Cross-reactivity with SIVmac in east African HIV-1-positive sera: evidence against double infection with HIV-1 and a SIVmac/HIV-2-like virus.

IgG antibodies reactive with simian immunodeficiency virus isolated from a rhesus monkey suffering from simian acquired immunodeficiency syndrome (SIVmac, strain 239, a virus which is very closely related to human immunodeficiency virus type 2-HIV-2) were found in 18 of 120 Swedish and 8 of 11 east African confirmed HIV-1 antibody positive (HIV-1 ab+) sera, both by enzyme immunoassay and electrophoretic immunoblotting (p = 1 x 10(-6). In electrophoretic immunoblotting most of the cross-reactivity of SIVmac-reactive sera occurred on p27, the major gag protein of SIVmac. The possibility that SIVmac antibody reactivity could be due to double infection with HIV-1 and a SIVmac-related virus was eliminated by the results of absorptions between sera of Swedish and west and east African origin and viral antigens (SIVmac and North American or African/Haitian strains of HIV-1) coupled to agarose beads. HIV-2 ab+ and SIVmac reactive west African sera recognized SIVmac epitopes unrelated to HIV-1, whereas HIV-1 ab+, SIVmac reactive east African, and Swedish sera recognized SIVmac epitopes cross-reactive with epitopes present in both African and North American HIV-1 strains. No unique SIVmac-reactive African HIV-1 epitopes could thus be defined. Neither did absorption of Swedish and African HIV-1-positive sera with different HIV-1 strains (1 Haitian, 2 Zairian, and 1 North American) give evidence for unique epitopes.     

Two Orphan Seven-Transmembrane Segment Receptors Which Are Expressed in CD4-positive Cells Support Simian Immunodeficiency Virus Infection

Clinical isolates of primate immunodeficiency viruses, including human immunodeficiency virus type 1 (HIV-1), enter target cells by sequential binding to CD4 and the chemokine receptor CCR5, a member of the seven-transmembrane receptor family. HIV-1 variants which use additional chemokine receptors are present in the central nervous system or emerge during the course of infection. Simian immunodeficiency viruses (SIV) have been shown to use CCR5 as a coreceptor, but no other receptors for these viruses have been identified. Here we show that two orphan seven-transmembrane segment receptors, gpr1 and gpr15, serve as coreceptors for SIV, and are expressed in human alveolar macrophages. The more efficient of these, gpr15, is also expressed in human CD4⁺ T lymphocytes and activated rhesus macaque peripheral blood mononuclear cells. The gpr15 and gpr1 proteins lack several hallmarks of chemokine receptors, but share with CCR5 an amino-terminal motif rich in tyrosine residues. These results underscore the potential diversity of seven-transmembrane segment receptors used as entry cofactors by primate immunodeficiency viruses, and may contribute to an understanding of viral variation and pathogenesis.     

Nonpathogenic SIV infection of African green monkeys induces a strong but rapidly controlled type I IFN response

African green monkeys (AGMs) infected with the AGM type of SIV (SIVagm) do not develop chronic immune activation and AIDS, despite viral loads similar to those detected in humans infected with HIV-1 and rhesus macaques (RMs) infected with the RM type of SIV (SIVmac). Because chronic immune activation drives progressive CD4⁺ T cell depletion and immune cell dysfunctions, factors that characterize disease progression, we sought to understand the molecular basis of this AGM phenotype. To this end, we longitudinally assessed the gene expression profiles of blood- and lymph node–derived CD4⁺ cells from AGMs and RMs in response to SIVagm and SIVmac infection, respectively, using a genomic microarray platform. The molecular signature of acute infection was characterized, in both species, by strong upregulation of type I IFN–stimulated genes (ISGs). ISG expression returned to basal levels after postinfection day 28 in AGMs but was sustained in RMs, especially in the lymph node–derived cells. We also found that SIVagm induced IFN-α production by AGM cells in vitro and that low IFN-α levels were sufficient to induce strong ISG responses. In conclusion, SIV infection triggered a rapid and strong IFN-α response in vivo in both AGMs and RMs, with this response being efficiently controlled only in AGMs, possibly as a result of active regulatory mechanisms.     

