Levels of soluble CD40 ligand (CD154) in serum are increased in human immunodeficiency virus type 1-infected patients and correlate with CD4(+) T-cell counts

CD40 ligand (CD40L or CD154) is a costimulatory molecule expressed mainly on activated CD4(+) T cells. Concentrations of the soluble form of CD40L (sCD40L) in serum were determined for a cohort of 77 human immunodeficiency virus type 1 (HIV-1)-infected patients before and after initiation of highly active antiretroviral treatment (HAART) by a quantitative enzyme-linked immunosorbent assay. Circulating sCD40L levels were higher by twofold in untreated patients than in healthy controls (means +/- standard deviations [SD]: 1.41 +/- 1.48 versus 0.69 +/- 0.59 ng/ml; P < 0.001). HIV-1-infected patients classified as CD4 T-cell category 1 had significantly higher sCD40L levels than patients classified as CD4 categories 2 and 3 (mean +/- SD: 2.08 +/- 1.46 ng/ml versus 1.57 +/- 1.58 [category 2] and 0.94 +/- 1.25 ng/ml [category 3]; P = 0.046), while no correlation with clinical categories A, B, and C was found. Individual serum sCD40L levels correlated with CD4(+) T-cell counts (P = 0.039) but not with viral load, gamma globulin levels, or acute-inflammatory-response markers. After 8 to 12 months of HAART, a further threefold increase of serum sCD40L levels, which paralleled the increase of CD4(+) T-cell counts, was observed. These novel findings suggest that sCD40L measurement in HIV-1-infected patients could serve as a new surrogate marker useful in the assessment of treatment efficacy, especially in settings where well-equipped laboratories and funding required for CD4(+) T-cell count and viral load measurements are not available.     

Contribution of CD8(+) T cells to gamma interferon production in human tuberculosis

The proportions of peripheral blood mononuclear cells (PBMC), CD4(+) T cells, and CD8(+) T cells that produce gamma interferon (IFN-gamma) in response to Mycobacterium tuberculosis were markedly reduced in tuberculosis patients, particularly in those with severe disease. Depletion of CD4(+) but not CD8(+) cells prior to stimulation of PBMC with M. tuberculosis abolished IFN-gamma production. These results show that (i) IFN-gamma production by CD8(+) and CD4(+) cells correlates with the clinical manifestations of M. tuberculosis infection and (ii) IFN-gamma production by CD8(+) cells depends on CD4(+) cells.     

T-helper-cell proliferative responses to whole-killed human immunodeficiency virus type 1 (HIV-1) and p24 antigens of different clades in HIV-1-infected subjects vaccinated with HIV-1 immunogen (Remune)

The discovery of multiple subtypes of human immunodeficiency virus type 1 (HIV-1) worldwide has created new challenges for the development of both therapeutic and preventive AIDS vaccines. We examined T-helper proliferative responses to HIV-1 clade A, B, C, G, and E whole-killed virus and to HIV-1 clade G and B core (p24) antigens in HIV-1-infected subjects taking potent antiviral drugs who received HIV immunogen (Remune) therapeutic vaccination. Subjects who were immunized mounted strong proliferative responses to both whole virus and core antigens of the different clades. These results suggest that a whole-killed immunogen may have broad applications as a therapeutic as well as a preventive vaccine in the current multiclade HIV-1 pandemic.     

Direct measurement of CD4+ and CD8+ T-cell responses to CMV in HIV-1-infected subjects

Data from murine models of chronic viral infection suggest that CD4+ T-cell responses to viral pathogens are important in sustaining the number and/or function of CD8+ cytotoxic T-cell (CTL) effectors. In this study, we used cytokine flow cytometry (CFC), staining with HLA-A*0201-peptide tetramers, and peptide stimulation with epitopic peptides to study functional CD4+ and CD8+ T-cell responses to cytomegalovirus (CMV) in human subjects coinfected with CMV and the human immunodeficiency virus, type 1 (HIV-1). We show that strong CD4+ and CD8+ T-cell responses to CMV antigens are sustained over time in HIV-1-infected individuals. Those who maintain a strong CD4+ T-cell response to CMV are also likely to maintain higher frequencies of CD8+ T cells capable of binding to HLA-A*0201-CMV pp65 (A2-pp65) tetramers as well as responses to pp65 peptide stimulation with effector cytokine production. These data support the hypothesis that declines in frequencies of CD4+ T-cell responses to CMV are associated with an inability to sustain high levels of CMV-specific CD8+ T-cell responses in HIV-1-infected subjects. These declines may precede the onset of CMV-associated end organ disease.     

