Antibody to HIV-1 Tat protein inhibits the replication of virus in culture

Summary The HIV-1 transactivator protein Tat is essential for viral replication. Tat is released from infected cells and can be taken up and transactivate HIV-LTR in LTR-CAT transfected cell lines. The present study shows that the addition of monoclonal antibody to Tat in IIIB and MN-infected cultures reduces the HIV antigen production in a concentration dependent manner. These data suggest that external Tat might be important in the replication of HIV, exerting the effect in a paracrine fashion. Using 1 µg/ml of anti-Tat antibody resulted in a decline of HIV antigen production to 33% and 45% of controls in IIIB and MN infected H9 cells, respectively. A time course experiment showed progressively increased inhibition of replication during 7 days of exposure to anti-Tat antibody, which could be due to increasing Tat concentration. The inhibitory effect of anti-Tat antibodies on the replication of HIV could play an important regulatory role during infection in vivo.     

Inhibition of HIV-1 virus replication using small soluble Tat peptides

Although the introduction of highly active antiretroviral therapy (HAART) has led to a significant reduction in AIDS-related morbidity and mortality, unfortunately, many patients discontinue their initial HAART regimen, resulting in development of viral resistance. During HIV infection, the viral activator Tat is needed for viral progeny formation, and the basic and core domains of Tat are the most conserved parts of the protein. Here, we show that a Tat 41/44 peptide from the core domain can inhibit HIV-1 gene expression and replication. The peptides are not toxic to cells and target the Cdk2/Cyclin E complex, inhibiting the phosphorylation of serine 5 of RNAPII. Using the Cdk2 X-ray crystallography structure, we found that the low-energy wild-type peptides could bind to the ATP binding pocket, whereas the mutant peptide bound to the Cdk2 interface. Finally, we show that these peptides do not allow loading of the catalytic domain of the cdk/cyclin complex onto the HIV-1 promoter in vivo.     

Double-stranded deoxyribonucleic acid binds to HLA class II molecules and inhibits HLA class II-mediated antigen presentation

CD4⁺ T cells proliferating in response to purified double-stranded deoxyribonucleic acid (dsDNA) have been recently demonstrated in peripheral blood mononuclear cells of patients with systemic lupus erythematosus. Their activation was inhibited by anti-HLA class II (HLA-II) monoclonal antibodies; thus, the existence of a molecular interaction between dsDNA and HLA-II is conceivable. In this report we show that dsDNA specifically bind to HLA-II. After preincubating cells with purified dsDNA or synthetic oligonucleotides, dsDNA was detected on the cell membrane and in the lysates of HLA-II⁺ but not of isogenic HLA-II⁻ cell lines. We demonstrate that dsDNA binding inhibits that of a specific peptide to HLA-II. Mixed lymphocyte reaction and antigen-specific T cell proliferation were inhibited by the preincubation of stimulator cells or antigen-presenting cells with dsDNA. These results suggest the existence of a novel mechanism of down-modulation of the CD4⁺ T cell function generated by lack of stimulation due to the HLA-II presenting molecules being “n;-occupied” by dsDNA.     

Improving immune function and controlling viral replication in HIV-1-infected patients with immune-based therapies

Restoring and preserving immune function is a key component to successfully managing HIV-1 disease. Phase II/III studies have evaluated the safety and immunologic effects of immune-based therapies, including granulocyte-macrophage colony-stimulating factor, interleukin-2, and an inactivated HIV-1 immunogen, as adjuncts to antiretroviral therapy. Addition of each of these immune-based therapies to a background antiretroviral regimen enhanced, to varying degrees, immunologic function and suppression of viral replication in HIV-1-infected patients, suggesting a potential role for immune-based therapies in the treatment of HIV-1 disease. Further studies are needed to better characterize specific immunologic and virologic effects in different patient populations and to determine their impact on clinical outcomes.     

