Lack of alternative coreceptor use by pediatric HIV-1 R5 isolates for infection of primary cord or adult peripheral blood mononuclear cells

Summary HIV-1 infection of neonates results in an extended acute period of virus replication, frequent neurological problems and reduced survival compared to adults. In adults, R5 viruses mainly infect CCR5⁺ CD4⁺ memory T-cells. In neonates, CCR5⁺ memory T-cells form a substantially smaller fraction of total lymphocytes. We therefore tested whether alternative coreceptors confer infection of lymphocytes by pediatric isolates. Pediatric HIV-1 R5 isolates failed to replicate in Δ32/Δ32 CCR5 PBMCs or in cord PBMCs treated with a CCR5 inhibitor. These results do not indicate a role for alternative coreceptors and provide support for CCR5 inhibitors in the therapy of HIV-1⁺ neonates. Correspondence: Paul R. Clapham, Program in Molecular Medicine, UMASS Medical School, 373 Plantation St, Biotech II Suite 315, Worcester, MA 01605, U.S.A.     

脐血和外周血单个核细胞培养上清影响HIV-1感染的体外实验研究

目的：体外研究脐血和外周血单个核细胞（CBMC和PBMC）培养上清对HIV-1感染的影响，为发现抗HIV-1的可溶性因子奠定基础。 方法： PHA刺激CBMC和PBMC后5 h和12 h收集上清，加入荧光标记的HIV-1ⅢB/H9和MT-2细胞培养体系中，2 h后在倒置荧光显微镜下观察其对细胞融合的影响；用Luminex 100TM分析仪检测收集上清中细胞因子含量。 结果： PHA刺激CBMC和PBMC后5 h和12 h的上清均可抑制HIV-1ⅢB/H9和MT-2细胞融合，同一时间收集的PBMC和CBMC上清对HIV-1ⅢB/H9和MT-2细胞融合的抑制作用无差异，但5 h上清的抑制作用强于12 h的上清；CBMC 5 h上清中促炎症细胞因子比PBMC 5 h上清为低，而CCR5配体MIP-1α和RANTES则比PBMC 5 h上清高，差异显著（P＜0.05）。 结论： 通过脐血和外周血单个核细胞可以制备有效抗HIV感染的可溶性因子，可能为艾滋病治疗药物提供新的来源。     

In vitro susceptibility to infection with SIVcpz and HIV-1 is lower in chimpanzee than in human peripheral blood mononuclear cells

This study was undertaken to evaluate and compare the susceptibility of chimpanzee versus human peripheral blood mononuclear cells (PBMCs) to infection with SIVcpz and HIV-1 non-syncitium inducing primary isolates. The results demonstrate clearly that chimpanzee PBMCs have a lower capacity to support viral replication as compared to human PBMCs. There was no experimental evidence that this difference was due to a lower availability of target cells for viral infection (PBMCs positive for CD4 and CCR5 molecules) or to a differential susceptibility to apoptosis (PBMCs positive for CD4 and CD95 molecules). A lower capacity of chimpanzee PBMCs to support SIVcpz and HIV-1 replication in vitro is related to a post-entry barrier to virus replication.     

Cultivation of HIV-1 virus on mononuclear peripheral blood cells of HIV-1 seropositive individuals

Using the method of co-cultivation with phytohaemagglutinin-stimulated lymphocytes from healthy donors, the author isolated the HIV-1 virus from peripheral mononuclear blood cells of three patients with the AIDS symptomatology and one patient with the ARC symptomatology. The presence of the virus in infected cells was proved by detection of the viral antigen p 24 in enzymatic immunoassays and in the immunofluorescence test. Three of the isolated strains were adapted to sensitive continual tissue cultures, where the isolates caused chronic infection of the cells associated with the development of a cytopathic effect. In the investigated patients no relationship was proved between viraemia and antigenaemia. The author discusses the importance of virus cultivation for laboratory diagnosis, epidemiology and research of HIV infection and AIDS.     

Analysis of the spontaneous in vitro anti-HIV-1 antibody secretion by peripheral blood mononuclear cells in HIV-1 infection.

