Correlation of immune activation with HIV-1 RNA levels assayed by real-time RT-PCR in HIV-1 subtype C infected patients in Northern India

BACKGROUND: Assays with specificity and cost effectiveness are needed for the measurement of HIV-1 burden to monitor disease progression or response to anti-retroviral therapy (ART) in HIV-1 subtype C infected patients. OBJECTIVES: The objective of this study was to develop and validate an affordable one step real-time RT-PCR assay with high specificity and sensitivity to measure plasma HIV-1 loads in HIV-1 subtype C infected patients. RESULTS: We developed an RT-PCR assay to detect and quantitate plasma HIV-1 levels in HIV-1 subtype C infected patients. An inverse correlation between plasma viral loads (PVL) and CD4+ T-cell numbers was detected at all CDC stages. Significant correlations were found between CD8+ T-cell activation and PVL, as well as with the clinical and immunological status of the patients. CONCLUSIONS: This RT-PCR assay provides a sensitive method to measure PVL in HIV-1 subtype C infected patients. Viral loads correlated with immune activation and can be used to monitor HIV care in India.     

High replication fitness and transmission efficiency of HIV-1 subtype C from India: Implications for subtype C predominance

HIV-1 subtype C has been the predominant subtype throughout the course of the HIV-1 epidemic in India regardless of the geographic region of the country. In an effort to understand the mechanism of subtype C predominance in this country, we have investigated the in vitro replication fitness and transmission efficiency of HIV-1 subtypes A and C from India. Using a dual infection growth competition assay, we found that primary HIV-1 subtype C isolates had higher overall relative fitness in PBMC than subtype A primary isolates. Moreover, in an ex vivo cervical tissue derived organ culture, subtype C isolates displayed higher transmission efficiency across cervical mucosa than subtype A isolates. We found that higher fitness of subtype C was not due to a trans effect exerted by subtype C infected PBMC. A half genome A/C recombinant clone in which the 3′ half of the viral genome of subtype A was replaced with the corresponding subtype C3′ half, had similar replicative fitness as the parental subtype A. These results suggest that the higher replication fitness and transmission efficiency of subtype C virus compared to subtype A virus from India is most probably not due to the envelope gene alone and may be due to genes present within the 5′ half of the viral genome or to a more complex interaction between the genes located within the two halves of the viral genome. These data provide a model to explain the asymmetric distribution of subtype C over other subtypes in India.     

Characterization of the main placental cytokine profiles from HIV-1-infected pregnant women treated with anti-retroviral drugs in France

Cytokines are involved in regulating HIV-1 infection. They are also placental environment major components. We assessed the potential impact of HIV-1 infection and/or anti-retroviral drugs on the placental cytokine profiles that may be involved in controlling HIV-1 placental dissemination. Placental explants were obtained after elective caesarean section from anti-retroviral-treated HIV-1-infected pregnant women and from HIV-1 non-infected pregnant women. The main placental cytokines were assessed for protein secretion in the supernatants of 24-h placental culture explants and/or in uncultured placental explants for mRNA expression levels. The cytokine profiles were different between the HIV-1-infected and the non-infected groups. Higher medians of leukaemia inhibiting factor (LIF), tumour necrosis factor (TNF)-alpha and interleukin (IL)-8 secretion were found in the 24-h culture supernatant of term placenta from HIV-1-infected women. High median levels of IL-16 and regulated upon activation normal T cell expressed and secreted (RANTES) levels were found in both groups. The mRNA expression medians were lower for TNF-alpha and IL-8 and higher for stromal cell-derived factor-1 (SDF-1) in uncultured placental explants from HIV-1-infected women. In the HIV-1-infected group, but not in the non-infected group, the secretion levels of TNF-alpha and IL-8, as well as their mRNA expression levels, were highly positively correlated; furthermore, their secretion levels were correlated positively with LIF and IL-10 secretion levels. We found no correlation between the cytokine levels and the immunovirological status of the HIV-1-infected mothers or the type or duration of treatment. These results highlight the potential impact of HIV-1 and of the anti-retroviral treatments on the placental cytokines pattern, independently of their anti-viral activity.     