Sequence-based typing of HLA class II DQB1

Due to the expanding number of known HLA class II DQB1 alleles, high-resolution oligotyping is becoming ineffective, therefore a sequence-based typing (SBT) strategy was developed to provide rapid and definitive typing of HLA-DQB1. HLA-DQB1*02, *03, *04, *05, and *06 alleles were individually amplified by polymerase chain reaction (PCR) using exon 2 group-specific primers. Forward and reverse PCR primers were tailed with M13 universal and M13 reverse sequences, respectively. Subsequent bi-directional cycle-sequencing was carried out using Cy5.5-labeled M13 universal primer and Cy5.0-labeled M13 reverse primer. Automated sequencing was performed in 30 min using a Visible Genetics, Inc. (VGI) MicroGene Clipper Sequencer. Full concordance was observed between this SBT method and oligotyping among 151 individuals.     

The local environment orchestrates mucosal decidual macrophage differentiation and substantially inhibits HIV-1 replication

Macrophages from the decidua basalis (dM), the main uterine mucosa during pregnancy, are weakly permissive to HIV-1 infection. Here, we investigated the mechanisms underlying this natural control. We show, by using freshly purified decidual macrophages and ex vivo human decidual explants, that the local decidual environment influences dM differentiation and naturally protects these cells from HIV-1 infection. Interferon (IFN)-γ, present in the decidual tissue, contributes to maintenance of the dM phenotype and restricts HIV-1 infection by mechanisms involving the cyclin-dependent kinase inhibitor p21Cip1/Waf1. We also found that activation of Toll-like receptors 7 and 8 expressed by dM reinforces the low permissivity of dM to HIV-1 by restricting viral replication and inducing secretion of cytokines in the decidual environment, including IFN-γ, that shape dM plasticity. A major challenge for HIV-1 eradication is to control infection of tissue-resident macrophages in the female reproductive tract. Our findings provide clues to the development of novel strategies to prevent HIV-1 macrophage infection.     

我国中部地区HIV感染者HLA Ⅰ类等位基因多态性与病毒载量的相关性研究

目的 分析我国中部地区既往采浆HIV感染者HLA Ⅰ类等位基因多态性及其与病毒载量的相关性.方法 应用PCR-SSP(序列特异性引物)技术对106例HIV感染者进行HLA Ⅰ类基因分型,分析HLA Ⅰ类等位基因多态性和单元型与血浆病毒载量的相关性.利用ELISPOT技术检测Gag蛋白特异性的CTL反应,并分析其与血浆病毒载量的相关性.结果 携带HLA Ⅰ类等位基因纯合子的感染者具有较高的病毒载量(P=0.0098);HLA-A*30、-B*13、-Cw*06、-Cw*14等位基因及A*30/B*13/Cw*06单元型与低病毒载量相关(P=0.0004,0.0103,0.0058,0.0371和0.0006);携带HLA-A*30/B*13/Cw*06单元型的感染者p2p7p1p6蛋白特异性的CTL反应与病毒载量负相关;携带HLA-Cw*14的感染者p17蛋白特异性的CTL反应与病毒载量负相关.结论 我国中部地区既往采浆HIV感染人群HLA-A*30、-B*13、-Cw*06、-Cw*14等位基因及A*30/B*13/Cw*06单元型携带者具有较低的病毒载量,其保护性与Gag蛋白特异性的CTL反应有关.     

HIV-1 proteins in infected cells determine the presentation of viral peptides by HLA class I and class II molecules and the nature of the cellular and humoral antiviral immune responses—A review