Dideoxynucleosides are less inhibitory in vitro against human immunodeficiency virus type 2 (HIV-2) than against HIV-1.

The antiviral activities of various dideoxynucleosides against LAV strains of human immunodeficiency viruses type 1 (HIV-1) and type 2 (HIV-2) were evaluated. Significantly more 3'-azido-3'-deoxythymidine was required to inhibit the replication of HIV-2 than HIV-1 in three human cell lines. HIV-2 also appeared more resistant than HIV-1 to other dideoxynucleosides. These results suggest that dideoxynucleosides may be less effective in vivo for HIV-2 infection and that a broader range of clinical isolates of human retroviruses should be examined for drug susceptibility in vitro.     

Efficient entry inhibition of human and nonhuman primate immunodeficiency virus by cell surface-expressed gp41-derived peptides

Membrane-anchored C-peptides (for example, maC46) derived from human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp41 effectively inhibit HIV-1 entry in cell lines and primary human CD4+ cells in vitro. Here we evaluated this gene therapy approach in animal models of AIDS. We adapted the HIV gp41-derived maC46 vector construct for use in rhesus monkeys. Simian immunodeficiency virus (SIV and SHIV) sequence-adapted maC46 peptides, and the original HIV-1-derived maC46 expressed on the surface of established cell lines blocked entry of HIV-1, SIVmac251 and SHIV89.6P. Furthermore, primary rhesus monkey CD4+ T cells expressing HIV sequence-based maC46 peptides were also protected from SIV entry. Depletion of CD8+ T cells from PBMCs enhanced the yield of maC46-transduced CD4+ T cells. Supplementation with interleukin-2 (IL-2) increased transduction efficiency, whereas IL-7 and/or IL-15 provided no additional benefit. Phenotypic analysis showed that maC46-transduced and expanded cells were predominantly central memory CD4+ T cells that expressed low levels of CCR5 and slightly elevated levels of CD62L, beta7-integrin and CXCR4. These findings show that maC46-based cell surface-expressed peptides can efficiently inhibit primate immunodeficiency virus infection, and therefore serve as the basis for evaluation of this gene therapy approach in an animal model for AIDS.     

Anti-human immunodeficiency virus effects of dextran sulfate are strain dependent and synergistic or antagonistic when dextran sulfate is given in combination with dideoxynucleosides.

The effects of three molecular weight ranges of dextran sulfate on five different human immunodeficiency virus (HIV) isolates (from patients with acquired immunodeficiency syndrome), alone and in combination with dideoxynucleosides, were investigated in vitro. The higher the molecular weight range of dextran sulfate, the more potent the activity as assessed by a quantitative syncytium formation assay. Although all five HIV isolates had similar susceptibilities to the inhibitory effects of dideoxynucleosides, the two clinical isolates of HIV (HIV type 1 [HIV-1] TM and SP) exhibited a pattern of reduced susceptibility to dextran sulfate when compared with the two cloned isolates (HIV-1 WMF and HIV-2 ROD) and a prototype laboratory strain (HIV-1 IIIB). In combination with dideoxynucleosides, the high-molecular-weight range of dextran sulfate (500,000) resulted in an antagonistic response directed against the two clinical isolates of HIV (HIV-1 TM and SP) when the antiviral concentrations of dextran sulfate were in the ineffective range. Additive or synergistic effects were seen with the other three HIV isolates and all five HIV isolates when the low-molecular-weight range of dextran sulfate (8,000) was used. The results of these studies raise issues on the impact of drug-resistant strains on disease progression and the use of dextran sulfate in combination with nucleoside analogs for the clinical management of HIV disease.     