We studied the spontaneous in vitro secretion of anti-HIV-1 antibodies by peripheral blood mononuclear cells (PBMC) from HIV-1-infected patients. Specific antibody production was detected in supernatants of PBMC cultures using an ELISA; HIV-1 specificity was confirmed by antigen adsorption and Western blotting. This antibody secretion was found to be an active phenomenon and was not due to a release of plasma antibodies passively adsorbed onto the cell membranes. In all positive supernatants, anti-HIV-1-secreted antibodies were directed against env-encoded antigens and many supernatants also contained antibodies to pol- and gag-encoded antigens. PBMC from all HIV-1-infected patients tested (140 adults and 18 infants) secreted anti-HIV-1 antibodies. This production was found during all the clinical stages of HIV-1 infection. Our results suggest that this spontaneous HIV-1-specific antibody secretion represents a marker of HIV-1 infection. Detection of these antibodies could be a valuable tool for early confirmation of HIV-1 infection in neonates born to HIV-1-seropositive mothers.     

Subpopulations of peripheral blood dendritic cells show differential susceptibility to infection with a lymphotropic strain of HIV-1

Blood dendritic cells (DC) express CD4 and are susceptible to HIV infection. By electron microscopy two morphologically distinct types of DC were identified in peripheral blood. Only one of these two types was susceptible to infection with a lymphotropic strain of HIV-1. By FACS two populations could be defined based on the expression of CD11c. The morphology of cultured FACS-purified CD11c negative DC was similar to that DC population shown to be susceptible to infection with the lymphotropic strain of HIV. Furthermore after several hours in culture CXCR4, the co-receptor for lymphotropic strains of HIV-1, was expressed at a significantly higher level on the CD11c negative DC than on the CD11c positive cells. This study suggests that there are subpopulations of DC that show differences in susceptibility to infection with some strains of HIV-1.     

抗CD4及抗CXCR4抗体阻断HIV—1感染细胞作用的研究

为探讨抗ＣＤ４ 及抗ＣＸＣＲ４ 抗体在阻断Ｉ型人免疫缺陷病毒（ＨＩＶ－ １） 感染应用中的意义, 本文应用上述两种抗体分别与ＳｕｐＴ１ 细胞及人外周血单个核细胞（ＰＢＭＣ） 共培育, 以封闭ＨＩＶ－ １ 在上述细胞上的受体。然后, 以ＨＩＶ１ ＮＬ４３ 病毒株感染上述细胞, 通过测定感染细胞上清中ＨＩＶ－ １ 的Ｐ２４ 蛋白含量, 观察上述抗体对ＨＩＶ－ １ 感染细胞的阻断作用。结果显示, 无论是抗ＣＤ４ 或抗ＣＸＣＲ４ 的抗体单独应用或是两者联合应用, 均可明显地抑制ＨＩＶ－ １ 感染细胞的作用。该结果为今后开拓ＡＩＤＳ的抗体治疗提供了理论基础。     

Increased susceptibility to HIV-1 of peripheral blood lymphocytes in acute infection with Epstein-Barr virus

Epstein-Barr virus (EBV) is an important pathogen in human immunodeficiency virus (HIV)-infected individuals that causes lymphoma and other lymphoproliferative disorders upon disease progression; however, interaction between the two viruses during acute infection is not well known. Expression of CCR5, a major coreceptor for HIV, was enhanced on CD4+ T cells from patients with acute EBV infection. Furthermore, susceptibility of those cells to R5-HIV-1, but not X4-HIV-1, was increased. EBV effects on CCR5 expression on or susceptibility to R5-HIV-1 of CD4+ T cells did not require coinfection of the same cell with the two viruses, because CD4+ T cells from patients with acute EBV infection were not infected with EBV. Considering that both HIV and EBV are transmitted by intimate contact, such possible interaction between the two viruses may have implications for viral transmission and the pathogenesis of HIV disease.     