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.     

Pregnancy and human herpesvirus 8 reactivation in human immunodeficiency virus type 1-infected women

To investigate the impact of pregnancy on human herpesvirus 8 (HHV-8) reactivation in human immunodeficiency virus type 1 (HIV-1)-infected women, the HHV-8 DNA presence and load were analyzed in peripheral blood mononuclear cells (PBMCs) and cervicovaginal secretions (CVSs) from 15 pregnant women coinfected with HIV-1 and HHV-8. HHV-8 detection was analyzed in relation to anti-HHV-8 antibodies and HIV-1-related parameters. Nucleotide sequence analysis of an ORFK1 hypervariable region of the HHV-8 strains was performed. HHV-8 was detected in maternal PBMCs (5/15 women) from the second trimester and in CVSs (5/15 women) mainly from the third trimester. The HHV-8 load significantly increased late in pregnancy in both maternal compartments and was associated with a significant increase in HIV-1 shedding in the genital tract. Antilytic antibodies were significantly more common in HHV-8 DNA-positive women. An elevated HHV-8 load was found in the PBMCs of an infant born to a mother with large amounts of HHV-8 in both compartments at delivery. Different ORFK1 subtypes were found in maternal samples, whereas the same subtype was identified in the mother-child pair. These data suggest that pregnancy may induce HHV-8 replication in HIV-1-infected women. An augmented HHV-8 load may, in turn, influence mother-to-child transmission, since one of the HIV-1-infected mothers with HHV-8 reactivation transmitted her ORFK1 subtype to the infant, who showed a high level of HHV-8 viremia indicative of a primary infection. This finding documents for the first time the perinatal transmission of a specific HHV-8 subtype. Vertical transmission may thus play a role in HHV-8 spread also in areas of subendemicity among HIV-1-infected women.     

A double-blind, adjuvant-controlled trial of human immunodeficiency virus type 1 (HIV-1) immunogen (Remune) monotherapy in asymptomatic, HIV-1-infected thai subjects with CD4-cell counts of >300

We examined the effect of a human immunodeficiency virus (HIV)-specific immune-based therapy in Thailand, where access to antiviral drug therapy is limited. A 40-week trial was conducted with 297 asymptomatic, HIV-infected Thai subjects with CD4-cell counts greater than 300 microl/mm(3). Subjects were randomized to receive either HIV type 1 (HIV-1) immunogen (Remune; inactivated HIV-1 from which gp120 is depleted in incomplete Freund's adjuvant or adjuvant control at 0, 12, 24, and 36 weeks at five different clinical sites in Thailand. Neither group received antiviral drug therapy. The a priori primary endpoint for the trial was changes in CD4-cell counts with secondary parameters of percent changes in CD8-cell counts (percent CD4, CD8, and CD4/CD8) and body weight. Subsets of subjects were also examined for changes in plasma HIV-1 RNA levels, Western blot immunoreactivity, and HIV-1 delayed-type hypersensitivity (DTH) skin test reactivity. There was a significant difference in changes in CD4-cell counts that favored the HIV-1 immunogen-treated group compared to those for the adjuvant-treated control group (P<0.05). On average, for HIV-1 immunogen-treated subjects CD4-cell counts increased by 84 cells by week 40, whereas the increase for the control group was 38 cells by week 40. This increase in CD4-cell count was associated with increased HIV-specific immunogenicity, as shown by Western blotting and enhanced HIV-1 DTH skin reactivity. No significant differences in adverse events were observed between the groups. The results of this trial suggest that HIV-1 immunogen is safe and significantly increases CD4-cell counts and HIV-specific immunity compared to those achieved with the adjuvant control in asymptomatic HIV-1-infected subjects not taking antiviral drugs.     

Epitopes of human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins recognized by antibodies in the sera of HIV-1-infected individuals.

Sera from human immunodeficiency virus (HIV)-infected study subjects and controls were analyzed by enzyme-linked immunosorbent assay using 10 synthetic peptides to identify epitopes of HIV envelope glycoproteins (ENVgp) that were recognized by antibodies. Two epitopes of HIV ENVgp, ENVP466 (amino acids 466-481) and ENVP497 (amino acids 497-509), were recognized by antibodies in the sera of most HIV-infected individuals. The frequency of individuals with detectable serum antibodies to these two epitopes was not associated with the stage of HIV disease. Purified antibodies to ENV497 had only very weak neutralizing activity against infectious HIV. These data suggest that a particular dominant type of antibody response to HIV's ENVgp has minimal protective effects. These and other studies to identify and stimulate immune responses to selected epitopes of HIV antigens may be useful in the design of vaccines to prevent or treat HIV infections.     