Use of a small molecule CCR5 inhibitor in macaques to treat simian immunodeficiency virus infection or prevent simian-human immunodeficiency virus infection

Human immunodeficiency virus type 1 (HIV-1) fuses with cells after sequential interactions between its envelope glycoproteins, CD4 and a coreceptor, usually CC chemokine receptor 5 (CCR5) or CXC receptor 4 (CXCR4). CMPD 167 is a CCR5-specific small molecule with potent antiviral activity in vitro. We show that CMPD 167 caused a rapid and substantial (4-200-fold) decrease in plasma viremia in six rhesus macaques chronically infected with simian immunodeficiency virus (SIV) strains SIVmac251 or SIVB670, but not in an animal infected with the X4 simian-human immunodeficiency virus (SHIV), SHIV-89.6P. In three of the SIV-infected animals, viremia reduction was sustained. In one, there was a rapid, but partial, rebound and in another, there was a rapid and complete rebound. There was a substantial delay (>21 d) between the end of therapy and the onset of full viremia rebound in two animals. We also evaluated whether vaginal administration of gel-formulated CMPD 167 could prevent vaginal transmission of the R5 virus, SHIV-162P4. Complete protection occurred in only 2 of 11 animals, but early viral replication was significantly less in the 11 CMPD 167-recipients than in 9 controls receiving carrier gel. These findings support the development of small molecule CCR5 inhibitors as antiviral therapies, and possibly as components of a topical microbicide to prevent HIV-1 sexual transmission.     

The Envelope Glycoprotein Ectodomains Determine the Efficiency of CD4+ T Lymphocyte Depletion in Simian– Human Immunodeficiency Virus–Infected Macaques

CD4⁺ T lymphocyte depletion in human immunodeficiency virus type 1 (HIV-1)–infected humans underlies the development of acquired immune deficiency syndrome. Using a model in which rhesus macaques were infected with chimeric simian–human immunodeficiency viruses (SHIVs), we show that both the level of viremia and the structure of the HIV-1 envelope glycoprotein ectodomains individually contributed to the efficiency with which CD4⁺ T lymphocytes were depleted. The envelope glycoproteins of recombinant SHIVs that efficiently caused loss of CD4⁺ T lymphocytes exhibited increased chemokine receptor binding and membrane-fusing capacity compared with those of less pathogenic viruses. These studies identify the HIV-1 envelope glycoprotein ectodomains as determinants of CD4⁺ T lymphocyte loss in vivo and provide a foundation for studying pathogenic mechanisms.     

Quantitative analysis of the human immunodeficiency virus type 1 (HIV- 1)-specific cytotoxic T lymphocyte (CTL) response at different stages of HIV-1 infection: differential CTL responses to HIV-1 and Epstein- Barr virus in late disease

Major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) are part of the cellular immune response to human persistent virus infections. Measurements of the frequency and specificity of human immunodeficiency virus type 1 (HIV-1)-specific CTL and their variation with time may indicate their relative importance in modulating the progression of HIV-1 infection. We have used limiting dilution analysis (LDA) to derive quantitative estimates of the frequency of HIV-1-specific CTL precursors in a cross-sectional study of 23 patients at different clinical stages of HIV-1 infection and to compare these with the frequency of CTL precursors specific for another persistent virus (Epstein-Barr virus [EBV]) in the same patients. Peripheral blood mononuclear cells (PBMC) were stimulated in vitro with autologous HIV-1-infected lymphoblasts and assayed for cytotoxicity in 51Cr release assays against autologous and MHC-mismatched lymphoblastoid B cells infected with recombinant vaccinia viruses expressing the three HIV-1 structural gene products. The frequency of MHC-restricted precursors was high in asymptomatic HIV-1-infected patients (env-specific CTL precursors up to 73/10(6) PBMC; gag-specific CTL precursors up to 488/10(6) PBMC), although the relative frequency against the different structural gene products varied from patient to patient. The HIV-1-specific CTL precursor frequency was reduced in patients who had more severe (< 400/microliters) CD4+ lymphocyte depletion, while in the majority of such patients the frequency of CTL precursors against EBV was maintained at levels observed in healthy controls. Direct CTL activity in unstimulated PBMC was observed in three of nine patients but no correlation was found between the presence of an activated CTL response and the magnitude of the CTL response detected after stimulation in LDA. Thus, CTL precursors were detected against all three HIV-1 structural gene products in patients with CD4+ lymphocyte counts > 400/microliters, at frequencies that are high compared with those reported for other persistent viruses. A CTL response directed against multiple protein antigens of HIV-1 may protect the patient against epitope variation. The fact that the EBV- specific CTL precursor frequencies were maintained in advanced HIV-1 infection suggests that there may be selective impairment of the HIV-1- specific CTL response associated with disease progression.     

