Studies of human immunodeficiency virus type 1 mucosal viral shedding and transmission in Kenya

If human immunodeficiency virus type 1 (HIV-1) vaccines are to be highly effective, it is essential to understand the virologic factors that contribute to HIV-1 transmission. It is likely that transmission is determined, in part, by the genotype or phenotype (or both) of infectious virus present in the index case, which in turn will influence the quantity of virus that may be exchanged during sexual contact. Transmission may also depend on the fitness of the virus for replication in the exposed individual, which may be influenced by whether a virus encounters a target cell that is susceptible to infection by that specific variant. Of interest, our data suggest that the complexity of the virus that is transmitted may be different in female and male sexual exposures.     

Relationship between human immunodeficiency virus type 1 (HIV-1)-specific memory cytotoxic T lymphocytes and virus load after recent HIV-1 seroconversion.

Human immunodeficiency virus type 1 (HIV-1)-specific memory, or precursor, cytotoxic T lymphocytes (CTL) in 14 subjects who had recently experienced seroconversion were evaluated with respect to virus set point, defined as plasma HIV-1 RNA level 6 months after seroconversion. Env-, Gag-, Pol-, and Nef-specific precursor CTL were detected in (51)Cr-release assays, using antigen-stimulated peripheral blood mononuclear cells as effectors and B cell lines infected with HIV-1-vaccinia recombinants as targets. All subjects tested had precursor CTL specific to at least 2 HIV-1 antigens. Detection of Env-specific precursor CTL was associated with a high set point (P=.0221). The number of antigens recognized tended to be greater in subjects with higher set points (rho=.45621; P=.1171). Gag-specific precursor CTL frequency correlated inversely with set point (rho=-.8478; P=.0003). Two heterozygotes for a 32-bp deletion in CCR5 had the lowest set points (P=.0220) and highest Gag precursor CTL frequencies (P=.0128). These data suggest that host factors that restrict viral replication may be important determinants of the level of HIV-1-specific precursor CTL.     

中国人类免疫缺陷病毒-1感染者细胞毒性T淋巴细胞特异性应答

目的 探讨中国人类免疫缺陷病毒（HIV）／艾滋病（AIDS）患者HIV特异性细胞毒性T淋巴细胞（CTL）应答的特征。方法 以HIV-1 B、C亚型构建的2个全基因组肽库作为抗原，通过酶联免疫斑点（ELISPOT）法检测HIV／AIDS患者HIV特异性CTL应答。结果 无论HIV-1 B亚型还是HIV-1 C亚型所构建肽库的应答效应和频率主要集中在Gag和Nef蛋白，其他蛋白也有不同程度的应答。HIV-1 B、C亚型间应答比较，整体范围大致相同，但单个肽段水平还存在着一定差异，B亚型应答频率最高的是Nef的GPKEPFRDYVDRFYKTLR（5／17，29．4％）和Gag的LWVYHTQGYFPDWQNY（5/17，29．4％），C亚型应答频率最高的是Gag的GPKEPFRDYVDRFFKTLR应答频率为35．29％。结论 中国人群CTL应答多集中在Gag和Nef蛋白，B、C亚型在单个肽段水平略有差异。提示中国人群的CTL应答研究对设计针对中国人群的HIV疫苗有较重要的意义。     

Human immunodeficiency virus type 1 and mucosal humoral defense

Mucosal sites serve as the principle venues through which primary human immunodeficiency virus type 1 (HIV-1) infections are transferred from donor to host. These moist tissues, which provide the interface with the external environment, also provide access to many of the secondary opportunistic infections that aggravate and may accelerate HIV-1 disease. Antibodies to HIV-1, particularly of the IgG rather than the IgA class, have been detected in virtually all mucosal fluids from HIV-1-infected patients. However, the ability of such patients to generate de novo humoral responses to new mucosal pathogens is impaired. Current studies are directed to characterizing the functional role of natural and infection-derived antibodies in control of HIV-1 infection as well as the impact of HIV-1 disease on mucosal B cell responses to immunization and infection.     

Mucosal candidiasis in transgenic mice expressing human immunodeficiency virus type 1

The availability of CD4C/HIV(MutA) transgenic (Tg) mice expressing human immunodeficiency virus type 1 in immune cells and developing an AIDS-like disease has provided the opportunity to devise a model of mucosal candidiasis that closely mimics the clinical and pathologic features of candidal infection in human AIDS. After intraoral infection with Candida albicans, oral burdens were strikingly elevated in the Tg mice, compared with non-Tg littermates (P<.05), during primary infection, a 6-10-week carrier state, and a marked terminal outgrowth preceding death. The chronic carrier state was absent in the non-Tg mice because of clearing of C. albicans. Candida hyphae penetrated the epithelium of the oral cavity, esophagus, and cardial-atrium fold of the stomach, accompanied by a mononuclear cell infiltrate. Immunohistochemical analysis suggested that decreased frequencies of major histocompatibility complex class II-expressing cells, combined with reduced CD4+ cells, may underlie the susceptibility to mucosal candidiasis in these Tg mice.     