Genetic characterization of HIV-1 from semen and blood from clade C-infected subjects from India and effect of therapy in these body compartments

Biologic and genetic differences between HIV-1 clade C in India and clade B in US suggest that the effect of anti-viral therapy in various body compartments may differ between these two clades. We examined the effect of therapy on viral loads in semen and blood of HIV-1-clade C infected subjects from India and evaluated whether HIV-1 in the semen is different from that in blood in these subjects. HIV-1 RNA was detected in semen and blood at all stages of the disease. Viral loads in semen and blood were strongly correlated with each other, but not with the CD4+ T cell count. Anti-viral treatment reduced viral load drastically in blood and semen within one month of post therapy. Genetic characterization of HIV-1 in the semen and blood demonstrated that they were highly compartmentalized. These data have important implications of sexual transmission of HIV-1 in clade C HIV-1 infected subjects.     

HIV-1流行毒株HIV DNA的研究

目的 了解HIV－1流行毒株中HIV DNA的变异特性。方法 用逆转录PCR（RT－PCR）,长片段PCR（Long-Distance PCR LD-PCR)、分子克隆、杂交和测序等对63例HIV－1感染者外周血单个核细胞（PBMC）HIV DNA进行研究。结果 57例标本可明确定型,其中B亚型43例,C亚型5例,E亚型9例。57例中31例既存在完整又存在不完整的HIV－1 DNA,19例只存在不完整的HIV－1 DNA片段,7例只有完整的HIV－1 DNA,缺失的最大范围为7759个碱基,最小范围71个碱基,以多片段缺损最为常见。结论 HIV DNA基因片段变异在HIV感染和流行传播中是非常重要的。     

Induction of HIV-1-specific T cell responses by administration of cytokines in late-stage patients receiving highly active anti-retroviral therapy.

Highly active anti-retroviral therapy (HAART) is associated with reduction in the morbidity and mortality of patients with advanced HIV-1 disease. The ability of such treatment to improve immune responses against HIV-1 and opportunistic pathogens is variable and limited. Addition of cytokine immunotherapy to this treatment may improve immune responses. IL-2 with or without granulocyte-macrophage colony-stimulating factor (GM-CSF) was administered to HIV-1+ individuals receiving HAART with undetectable viral loads, and CD4 counts < 100 cells/microl. In one patient presenting with Mycobacterium avium complex (MAC) infection, we evaluated the effect of cytokine immunotherapy on lymphocyte phenotype; plasma viral load; proliferative responses to mitogens, recall and HIV-1 antigens; cytokine production and message in response to non-specific and specific stimuli; and natural killer (NK) cell activity. Proliferation assays were performed in two similar patients. Before cytokine immunotherapy the predominant CD8+ population was mainly CD28-. No proliferation or IL-2 production was seen in response to mitogens, recall or HIV-1 antigens; and no HIV-1 peptide-specific interferon-gamma (IFN-gamma)-secreting cells were present. Low levels of IL-4 were detected in response to antigens to which patients had been exposed, associated with up-regulated expression of costimulatory molecules influenced by IL-4. Following IL-2 administration, loss of IL-4 was associated with increased NK cell activity and HIV-1 peptide-specific and non-specific IFN-gamma-producing cells. Proliferative responses associated with IL-2 production and responsiveness were only seen after subsequent concomitant administration of GM-CSF with IL-2. These changes mirrored clinical improvement. An imbalance of lymphocyte subsets may account for immune unresponsiveness when receiving HAART. Restoration of responses following immunotherapy suggests a shift towards a lymphocyte profile with anti-pathogen activity.     