The goals of molecular virology and immunology during the second half of the 20th century have been to provide the conceptual approaches and the tools for the development of safe and efficient virus vaccines for the human population. The success of the vaccination approach to prevent virus epidemics was attributed to the ability of inactivated and live virus vaccines to induce a humoral immune response and to produce antiviral neutralizing antibodies in the vaccinees. The successful development of antiviral vaccines and their application to most of the human population led to a marked decrease in virus epidemics around the globe. Despite this remarkable achievement, the developing epidemics of HIV-caused AIDS (accompanied by activation of latent herpesviruses in AIDS patients), epidemics of Dengue fever, and infections with respiratory syncytial virus may indicate that conventional approaches to the development of virus vaccines that induce antiviral humoral responses may not suffice. This may indicate that virus vaccines that induce a cellular immune response, leading to the destruction of virus-infected cells by CD8⁺ cytotoxic T cells (CTLs), may be needed. Antiviral CD8⁺ CTLs are induced by viral peptides presented within the peptide binding grooves of HLA class I molecules present on the surface of infected cells. Studies in the last decade provided an insight into the presentation of viral peptides by HLA class I molecules to CD8⁺ T cells. These studies are here reviewed, together with a review of the molecular events of virus replication, to obtain an overview of how viral peptides associate with the HLA class I molecules. A similar review is provided on the molecular pathway by which viral proteins, used as subunit vaccines or inactivated virus particles, are taken up by endosomes in the endosome pathway and are processed by proteolytic enzymes into peptides that interact with HLA class II molecules during their transport to the plasma membrane of antigen-presenting cells. Such peptides are identified by T-cell receptors present on the plasma membrane of CD4⁺ T helper cells. The need to develop viral synthetic peptides that will have the correct amino acid motifs for binding to HLA class I A, B, and C haplotypes is reviewed.The development of HIV vaccines that will stimulate, in an uninfected individual, the humoral (antibody) and cellular (CTL) immune defenses against HIV and HIV-infected cells, respectively, and may lead to protection from primary HIV infection are discussed. The need to eliminate the release of HIV virions from infected cells introduced by an infected donor to an uninfected recipient may require both the humoral and cellular immune responses. However, such CTLs may fail to identify HIV-infected cells with integrated HIV proviral DNA that do not express viral genes and proteins. Based on reported results on the immunization of monkeys with uninfected cells, which prevented infection with SIV grown in the same type of cells, it may be possible to consider immunization of specific human populations against HLA haplotypes prevalent in HIV-infected donors. Since HIV virions may carry the HLA class I molecules present in the infected donors' cells, synthesis of CTLs to the mutated amino acid sequence in peptide binding grooves of the foreign HLA haplotypes may induce anti-HLA CTLs in the immunized individual, which may destroy HIV-infected, virus synthesizing donor cells, as well as donor cells containing latent proviral DNA. Such anti-foreign HLA CTLs may prevent the release of virions from the infecting donor's cells. The importance of HLA haplotypes for protection against HIV will be discussed.Dedicated to the memory of Dr. Albert Sabin (1906–1993).     

Modified oligopeptides designed to interact with the HIV-1 proteinase inhibit viral replication

Summary The human immunodeficiency virus 1 (HIV-1) codes for a proteinase that cuts viral proteins at specific sites. We have tested 13 modified oligopeptides related to these cleavage sites to see if they inhibit viral replication. To indicate whether a decrease in replication could be due to a general inhibition of cell metabolism, we also measured the effect of the peptides on cellular protein synthesis. Three of the peptides tested (Ac-Gln-Asn-Sta-Val-NH₂, Ac-Gln-Asn-Sta-Val-Val-NH₂, and Ac-Glu-Asn-Sta-Ile-NH₂) inhibited HIV-1 replication at concentrations that did not inhibit protein synthesis. Ac-Gln-Asn-Sta-Val-NH₂ was the most potent, causing an approximately 40% decrease in viral replication, measured as the synthesis of HIV-1 antigens and the formation of infectious particles.     

Safety and immunogenicity of HIV-1 Tat toxoid in immunocompromised HIV-1-infected patients

OBJECTIVES: To antagonize the deleterious effects of the HIV-1 toxin extracellular Tat on uninfected immune cells, we developed a new strategy of anti-HIV-1 vaccine using an inactivated but immunogenic Tat (Tat toxoid). Tat toxoid has been assayed for safety and immunogenicity in seropositive patients. METHOD: The phase I vaccine clinical trial testing Tat toxoid preparation in Seppic Isa 51 oil adjuvant was performed on 14 HIV-1-infected asymptomatic although biologically immunocompromised individuals (500-200 CD4+ cells/mm3). RESULTS: Following as many as 8 injections, no clinical defects were observed. All patients exhibited an antibody (Ab) response to Tat, and some had cell-mediated immunity (CMI) as evaluated by skin test in vivo and T-cell proliferation in vitro. CONCLUSION: These results provide initial evidence of safety and potency of Tat toxoid vaccination in HIV-1-infected individuals.     