Simian-Tropic HIV as a Model To Study Drug Resistance against Integrase Inhibitors

Drug resistance represents a key aspect of human immunodeficiency virus (HIV) treatment failure. It is important to develop nonhuman primate models for studying issues of drug resistance and the persistence and transmission of drug-resistant viruses. However, relatively little work has been conducted using either simian immunodeficiency virus (SIV) or SIV/HIV recombinant viruses for studying resistance against integrase strand transfer inhibitors (INSTIs). Here, we used a T-cell-tropic SIV/HIV recombinant virus in which the capsid and vif regions of HIV-1 were replaced with their SIV counterparts (simian-tropic HIV-1 [stHIV-1]_(SCA,SVIF)) to study the impact of a number of drug resistance substitutions in the integrase coding region at positions E92Q, G118R, E138K, Y143R, S153Y, N155H, and R263K on drug resistance, viral infectivity, and viral replication capacity. Our results show that each of these substitutions exerted effects that were similar to their effects in HIV-1. Substitutions associated with primary resistance against dolutegravir were more detrimental to stHIV-1_(SCA,SVIF) infectiousness than were resistance substitutions associated with raltegravir and elvitegravir, consistent with data that have been reported for HIV-1. These findings support the role of stHIV-1_(SCA,SVIF) as a useful model with which to evaluate the role of INSTI resistance substitutions on viral persistence, transmissibility, and pathogenesis in a nonhuman primate model.     

Hydroxyurea Enhances the Activities of Didanosine, 9-[2-(Phosphonylmethoxy)ethyl]adenine, and 9-[2-(Phosphonylmethoxy)propyl]adenine against Drug-Susceptible and Drug-Resistant Human Immunodeficiency Virus Isolates

We assessed the effects of hydroxyurea (HU) at a concentration of 50 microM on the in vitro activities of 2',3'-dideoxyinosine (ddI), 9-[2-(phosphonylmethoxy)ethyl]adenine (PMEA), and 9-[2-(phosphonylmethoxy)propyl]adenine (PMPA) against a wild-type human immunodeficiency virus (HIV) type 1 (HIV-1) laboratory isolate and a panel of five well-characterized drug-resistant HIV isolates. Fifty micromolar HU significantly increased the activities of ddI, PMEA, and PMPA against both the wild-type and the drug-resistant HIV-1 isolates. In fixed combinations, both ddI and PMEA were synergistic with HU against wild-type and drug-resistant viruses.     

Biological comparison of wild-type and zidovudine-resistant isolates of human immunodeficiency virus type 1 from the same subjects: susceptibility and resistance to other drugs.

We used a viral endpoint dilution assay to show changes in the proportion of zidovudine (azidothymidine; AZT)-resistant viruses within a heterogeneous mixture of human immunodeficiency virus type 1 (HIV-1) quasispecies isolated from patients on long-term AZT therapy. Several HIV-1 isolates, which could replicate in 10 microM AZT, were susceptible to both 2',3'-dideoxycytidine and a novel cytosine analog BCH-189, in which a sulfur atom replaces the 3' carbon of the pentose ring. In certain instances, cross-resistance was seen with 3'-didehydro-2',3'-dideoxythymidine. Although most strains of AZT-resistant HIV-1 displayed reduced susceptibility to 3'-azido-2',3'-dideoxyuridine, two strains were identified for which this was not the case.     