Molecular genotyping of HIV-1 in 61 patients with AIDS from Lomé, Togo

To study the distribution of HIV types and genotypes, in Lomé, Togo, a random population of patients who met the clinical criteria of the Bangui definition of AIDS and were positive with two independent screening assays for antibodies to HIV-1 group M, HIV-2, and HIV-1 group O was selected. HIV RNA from serum samples was reverse-transcribed and amplified with degenerate primers annealing to conserved regions of the HIV-1, HIV-2, and HIV-O gag gene. Amplicons were directly sequenced using an automated sequencer. A 262–271-bp (strain-dependent) fragment of the gag gene from each patient was phylogenetically analyzed and compared to the corresponding gag sequences of published HIV-1 sequences of known African genotypes. Genotype A was found in 48 of 60 patient amplicons (80%), subdivided into two clusters. Ten patients (16.7%) were HIV-1 genotype G; one was genotype D and one genotype H. HIV-1 genotype B was not found. Amplicons from two patients contained sequence ambiguities, requiring cloning and sequencing of the gag insert. One patient (T52) was apparently infected with HIV-1 genotypes A and G; whereas HIV-1 from patient T139 was of genotype A, with 2/10 clones having a three-codon insertion at nucleotide position 1142 of the gag gene. HIV-1 genotype A is dominant in Togo; genotype G is frequent and genotype B has not been found. J. Med. Virol. 57:25–30, 1999. © 1999 Wiley-Liss, Inc.     

Phenotytic Knockout of HIV-1 Chemokine Coreceptor CXCR4 and CCR5 by Intrakines for Blocking HIV-1 Infection

Acquired immunodeficiency syndrome (AIDS) and humanimmunodeficiency virus (HIV) infection continues to bemajor global health concerns. Although highly active an-tiretroviral therapy (HAART) has led to profound and pro-longed reductions in circulating viru…     

Functional Variability of Rev Response Element in HIV-1 Primary Isolates

We have previously studied sequence heterogeneity of HIV-1 Rev response element (RRE), and showed uneven variations in different stem–loops of both primary sequence and secondary structure. Here we studied the functional variation of RRE clones from a set of 10 primary isolates, and demonstrated a variation in the function of these RRE clones on the expression of Gag proteins from a truncated HIV-1 genome. The difference in Gag level was, in part, if not exclusively, resulted from the differential efficiency of RNA transport and enhancing of translation. These data suggested that variation of HIV-1 RRE may play a role in regulation of viral replication rate in HIV-1 primary isolates.     

Molecular determinants of HIV-1 subtype C coreceptor transition from R5 to R5X4

The molecular mechanism(s) underlying transition from CCR5 to CXCR4 usage of subtype C viruses remain largely unknown. We previously identified a subtype C HIV-1 infected child whose virus demonstrated CXCR4 usage along with CCR5 upon longitudinal follow-up. Here we delineated the molecular determinants of Env involved in expanded coreceptor usage. Residue changes in three positions of Env V3 domain are critical for the dual tropic phenotype. These include: substitution of arginine at position 11, MG or LG insertion between positions 13 and 14, and substitution of threonine at the position immediately downstream of the GPGQ crown. Introducing these mutations into V3 region of a heterologous R5 virus also conferred dual tropism. Molecular modeling of V3 revealed a possible structural basis for the dual tropic phenotype. Determining what defines a subtype C X4 virus will lead to a better understanding of subtype C HIV-1 pathogenesis, and will provide important information relevant to anti-retroviral therapy.     

Inhibition of HIV-1 expression by HIV-2

HIV-1 and HIV-2 are co-endemic in certain geographic areas. HIV-2 is more weakly pathogenic than HIV-1, and progression to AIDS occurs less frequently and over a longer period of time. Recent epidemiologic studies suggest that individuals infected with HIV-2 have a lower risk of HIV-1 infection. Both immune mechanisms and various modes of viral interference have been proposed to account for these results. Our findings, described in this paper, suggest that HIV-2 inhibits HIV-1 replication. To study the molecular interactions between HIV-1 and HIV-2, proviral clones were transfected alone or in combination into the human T cell line CEM. LTR-CAT indicator constructs were included for the purpose of monitoring viral promoter activity. Viral replication in transfected cells was monitored by p24 antigen capture assay of cell culture supernatants and Western blot analysis of cell extracts. HIV-2 inhibited HIV-1 replication as determined by intracellular and extracellular p24 antigen levels. Similar results were obtained with simultaneous virus infection using HIV-1 and HIV-2, rather than transfections of proviral DNA. Using cotransfection of HIV-1 and HIV-2 LTR indicator gene constructs, the mechanism of inhibition was found to be suppression of the HIV-1 LTR by HIV-2. The inhibitory effect of HIV-2 is not due to Tat-2, but appears to discriminate between the HIV-1 and HIV-2 LTRs based on differences in the Tat activation response element, TAR. These results suggest both a molecular mechanism for HIV-2 interference with HIV-1 replication and a potential molecular approach to therapy.     