Viral-specific cytotoxic T lymphocytes lyse human immunodeficiency virus-infected primary T lymphocytes by the granule exocytosis pathway.

Cytotoxic T lymphocytes (CTL) lyse antigen-bearing target cells by two distinct pathways. Whereas granule exocytosis targets any antigen-bearing cell, fas-mediated cytotoxicity kills only fas-expressing cells and does not require antigen expression. Fas pathway activation can potentially lead to lysis of uninfected bystander cells. We examined the relative usage of the two pathways by CTL clones and cell lines directed against four different human immunodeficiency virus (HIV) proteins in lysing primary HIV-infected targets. Although fas was expressed on HIV-infected primary CD4(+) T cells, their lysis by antigen-specific CD8(+) CTL was only by the granule pathway. Fas ligand (fasL) was not detectable on antigen-specific CD8 clones, T-cell lines, or circulating HIV-specific CD8 T cells from HIV-infected donors, stained with a tetrameric HLA-A2-HIV-peptide complex. FasL expression by HIV-specific CTL clones was not activated by exposure to HIV-presenting cells, but was after unphysiological stimulation with phorbol myristate acetate (PMA). CTL clones did not lyse bystander Jurkat cells, but HIV-infected primary CD4(+) T cells lysed uninfected bystander cells by the fas-mediated pathway. These results suggest that HIV-specific CD8(+) CTL do not cause HIV immunopathology by lysing bystander cells. On the contrary, fas-mediated lysis of uninfected cells by HIV-infected cells may contribute to CD4 decline.     

CD8+ cytotoxic T lymphocyte responses to lytic proteins of human herpes virus 8 in human immunodeficiency virus type 1-infected and -uninfected individuals

T cell immunity to lytic proteins of herpesviruses is important in host control of infection. We have characterized the cytotoxic T lymphocyte (CTL) response to 5 human herpesvirus 8 (HHV-8) homologues of lytic proteins in HHV-8-seropositive individuals. HLA class I-restricted, CD8(+) CTL responses to >/=1 HHV-8 lytic protein were detected in all 14 HHV-8-seropositive study subjects tested, with or without human immunodeficiency virus type 1 (HIV-1) infection, but not in any of 5 HHV-8-seronegative individuals. Seven of these study subjects with both HHV-8 and HIV-1 infection had greater anti-CTL reactivity to glycoprotein H (open-reading frame 22) than did the 7 study subjects infected only with HHV-8. Moreover, there was a strong, inverse correlation between HIV-1 load and glycoprotein H-specific CTL lysis in the study subjects infected with both viruses. CTL reactivity to HHV-8 lytic proteins may be involved in host control of HHV-8-related diseases, such as Kaposi's sarcoma.     

Accumulation of specific amino acid substitutions in HLA-B35-restricted human immunodeficiency virus type 1 cytotoxic T lymphocyte epitopes.

HLA is one of the genetic factors that influence the clinical course of HIV-1 infection, and patients with HLA-B35 are prone to rapid disease progression. Nine viral epitopes that are recognized by cytotoxic T lymphocytes (CTLs) in an HLA-B35-restricted manner were determined. To examine how HIV-1 sequences are selected by CTLs in vivo, we sequenced the nine CTL epitopes of the virus in patient plasma. Here we show that certain amino acid substitutions at three epitopes were observed with significantly higher frequency in HLA-B35-positive patients than in HLA-B35-negative patients. By performing experiments with CTL clones established from the HLA-B35-positive patients, it was determined that one of the three substitutions was probably an escape mutation. However, concerning the other two epitopes, representative CTL clones killed target cells pulsed with mutant peptides as efficiently as those pulsed with wild-type peptides, suggesting that CTLs that can be established in vitro are not functioning properly in vivo. Amino acid sequence drift in all HLA-B35-restricted epitopes was rare during the observation period (1 year). Our results may have relevance in understanding the rapid clinical progression in HLA-B35-positive patients.     

