We report here that the naturalglycoproteins bovine fetuin and asialofetuin, human oroso-mucoidand a-fetoprotein, and mannan, which all specifically interactwith Env, inhibited infection of primary mac-rophages by macrophage-tropicHIV-1 strains, whereas dextran had no such effect This activitywas conserved if fetuin, asialofetuin, or orosomucoid were heat-treated,which rules out the role of their three-dimensional structure.Orosomucoid and mannan partially inhibited Env binding to macrophagesbut not to U937 or CEM cells. This indicates that Env does notbind in the same manner to primary macrophages and to immortalizedCD4⁺ cells, and that orosomucoid and mannan act at CD4-independentstages of virus binding to macrophages. Mannan also inhibitedEnv binding to surface glycopeptides obtained after trypsintreatment of macrophages. Furthermore, orosomucoid and fetuininteracted with, and they inhibited the binding of a V3 loopB clade consensus peptide to several macrophage membrane proteins,including two 36 and 42 kDa proteins. These data indicate thatthese glycoproteins interfere with post-binding events duringHIV-1 infection of primary macrophages. In contrast, the compoundsdid not affect infection of U937 or CEM cells by T-cell tropicHIV-1_LAI nor infection of peripheral blood lymphocytes by HIV-1_LAIor HIV-1_BA-L. Thus, carbohydrate-specific inhibition of HIVinfection depends on the cell type more than on the viral strain,and differences in the glycan structure of cell-type-specificcofactors may be important for HIV entry into cells. HIV macrophage lymphocyte inhibition glycoprotein     

T cell responses to human endogenous retroviruses in HIV-1 infection

Human endogenous retroviruses (HERVs) are remnants of ancient infectious agents that have integrated into the human genome. Under normal circumstances, HERVs are functionally defective or controlled by host factors. In HIV-1-infected individuals, intracellular defense mechanisms are compromised. We hypothesized that HIV-1 infection would remove or alter controls on HERV activity. Expression of HERV could potentially stimulate a T cell response to HERV antigens, and in regions of HIV-1/HERV similarity, these T cells could be cross-reactive. We determined that the levels of HERV production in HIV-1-positive individuals exceed those of HIV-1-negative controls. To investigate the impact of HERV activity on specific immunity, we examined T cell responses to HERV peptides in 29 HIV-1-positive and 13 HIV-1-negative study participants. We report T cell responses to peptides derived from regions of HERV detected by ELISPOT analysis in the HIV-1-positive study participants. We show an inverse correlation between anti-HERV T cell responses and HIV-1 plasma viral load. In HIV-1-positive individuals, we demonstrate that HERV-specific T cells are capable of killing cells presenting their cognate peptide. These data indicate that HIV-1 infection leads to HERV expression and stimulation of a HERV-specific CD8+ T cell response. HERV-specific CD8+ T cells have characteristics consistent with an important role in the response to HIV-1 infection: a phenotype similar to that of T cells responding to an effectively controlled virus (cytomegalovirus), an inverse correlation with HIV-1 plasma viral load, and the ability to lyse cells presenting their target peptide. These characteristics suggest that elicitation of anti-HERV-specific immune responses is a novel approach to immunotherapeutic vaccination. As endogenous retroviral sequences are fixed in the human genome, they provide a stable target, and HERV-specific T cells could recognize a cell infected by any HIV-1 viral variant. HERV-specific immunity is an important new avenue for investigation in HIV-1 pathogenesis and vaccine design.     

Human immunodeficiency virus type 1 (HIV-1) diversity at time of infection is not restricted to certain risk groups or specific HIV-1 subtypes

African women frequently acquire several genetically distinct human immunodeficiency virus type 1 (HIV-1) variants from a heterosexual partner, whereas the acquisition of multiple variants appears to be rare in men. To determine whether newly infected individuals in other risk groups acquire genetically diverse viruses, we examined the viral envelope sequences in plasma samples from 13 women and 4 men from the United States infected with subtype B viruses and 10 men from Kenya infected with non-subtype B viruses. HIV-1 envelope sequences differed by more than 2% in three U.S. women, one U.S. man, and one Kenyan man near the time of seroconversion. These findings suggest that early HIV-1 genetic diversity is not exclusive to women from Africa or to infection with any particular HIV-1 subtype.     

Frequent and strong antibody-mediated natural killer cell activation in response to HIV-1 Env in individuals with chronic HIV-1 infection

Natural killer (NK) cells play a critical role in the control of HIV-1 infection, and NK cells that respond to HIV-1 peptides have been recently described. However, the mechanisms by which NK cells recognize HIV-1 antigens are not fully understood. We investigated NK cell activation in response to HIV-1 peptides during early and chronic HIV-1 clade B infection using a whole-blood assay and multiparameter flow cytometry. Antibody-mediated NK cell activation in response to HIV-1 peptides was not detected in HIV-1-uninfected individuals. In contrast, 79% of individuals with chronic infection and 22% of individuals with early infection had detectable gamma interferon (IFN-γ) NK cell responses to HIV-1 antigens (P < 0.00001). IFN-γ- and tumor necrosis factor alpha (TNF-α)-producing NK cells most frequently targeted Env gp120 (median of 4% and range of 0 to 31% of all NK cells). NK cells rarely targeted other HIV-1 proteins such as Gag, Pol, and Nef. Antibody-mediated NK cell responses to peptides mapped predominantly to Env protein, required the presence of plasma or plasma IgG, and resulted in lower CD16 expression on NK cells, suggesting an antibody-mediated activation of NK cells. Further studies are needed to assess the consequences of these antibody-mediated NK cell responses for HIV-1 disease progression and vaccine-induced protection from infection.