Therapeutic immunization with human immunodeficiency virus type 1 (HIV-1) peptide-loaded dendritic cells is safe and induces immunogenicity in HIV-1-infected individuals

Treatments for human immunodeficiency virus type 1 (HIV-1)-positive individuals that augment HIV-1 suppression and have potential for achieving long-term control of HIV-1 viremia in the absence of antiretroviral therapy (ART) are urgently needed. We therefore conducted a phase I, clinical safety trial of a dendritic cell (DC)-based vaccination strategy as immunotherapy for HIV-1-positive individuals on ART. We studied 18 HIV-1-positive subjects on ART who underwent leukapheresis to obtain peripheral blood mononuclear cells for DC generation from monocytes cultured with cytokines. Mature DC were pulsed with three HIV-1 HLA*A0201 Gag, Env, and Pol peptides and one influenza A virus matrix protein peptide. The vaccine was administered to donors randomized to receive two vaccinations, either intravenously or subcutaneously. The primary end points were safety and tolerability of two doses of peptide-DC vaccine (3 million versus 10 million). Secondary end points included gamma interferon (IFN-γ) enzyme-linked immunospot assay responses and clinical correlates of an immune response to vaccination. Autologous DC-peptide vaccine was safe, well tolerated, and feasible for use in all participants. Adverse events were rare. Although the trial was not powered to assess an immunologic response, a significantly increased frequency of HIV-1 peptide-specific IFN-γ-positive cells was observed 2 weeks following the second vaccine, with three individuals responding to all four peptides. DC vaccination was safe, was feasible, and showed promise of immunogenicity in ART-treated, HIV-1-positive individuals. Additional studies of DC immunization strategies for HIV-1 infection are warranted.     

Chemokine receptors as fusion cofactors for human immunodeficiency virus type 1 (HIV-1)

CD4 is the primary cellular receptor for human immunodeficiency virus type 1 (HIV-1), but is not sufficient for entry of HIV-1 into cells. After a decade-long search, the cellular coreceptors that HIV-1 requires in conjunction with CD4 have been identified as members of the chemokine receptor family of seven-transmembrane G-protein coupled receptors. The discovery of distinct chemokine receptors that support entry of T-cell tropic (CXCR-4) and macrophage tropic HIV-1 strains (CCR-5) explains the differences in cell tropism between viral strains, the inability of HIV-1 to infect most nonprimate cells, and the resistance of a small percentage of the population to HIV-1 infection. Further understanding of the role of chemokine receptors in viral entry may also help explain the evolution of more pathogenic forms of the virus, viral transmission, and HIV-induced pathogenesis. These recent discoveries will aid the development of strategies for combating HIV-1 transmission and spread, the understanding of HIV-1 fusion mechanisms, and the possible development of small animal models for HIV-1 drug and vaccine testing.     

Blocking-ELISA for detection of specific antibodies to the glycoproteins of the human immunodeficiency virus type 1 (HIV-1)

A blocking ELISA was developed to confirm the specificity of screening tests for anti-HIV-1 antibodies. A murine monoclonal antibody (McAb) raised against recombinant gp160 was used in combination with a commercial technique (ELA-VIA-1). After determining the optimal experimental conditions, the assay was applied to 92 samples presenting different reactivities by Western blot (WB) analysis. All the sera containing antibodies to gp160/gp120 (53) were positive in our assay. The six patients who sero converted showed a low positivity by ELAVIA-1 (optical density near the cutoff value) reacted by blocking-ELAVIA-1 with an McAb binding inhibition greater than 85%. By contrast, negative samples (29) and specimens that exhibited reactivity only against gag-proteins (10) were not detected (McAb binding inhibition smaller than 15%). This sensitive and specific blocking-ELAVIA-1 represents a convenient alternative to WB as a confirmatory test. The technique is time-saving and inexpensive and can easily be integrated with a screening test for diagnostic or epidemiologic studies on HIV-1 infection.     