Coreceptor use in nonhuman primate models of HIV infection

SIV or SHIV infection of nonhuman primates (NHP) has been used to investigate the impact of coreceptor usage on the composition and dynamics of the CD4+ T cell compartment, mechanisms of disease induction and development of clinical syndrome. As the entire course of infection can be followed, with frequent access to tissue compartments, infection of rhesus macaques with CCR5-tropic SHIVs further allows for study of HIV-1 coreceptor switch after intravenous and mucosal inoculation, with longitudinal and systemic analysis to determine the timing, anatomical sites and cause for the change in envelope glycoprotein and coreceptor preference. Here, we review our current understanding of coreceptor use in NHPs and their impact on the pathobiological characteristics of the infection, and discuss recent advances in NHP studies to uncover the underlying selective pressures for the change in coreceptor preference in vivo.     

Clinical performance of a polymerase chain reaction testing algorithm for diagnosis of HIV-1 infection in peripheral blood mononuclear cells

The clinical performance of a modified polymerase chain reaction (PCR) testing algorithm was evaluated for confirming the presence of HIV-1 proviral DNA in peripheral blood mononuclear cells. A whole cell lysate, rather than phenol-purified DNA, was used for PCR amplification, under systematically optimized conditions designed and verified within each PCR run to detect as few as 10 copies of proviral DNA. A sequential testing algorithm was designed requiring reactivity in duplicate (with corresponding non-reactivity in negative controls) with at least two sets of primers, before reporting a specimen as HIV-1-positive. In 196 specimens from patients staged according to the Walter Reed staging system, the PCR test sensitivity and the coculture isolation rate (in parentheses) were found to be: 97% (71%), 100% (85%), and 100% (76%) in stage 1, stage 2, and stage 3 specimens, respectively; and 100% (100%) in stage 4, 5, and 6 specimens. Results were uniformly negative for PCR and coculture isolation from 21 blind negative specimens and 105 (negative) donor leukopacks. These data indicate that this PCR testing algorithm is more accurate than tissue culture isolation methods, especially with early stage patients, and results in detection of HIV-1 in virtually 100% of seropositive individuals, with no false positives.     

Genetic determinants of HIV-1 infection and progression to AIDS: susceptibility to HIV infection

Interindividual variability in susceptibility to HIV-1 infection, its transmission, disease progression, and response to antiviral therapy has been attributed to host determinants and variability in multiple genes. Although most people exposed to the virus go on to develop full-blown disease at variable intervals, a proportion of them, labeled as long-term nonprogressors or exposed uninfected, possess 'natural resistance' to infection. A better understanding of genetic and immunologic basis of such a natural resistance to infection would bear important implications in designing therapeutic vaccine designs. The genetic variants that could influence susceptibility to HIV-1 and limit AIDS vary in different populations and among individuals. Meta-analyses of large cohort studies have identified numerous 'AIDS restriction genes' that regulate HIV cell entry (particularly chemokine coreceptors and their ligands), acquired and innate immunity (major histocompatibility complex, killer cell immunoglobulin-like receptor, and cytokines), and others [tripartite interaction motif 5 α (TRIM5α) and apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G] that influence outcome of HIV infection. Studies carried out in the Indian population with regard to genetic polymorphisms in chemokine receptors have shown that (i) the protective CCR5 Δ32 variant is rare, (ii) CCR5HHE carrying *59402A is associated with increased likelihood of infection and development of AIDS, and (iii) the Indian population generally has low CCL3L1 copy numbers (∼2.3). These data have implications in developing screening tests that could identify people at higher or lower risk of infection and rate of disease progression, predict vaccine responsiveness in clinical trials and understand the pathogenic mechanisms.     

Mu-opioid modulation of HIV-1 coreceptor expression and HIV-1 replication

A substantial proportion of HIV-1-infected individuals are intravenous drug users (i.v.DUs) who abuse opiates. Opioids induce a number of immunomodulatory effects that may directly influence HIV-1 disease progression. In the present report, we have investigated the effect of opioids on the expression of the major HIV-1 coreceptors CXCR4 and CCR5. For these studies we have focused on opiates which are ligands for the mu-opioid receptor. Our results show that DAMGO, a selective mu-opioid agonist, increases CXCR4 and CCR5 expression in both CD3(+) lymphoblasts and CD14(+) monocytes three- to fivefold. Furthermore, DAMGO-induced elevation of HIV-1 coreceptor expression translates into enhanced replication of both X4 and R5 viral strains of HIV-1. We have confirmed the role of the mu-opioid receptor based on the ability of a mu-opioid receptor-selective antagonist to block the effects of DAMGO. We have also found that morphine enhances CXCR4 and CCR5 expression and subsequently increases both X4 and R5 HIV-1 infection. We suggest that the capacity of mu-opioids to increase HIV-1 coreceptor expression and replication may promote viral binding, trafficking of HIV-1-infected cells, and enhanced disease progression.     