Detection of human T cell leukemia virus type I and human immunodeficiency virus in cultured lymphocytes of a Zairian man with AIDS

We report the detection of human T cell leukemia virus type I (HTLV-I) and human immunodeficiency virus (HIV) in the cultured lymphocytes of a 45-year-old Zairian man with AIDS. HIV was successfully isolated and analyzed by SDS-PAGE and competition radioimmunoassay. However, by the culture techniques used, HTLV-I could not be separated from the HIV. Western blot analysis of the patient's serum showed the presence of both HTLV-I- and HIV-specific antibodies. The finding of this dual infection may explain reports that greater than or equal to 30% of patients with AIDS are positive for antibodies to HTLV-I.     

Gene inoculation generates immune responses against human immunodeficiency virus type 1.

Recently, immunization techniques in which DNA constructs are introduced directly into mammalian tissue in vivo have been developed. In theory, gene inoculation should result in the production of antigenic proteins in a natural form in the immunized host. Here we present the use of such a technique for the inoculation of mice with a human immunodeficiency virus type 1 (HIV-1) envelope DNA construct (pM160). Mice were injected intramuscularly with pM160 and were subsequently analyzed for their anti-HIV envelope immune responses. Antisera collected from inoculated animals reacted with the recombinant HIV-1 envelope in ELISA and immunoprecipitation assays. The antisera also contained antibodies that were able to neutralize HIV-1 infection and inhibit HIV-1-mediated syncytium formation in vitro. Furthermore, splenic lymphocytes derived from pM160-inoculated animals demonstrated HIV-envelope-specific proliferative responses. The gene inoculation technique mimics features of vaccination with live attenuated viruses and, therefore, may ultimately prove useful in the rapid development of safe and efficacious vaccines as it provides for production of relevant antigen in vivo without the use of infectious agents.     

Human immunodeficiency virus type 1 Gag proteins are processed in two cellular compartments.

The structural proteins of the retroviral capsid are translated as a polyprotein (the Gag precursor) that is cleaved by a virally encoded protease. Processing of the human immunodeficiency virus type 1 Gag precursor Pr55 was analyzed through a combination of pulse-chase labeling, cell fractionation, and immunoprecipitation. We observed a membrane-associated processing pathway for the Gag precursor that gives rise to virions. In addition, we found that a significant amount of processing occurs in the cytoplasm of infected cells resulting in the intracellular accumulation of appropriately processed viral proteins. This observation suggests the viral protease is active in the cytoplasmic compartment of the cell. Processing of the Gag protein was blocked in both compartments by the addition of a viral protease inhibitor. A comparison of the amount of cytoplasmic processing seen in lytically infected cells with that seen in chronically infected cells showed that cytoplasmic processing was associated with the lytic infection. These observations raise the possibility that activation of the human immunodeficiency virus type 1 protease in the cytoplasm of lytically infected cells might result in the cleavage of cellular proteins and thus contribute to cytotoxicity.     

A Rev-inducible mutant gag gene stably transferred into T lymphocytes: an approach to gene therapy against human immunodeficiency virus type 1 infection.

One strategy for somatic gene therapy for human immunodeficiency virus type 1 (HIV-1) infection is based on the regulated expression of dominant negative mutants of the HIV-1 gag gene. To limit expression of the mutant Gag polypeptide to HIV-1-infected cells, we have constructed a replication-defective retroviral vector that contains a Rev-responsive element. By using this construct we have obviated problems that can be associated with constitutive expression of an exogenous gene, an important step toward developing a human therapy. In uncloned T lymphocytes infected (transduced) with this retroviral construct, HIV-1 replication was inhibited by 94% with a concomitant decrease in the cytopathic effects of the virus. In addition, simian immunodeficiency virus (SIV) replication was also shown to be significantly inhibited, suggesting that this mutant Gag protein may have antiviral efficacy against a broad range of primate lentiviruses and that an SIV/macaque model can be used for further in vivo studies. These results have important implications in assessing the potential of somatic gene therapy in the treatment of HIV-1 infection.     