Natural polymorphisms in the human immunodeficiency virus type 2 protease can accelerate time to development of resistance to protease inhibitors

Human immunodeficiency virus type 2 (HIV-2) contains numerous natural polymorphisms in its protease (PR) gene that are implicated in drug resistance in the case of HIV-1. This study evaluated emergent PR resistance in HIV-2. Three HIV-2 isolates were selected for resistance to amprenavir (APV), nelfinavir (NFV), indinavir (IDV), and tipranavir (TPV) in cell culture. Genotypic analysis determined the time to the appearance of protease inhibitor (PI)-associated mutations compared to HIV-1. Phenotypic drug susceptibility assays were used to determine the levels of drug resistance. Within 10 to 15 weeks of serial passage, three major mutations--I54M, I82F, and L90M--arose in HIV-2 viral cultures exposed to APV, NFV, and IDV, whereas I82L was selected with TPV. After 25 weeks, other cultures had developed I50V and I84V mutations. In contrast, no major PI mutations were selected in HIV-1 over this period except for D30N in the context of NFV selective pressure. The baseline phenotypes of wild-type HIV-2 isolates were in the range observed for HIV-1, except for APV and NFV for which a lower degree of sensitivity was seen. The acquisition of the I54M, I84V, L90M, and L99F mutations resulted in multi-PI-resistant viruses, conferring 10-fold to more than 100-fold resistance. Of note, we observed a 62A/99F mutational motif that conferred high-level resistance to PIs, as well as novel secondary mutations, including 6F, 12A, and 21K. Thus, natural polymorphisms in HIV-2 may facilitate the selection of PI resistance. The increasing incidence of such polymorphisms in drug-naive HIV-1- and HIV-2-infected persons is of concern.     

PD 404,182 is a virocidal small molecule that disrupts hepatitis C virus and human immunodeficiency virus

We describe a virucidal small molecule, PD 404,182, that is effective against hepatitis C virus (HCV) and human immunodeficiency virus (HIV). The median 50% inhibitory concentrations (IC(50)s) for the antiviral effect of PD 404,182 against HCV and HIV in cell culture are 11 and 1 μM, respectively. The antiviral activity of PD 404,182 is due to the physical disruption of virions that is accompanied to various degrees (depending on the virus and exposure temperature/time) by the release of viral nucleic acids into the surrounding medium. PD 404,182 does not directly lyse liposomal membranes even after extended exposure, and it shows no attenuation in antiviral activity when preincubated with liposomes of various lipid compositions, suggesting that the compound inactivates viruses through interaction with a nonlipid structural component of the virus. The virucidal activity of PD 404,182 appears to be virus specific, as little to no viral inactivation was detected with the enveloped Dengue and Sindbis viruses. PD 404,182 effectively inactivates a broad range of primary isolates of HIV-1 as well as HIV-2 and simian immunodeficiency virus (SIV), and it does not exhibit significant cytotoxicity with multiple human cell lines in vitro (50% cytotoxic concentration, >300 μM). The compound is fully active in cervical fluids, although it exhibits decreased potency in the presence of human serum, retains its full antiviral potency for 8 h when in contact with cells, and is effective against both cell-free and cell-associated HIV. These qualities make PD 404,182 an attractive candidate anti-HIV microbicide for the prevention of HIV transmission through sexual intercourse.     

Anti-human immunodeficiency virus type 1 activity of an anti-CD4 immunoconjugate containing pokeweed antiviral protein.