Lack of associations between HLA class II alleles and resistance to HIV-1 infection among white, non-Hispanic homosexual men

HLA class II alleles were molecularly typed for 100 high-risk seronegative men and 184 low-risk seroconverters from the Multicenter AIDS Cohort Study (MACS). Seven resistant individuals homozygous for CCR5 Delta32 deletions were excluded from analysis. In the univariate analysis, no significant HLA class II associations with resistance/susceptibility to HIV type 1 infection were identified. However, the transporter associated with antigen presentation 2 (TAP2) Ala 665 variant associated with resistance in earlier analyses in the MACS was in linkage disequilibrium with some HLA class II alleles. After adjusting for the established associations with HLA-A*0205 subgroup and TAP2 Ala 665 variant, no HLA class II alleles were independently associated with resistance/susceptibility to HIV-1 infection. Other genetic factors in the HLA class II-TAP region of the major histocompatibility complex might be involved.     

Impact of suppression of viral replication by highly active antiretroviral therapy on immune function and phenotype in chronic HIV-1 infection

We compared immune phenotypes, lymphocyte proliferation (LP), and delayed type hypersensitivity (DTH) responses in 28 male antiretroviral treatment-naive and experienced HIV-1-infected patients, matched pair-wise according to age and CD4+ T-lymphocyte count. Median CD4+ T-lymphocyte counts were 441 cells/microL and 483 cells/microL and median CD4+ T-lymphocyte nadirs were 435 cells/microL and 150 cells/microL in both groups, respectively. Absolute numbers of circulating T-lymphocyte subpopulations and proportions of naive and memory T-lymphocytes were comparable in the two groups. Untreated patients had greater proportions of activated CD4+ (p <.05) and CD8+ (p <.01) T-cells expressing human leukocyte antigen (HLA)DR and CD38 and fewer CD8+ cells expressing CD28 (p <.05). DTH and LP responses were comparable in both groups except for HIVp24, LP responses, and mumps DTH responses, which were of greater magnitude in the group treated with highly active antiretroviral therapy (HAART) (p <.05). Thus, HIV-1-infected patients who experienced substantial increases in CD4+ T-lymphocyte counts after suppression of viral replication on HAART had fewer activated lymphocytes and similar immune function when compared with findings in untreated patients with similar CD4+ T-cell counts. HIV replication has minimal real-time effect on CD4+ T-cell function in response to non-HIV antigens but helper T-cell responses to HIV-gag antigen are impaired during ongoing viral replication and may be restored by antiretroviral therapy.     

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.     

Molecular pathway involved in HIV-1-induced CNS pathology: role of viral regulatory protein, Tat

The broad range of histological lesions associated with HIV-1 are somewhat subtle relative to the clinical manifestations that occur as a result of HIV infection. Although it is clear that HIV has a causative role in CNS disease, dementia appears to be a consequence of the infiltration of inflammatory cells and cytokine dysregulation rather than the amount of virus in CNS. The HIV transregulatory protein Tat plays an important intracellular as well as extracellular role in the dysregulation of cytokines. The cytokines and possibly chemokines that are induced by Tat modify the action of astrocytes such that the survival of neurons is compromised. Pathogenetic alteration induced by Tat involves a series of interactions between circulating monocyte/macrophages, endothelial cells, and astrocytes. Cytokine dysregulation induced by viral infection and extracellular Tat leads to alterations in expression of adhesion molecules and promotes migration of non-infected inflammatory cells into the CNS compartment. We demonstrate here that recombinant HIV-1 Tat protein introduced by stereotaxic injection into mouse brain can induce pathologically relevant alterations including macrophage invasion as well as astrocytosis. The mechanism of destruction of the CNS by Tat appears to involve autocrine and paracrine pathways that depend not only on Tat, but cytokine and chemokine signaling pathways that are altered by viral infection. In this review, we discuss various pathogenic effects of Tat in brain cells and provide experimental evidence for an increased TNF-alpha level in CSF in mice injected intracerebrally with Tat protein.