Genetic composition of replication competent clonal HIV-1 variants isolated from peripheral blood mononuclear cells (PBMC), HIV-1 proviral DNA from PBMC and HIV-1 RNA in serum in the course of HIV-1 infection

The HIV-1 quasispecies in peripheral blood mononuclear cells (PBMC) is considered to be a mix of actively replicating, latent, and archived viruses and may be genetically distinct from HIV-1 variants in plasma that are considered to be recently produced.Here we analyzed the genetic relationship between gp160 env sequences from replication competent clonal HIV-1 variants that were isolated from PBMC and from contemporaneous HIV-1 RNA in serum and HIV-1 proviral DNA in PBMC of four longitudinally studied therapy naïve HIV-1 infected individuals.Replication competent clonal HIV-1 variants, HIV-1 RNA from serum, and HIV-1 proviral DNA from PBMC formed a single virus population at most time points analyzed. However, an under-representation in serum of HIV-1 sequences with predicted CXCR4 usage was sometimes observed implying that the analysis of viral sequences from different sources may provide a more complete assessment of the viral quasispecies in peripheral blood in vivo.     

Abrogation of Vif function by peptide derived from the N-terminal region of the human immunodeficiency virus type 1 (HIV-1) protease

The human immunodeficiency virus type 1 (HIV-1) auxiliary gene vif is essential for virus propagation in peripheral blood lymphocytes, macrophages, and in some T-cell lines. Previously, it was demonstrated that Vif inhibits the autoprocessing of truncated HIV-1 Gag-Pol polyproteins expressed in bacterial cells, and that purified recombinant Vif and Vif-derived peptides inhibit and bind HIV-1 protease (PR). Here we show that Vif interacts with the N-terminal region of HIV-1 PR, and demonstrate that peptide derived from the N-terminal region of PR abrogates Vif function in non-permissive cells. Specifically, we show that (i) Vif protein binds HIV-1 PR, but not covalently linked tethered PR-PR; (ii) the four amino acids residing at the N terminus of HIV-1 PR are essential for Vif/PR interaction; (iii) synthetic peptide derived from the N terminus of HIV-1 PR inhibits Vif/PR binding; and (iv) this peptide inhibits the propagation of HIV-1 in restrictive cells. Based on these data, we suggest that Vif interacts with the dimerization sites of the viral protease, and that peptide residing at the N terminus of PR abrogates Vif function(s).     

Early reduction of the over-expression of CD40L, OX40 and Fas on T cells in HIV-1 infection during triple anti-retroviral therapy: possible implications for lymphocyte traffic and functional recovery.

Fas, CD40L and OX40 are members of the tumour necrosis factor (TNF) receptor superfamily with critical roles in T cell activation and death, B cell function, dendritic cell maturation and leucocyte traffic regulation. The aim of this study was to evaluate the effects of anti-retroviral therapy (HAART) on CD40L, OX40 and Fas expression on freshly isolated peripheral blood T cells by three-colour flow cytometry and compare them with lymphoproliferative responses, peripheral blood cell counts and viral load. Fourteen asymptomatic HIV-1+ patients treated with Lamivudine, Stavudine and Nelfinavir were prospectively investigated sequentially for 48 weeks. At baseline, patients exhibited significantly enhanced proportions and counts of CD40L+ and OX40+ cells within the CD4 subset which were corrected by weeks 8-16 of HAART. Interestingly, in the five patients showing viral load rebound during therapy in spite of increasing CD4 counts, the reduction of the levels of these costimulatory molecules was similarly maintained. Therapy induced a decrease in the over-expression of Fas, particularly in the CD4 subset where normal levels were reached at week 8. This reduction occurred in parallel with the major recovery of lymphoproliferative responses. Higher basal levels and lower reduction of Fas were associated with suboptimal suppression of viraemia. In conclusion, this previously undescribed increased expression of CD40L and OX40 may play a role in the HIV-associated pan-immune activation and represent a possible target for immunointervention, as suggested for several immunologically mediated diseases. Moreover, HAART induced an early correction of the over-expression of Fas, CD40L and OX40 in CD4 T cells which could be involved in the recovery of the cell traffic disturbances and in the T cell renewal capacity.     