The ability of an alpha CD4-pokeweed antiviral protein (PAP) immunoconjugate to inhibit replication of human immunodeficiency virus type 1 (HIV-1) was evaluated in vitro with 22 clinical HIV-1 strains obtained from four seropositive asymptomatic individuals, three patients with AIDS-related complex, and four patients with AIDS. Fifteen isolates were from zidovudine-untreated individuals, whereas seven isolates were obtained after 24 to 104 weeks of therapy with zidovudine, alone or alternating with zalcitabine. Mean zidovudine 50% inhibitory concentrations (IC50s) were 126 nM (range, 1 to 607 nM) for isolates from zidovudine-untreated individuals and 2,498 nM (range, 14 to 6,497 nM) for strains from patients treated with antiretroviral agents. Mean alpha CD4-PAP IC50s were 48 x 10(-3) nM (range, 0.02 x 10(-3) to 212 x 10(-3) nM) for isolates from zidovudine-untreated individuals, and 16 x 10(-3) nM (range, 2 x 10(-3) to 28 x 10(-3) nM) for isolates from treated patients. Overall, higher concentrations of alpha CD4-PAP were necessary to inhibit HIV-1 strains from untreated individuals at more advanced stages of disease. Seventeen isolates were susceptible to zidovudine (mean IC50, 117 nM), and five were resistant to zidovudine (mean IC50, 3,724 nM). Mean alpha CD4-PAP IC50s were 43 x 10(-3) nM for zidovudine-susceptible isolates and 19 x 10(-3) nM for isolates resistant to zidovudine. All HIV-1 strains had IC50s greater than 0.5 nM for unconjugated PAP, the alpha CD19-PAP immunoconjugate, and monoclonal antibody alpha CD4. At concentrations as high as 5,000 nM, alphaCD4-PAP did not inhibit colony formation by normal bone marrow progenitor cells(BFU-E, CFU-GM , and CFU-GEMM) or myeloid cell lines (KG-1 and HL-60) and did not decrease cell viabilities of T-cell (Jurkat) or B-cell (FL-112 and Raji) precursor lines. Overall, alphaCD4-PAP demonstrated more potent anti-HIV-1 activity than zidovudine and inhibited replication of zidovudine-susceptible and zidovudine-resistant viruses at concentrations that were not toxic to lymphohematopoietic cell populations.     

Susceptibilities of simian immunodeficiency virus to protease inhibitors

We used a focal infectivity assay with HeLa H1-JC.37 cells to directly compare susceptibilities of simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) to protease inhibitors. SIVmac239 was inhibited by indinavir, saquinavir, and ritonavir, with 50% effective concentrations (means +/- standard deviations) of 39 +/- 8, 55 +/- 3, and 13 +/- 5 nM, respectively. The corresponding values for inhibition of HIV-1 were 66 +/- 4, 47 +/- 10, and 25 +/- 14 nM, respectively.     

An underestimated lentivirus model: what can HIV-2 research contribute to the development of an effective HIV-1 vaccine?

The development of an HIV-1 vaccine that would be effective against all existing subtypes and circulating recombinant forms remains one of the great scientific and public health challenges of our generation. One of the major barriers to HIV-1 vaccine development is a lack of understanding of the correlates of protective immunity against the virus. In this context, research has focused on the rare phenomenon of spontaneous control of HIV-1 infection, in groups referred to as 'long-term nonprogressors' and 'elite controllers', together with models of nonprogressive sooty mangabey simian immunodeficiency (SIV) infection in African nonhuman primate hosts such as sooty mangabeys and African green monkeys, in which the majority of animals tolerate high levels of viral replication without development of immunodeficiency or disease. Much less attention has been given to humans infected with the nonpandemic strain HIV-2, derived from the SIV in West Africa, most of whom behave as long-term nonprogressors or viral controllers, while a minority develop disease clinically indistinguishable from AIDS caused by HIV-1. This apparent dichotomous outcome is, based on the evidence accumulated to date, more clearly related to the host immune response than the good clinical outcome of HIV-1 controllers. We propose that complementing research into HIV-1 controllers and nonpathogenic SIV models with the prioritization of HIV-2 research could enhance the HIV-1 vaccine research effort. The absence of disease progression or detectable plasma viral replication in the presence of an effective immune response in most patients living with HIV-2 represents an opportunity to unravel the virus' evolutionary adaptation in human hosts and to establish the correlates of such a protective response.