Leukocyte function-associated antigen-1 expression on peripheral blood mononuclear cell subsets in HIV-1 seropositive patients

In order to further investigate immune dysfunctions in HIV-1 infection, we studied the intensity of leukocyte function-associated antigen-1 (LFA-1) expression using a novel application of immunofluorescence analysis in 14 adults and 5 children seropositive for HIV-1 and in 14 healthy adults and 5 healthy children seronegative for HIV-1. While almost all lymphocytes in human peripheral blood expressed LFA-1 and while the percentage of the LFA-1 positive cells was not modified during the course of the HIV-1 infection in both adults and children, our results showed an increase of the LFA-1 expression on selected peripheral blood mononuclear cell subsets. Some LFA-1-labeled functional peripheral blood mononuclear cell subsets such as the CD16, CD14, CD3, and CD8 lymphocyte subpopulations expressed higher levels of the LFA-1 molecule during the HIV-1 infection. The LFA-1 dim cell subsets (CD4 cells) and the LFA-1 low cell subpopulation (CD19 lymphocytes) were not affected by the HIV-1 infection. Moreover, in the CD8 and CD3 cell subsets displaying a heterogeneous LFA-1 expression (dim and bright), we also observed a decrease of the LFA-1 dim/LFA-1 bright cell ratio.     

Selective decrease in human immunodeficiency virus type 1 (HIV-1)-induced alpha interferon production by peripheral blood mononuclear cells during HIV-1 infection.

We previously reported that human immunodeficiency virus type 1 (HIV-1), herpes simplex virus (HSV), and Sendai virus induce higher levels of alpha interferon (IFN-alpha) in blood dendritic cells than in monocytes of healthy donors. In the present study, the levels of IFN-alpha induced by T-cell tropic (IIIb and RF) and monocytotropic (BaL) strains of HIV-1 and by HSV were significantly decreased in peripheral blood mononuclear cells (PBMCs) derived from subjects with asymptomatic and symptomatic HIV-1 infection. In contrast, Sendai virus, a paramyxovirus that induces proportionally more IFN-alpha in monocytes and B cells than do either HIV-1 or HSV, stimulated normal levels of IFN-alpha in PBMCs from the HIV-1-infected men. The IFN-alpha produced by PBMCs from the HIV-1-seropositive subjects was partially acid labile, whereas the IFN-alpha produced by PBMCs from the HIV-1-seronegative donors was acid stable. We hypothesize that there is a selective defect in IFN-alpha production by peripheral blood dendritic cells, whereas the host retains the IFN-alpha-producing capacity of monocytes and B lymphocytes. The loss of IFN-alpha production in response to HIV-1, herpesviruses, and possibly other pathogens could contribute to the progression of HIV-1 infection and to the development of AIDS.     

A quantitative PCR method for the assay of HIV-1 provirus load in peripheral blood mononuclear cells

A set of plasmids designed for the construction of recombinant adenoviral vectors is described, which contain two expression cassettes, one in the early region 1 (E1) and the other in the early region 3 (E3). Two cloning steps in E. coli and a transfection of the resulting cosmid into 293 cells are sufficient to recover the recombinant virus. The method has been optimised to facilitate the introduction of the genes of interest in their respective regions and the reconstitution of the entire sequence of the recombinant adenoviral DNA in E. coli. The vectors are easy to handle and generate homogenous virus preparations. To illustrate the efficiency of the method, an adenovirus was constructed expressing the E. coli beta-galactosidase deltaM15 mutant in the E1 region and the complementing lacZ alpha-peptide in the E3 region.