Studies on the pathogenesis of type I diabetes mellitus--destruction of pancreatic beta cells by cytotoxic T lymphocytes in nonobese diabetic mice

Proliferation of islet-associated leukocytes occurred when isolated islets from 20 week-old female Non-obese Diabetic (NOD) mice were cultured with 10 U/ml recombinant interleukin-2 (rIL-2) for 7 days. Co-culture of these lymphocytes with freshly-isolated islets from 6-8 week-old NOD donors in the presence of 1 U/ml rIL-2 produced islet structural deformation within 24 h and islet cytolysis within 48 h. Three lines of evidence suggest that leukocytes were cytotoxic T lymphocytes (CTL) specific for islet cells. First, these proliferating cells adhered to NOD islets at 6 h and specifically killed islets after 48 h of culture, but the cytoadherence of these cells to the other organs including thyroid, pancreatic exocrine glands and liver from NOD mice could not be observed and the shape of tissue clumps hardly deformed after culture for 48 h. The accumulated insulin release from NOD islets to the medium after 6 h of culture was significantly increased in the presence of islet-derived cells compared with the insulin release in the absence of cells. On the contrary, lactic dehydrogenase activity released from liver and amylase activity from pancreatic exocrine glands showed on difference between with and without these cells for 6h of culture. Second, a flow cytometric analysis showed that these cells consisted of 96%Thy1.2, 70%Lyt2, and 8%L3T4-positive cells. After treatment with monoclonal anti-Thy1.2 or Lyt2 antibody and complement, these cells lost their activity to destroy NOD islets. However, these cells still had a full killing activity after the depletion of L3T4-positive cells. Third, islets of NOD (H-2 genotype KdDb), B10.GD (H-2KdDb), BALB/cA (H-2d), and DBA/2N (H-2d) were susceptible to destructive activity of these cells, whereas islets of NON (H-2b), C57BL/6N (H-2b), C57BL/10J (H-2b), and C3H/He (H-2k) mice remained intact. Furthermore, anti-Kd monoclonal antibody could prevent islet-specific cytolysis of these cells. These results suggest that CTL expressing Thy1.2 and Lyt2 phenotypes appear to recognize islet cell antigen with restriction of major histocompatibility complex (MHC) class H-2Kd and then destroy pancreatic beta cells in NOD mice.     

Complement and virus-specific antibody-dependent infection of normal B lymphocytes by human immunodeficiency virus type 1

We tested the susceptibility of human purified, normal B lymphocytes to human immunodeficiency virus type 1 (HIV-1) infection, in the presence or absence of complement-sufficient serum and of virus-specific antibodies. Virus replication was detected when cells were infected in the presence of both complement and anti-HIV antibodies (C'-ADE conditions), by day 2 postinfection. Similar results were obtained when B lymphocytes were purified either from peripheral blood (three healthy donors) or from tonsils (four individuals with chronic tonsillitis). HIV infection was shown by polymerase chain reaction (PCR) detection of proviral sequences (gag and pol genes), by p24 antigen synthesis, and by cocultivation assay with MT2 cells. The higher p24 production was obtained when B cells were preactivated for 2 days by phorbol 12- myristate 13-acetate (PMA) before infection and then cultured in the presence of low-molecular weight B-cell growth factor (LMW-BCGF). Expression of virus envelope glycoprotein (gp) 120 could also be detected on a subpopulation of B cells (CD19+, CD22+) by flow cytometry. Blocking experiments with monoclonal antibodies (MoAbs) against CD4, CD21 (complement receptor 2 [CR2]), CD35 (CR1), CD19, and CD5 surface molecules indicated that infection of B cells involves CD4, CD21, and CD35 antigens. Indeed, blocking of CD4 receptor inhibited 10% of p24 production, and blocking of both CD21 and CD35 led to extinction of p24 signal. CR-dependent pathway is thus a major route for C'-ADE of HIV infection in normal B cells. Our results emphasize the importance of studying interactions between HIV and the complement system for better understanding infection mechanisms and the major dysfunctions of B cells in HIV-infected individuals.     

In vivo cytolysis and fusion of human immunodeficiency virus type 1-infected lymphocytes in lymphoid tissue

Lymphoid tissue was examined to see whether in vivo cytopathic effects of human immunodeficiency virus (HIV) infection on lymphocytes could be detected. Transmission electron microscopy of mechanical suspensions prepared from lymph nodes showed both replication and phagocytosis of HIV particles by macrophages. Phagosomes contained cellular debris and virions, some of which were undergoing digestion. Neutrophils also contained HIV particles intermixed with cellular debris in phagosomes. Immunohistochemistry revealed whole Gag p24-positive lymphocytes and p24-positive cellular debris within the cytoplasm of paracortical macrophages. Lysing p24-positive lymphocytes were also seen. In the paracortex, p24-positive multinucleated lymphocytes with up to 5 nuclei were seen. In situ hybridization for HIV RNA in combination with immunohistochemistry for HAM56, a macrophage-specific marker, revealed colabeled cells. Thus, HIV-positive lymphocytes undergo lysis in lymphoid tissue. The cellular debris is phagocytized by macrophages, which themselves can replicate HIV. HIV-positive lymphocytes fuse in lymph nodes to form multinucleated cells.