淮安市经异性性传播的HIV感染者中HIV-1病毒基因亚型特征分析

目的 了解淮安市经异性性传播HIV感染者中HIV-1病毒基因亚型分布及其流行特征.方法 从60名异性性传播HIV-1感染者血液中提取DNA或RNA,用巢式PCR或RT-PCR方法扩增gag、env基因区片段,测定序列并分析.结果 60份标本中,确定了48份标本的基因亚型,发现有4种HIV-1亚型和重组型.其中CRF01_AE亚型占62.5％,CRF07_BC亚型占22.9％,CRF08_BC亚型占6.3％,B亚型占6.3％.结论淮安市异性性传播感染HIV者中,流行着CRF01_AE、CRF07_BC、CRF08_BC、B亚型这4种亚型病毒株,CRF01_AE为主要流行亚型.     

北京市男男性接触HIV-1感染者膜蛋白基因序列测定及亚型分析

目的 了解北京市男男性接触人群(MSM)中HIV-1的最新流行趋势及膜蛋白V3环序列特征.方法 巢式聚合酶链式反应(n-PER)扩增2007年提取的北京市男男性接触HIV感染者基因组DNA样品,对膜蛋白基因C2-V3区测序,进行病毒亚型及V3环序列特点分析.结果 11例样本中,4例是欧美B亚型,5例是AE重组亚型,1例是BC重组亚型,1例是01B重组亚型.V3环顶端四肽以GPGQ和GPGR为主.结论 北京市男男性接触HIV-1感染者中重组亚型呈蔓延流行趋势.     

淋巴细胞HIV-1辅受体表型剔除阻断病毒感染的研究

目的　观察HIV 1辅受体CCR5和CXCR4表型剔除对HIV 1DP1株感染的阻断作用。方法　用含有pLNCX R K S K的重组逆转录病毒液感染原代人PBLs(外周血淋巴细胞 ) ,抗体 免疫磁珠法分离筛选转化成功PBLs,流式细胞仪检测筛选效率 ;HIV 1DP1株攻击转化PBLs ,进行合胞体形成和p2 4抗原分泌检测。结果　抗 NGFR 免疫磁珠法获得了转化成功的PBLs ,流式细胞仪检测发现pLNCX R K S K转染组 77.4%的PBLsNGFR(神经生长因子受体 )标记物为阳性 ;HIV 1DP1株攻击后 ,未转染组和pLNCX转染组可以见到明显的合胞体形成 ,而在pLNCX R K S K转化PBLs没有见到合胞体形成 ;pLNCX R K S K转染组在感染后第 4、7和 10天皆可发现显著的p2 4抗原分泌抑制 ,抑制率分别为 15%、43 %、19%。结论　细胞内趋化因子通过与CCR5和CXCR4细胞内结合 ,使HIV 1两类主要辅受体难以在PBLs表面表达 ,从而可以达到阻断HIV 1病毒感染的目的     

Genotypic resistance profile of HIV-1 protease gene: a preliminary report from Vellore, south India

HIV-1 subtypes other than B are responsible for most new HIV infections worldwide; virus sequence data for drug resistance is described only from a limited number of non-B subtype HIV-1. This study is on mutations and polymorphisms of HIV-1 protease gene that can predict drug resistance in subtype C. The genotypic resistance assay was carried out on 38 HIV-1 strains with their plasma RNA and in nine, the proviral protease gene was sequenced. The treatment naïve strains showed minor resistance mutations, there were no major resistance mutations in the protease gene. We suggest the use of resistance testing to monitor individuals on therapy and also before initiation of therapy, gathering more sequence information for a data bank of Indian strains.