Influence of HCV genotype and co-infection with human immunodeficiency virus on CD4(+) and CD8(+) T-cell responses to hepatitis C virus

The role of T-cells in clearance of hepatitis C virus (HCV) during acute infection is critical. The relevance of the immunological response in the control of HCV replication is less clear in chronic HCV infection. HCV-specific T-cell responses were examined in 92 interferon-naive individuals with chronic hepatitis C. A panel of 441 overlapping peptides spanning all expressed HCV proteins was used to measure HCV-specific T-cell responses, using flow cytometry after stimulating peripheral blood mononuclear cells (PBMCs) with different pools of these peptides. Most patients showed responses to at least one HCV protein, with NS5B for CD8(+) responses and E2 for CD4(+) responses identified most frequently. Both the prevalence and breadth of CD4(+) and CD8(+) responses were lower in co-infected patients, independently of the HCV genotype.     

Antigen-specific beta-chemokine production and CD8 T-cell noncytotoxic antiviral activity in HIV-2-infected individuals

Human immunodeficiency virus-2 (HIV-2) is less pathogenic than HIV-1, and the disease progression in HIV-2-infected individuals seems to be similar to that seen in HIV-1-infected long-term nonprogressors. Cell-mediated immune responses and the production of noncytotoxic CD8+ T-cell antiviral factors (CAF) and beta-chemokines have been correlated to protection against HIV-1 and associated with asymptomatic infection and slower disease progression. We investigated the antigen-induced beta-chemokine production in HIV-2-infected patients living in Sweden and in Guinea-Bissau. We also compared in vitro CD8+ T-cell-mediated noncytotoxic antiviral activity against beta-chemokine-sensitive R5 virus (HIV-1Bal) and beta-chemokine-insensitive X4 virus (HIV-1IIIB) in HIV-2-infected patients with that in HIV-1-infected patients. HIV-2-specific beta-chemokine production was demonstrated in a majority of the HIV-2-infected subjects. CD8+ T cells of both HIV-1 and HIV-2-infected individuals suppressed R5 virus replication in vitro in a similar manner, while the inhibition of X4 virus replication seemed to be more frequent and of a higher magnitude among HIV-2-infected patients compared to HIV-1-infected subjects. Taken together, our results indicate that the production of CD8+ T-cell noncytotoxic antiviral factors may contribute to the low transmission of the virus and slower disease progression in HIV-2-infected patients.     

Low T-cell responsiveness to activation via CD3/TCR is a prognostic marker for acquired immunodeficiency syndrome (AIDS) in human immunodeficiency virus-1 (HIV-1)-infected men

In a cohort of 300 HIV-1-infected homosexual men, studied longitudinally, the prognostic value of T-cell proliferative responses for development of AIDS was analyzed. In 15 persons we observed that, at seroconversion, T-cell reactivity to anti-CD3 monoclonal antibody (Mab) and phytohemagglutinin (PHA) was decreased to 20 and 60% of the normal values, respectively. After seroconversion, within 3 months, the response to anti-CD3 Mab and PHA returned to 60 and 80%, respectively, of the normal magnitude and declined thereafter. To investigate whether low T-cell reactivity correlated with progression to AIDS, groups of progressors and nonprogressors were compared. In individuals who progressed to AIDS, already 12 months before diagnosis, responses to anti-CD3 Mab were virtually absent, whereas at that time CD4+ cell numbers and reactivity to horse anti-human lymphocyte serum and PHA were still comparable to values observed for nonprogressors and the asymptomatic population as a whole. In a disease-free survival analysis, low anti-CD3 Mab reactivity predicted progression to AIDS. Our findings demonstrate that low T-cell responsiveness to anti-CD3 Mab is an early marker of progression toward AIDS in HIV-1 infected individuals.     

Studies of spontaneous proliferation of mononuclear cells in human immunodeficiency virus (HIV-1)-infected individuals

We evaluated the ability of mononuclear cell populations from 56 hemophiliac subjects, 69 homosexuals, and 32 control subjects to proliferate spontaneously in vitro. Levels of spontaneous proliferation (SP) were not significantly different among homosexuals or hemophiliac subjects when they were grouped as asymptomatic human immunodeficiency virus (HIV) seronegative, asymptomatic seropositive, persistent generalized lymphadenopathy, acquired immunodeficiency syndrome-related complex, or acquired immunodeficiency syndrome groups. There was, however, a stepwise increase in levels of SP corresponding with progression of disease in both patient groups. No correlations were noted between SP and CD8+ cell populations or mitogenic responses in study subjects, but SP correlated inversely to percentages and numbers of CD4+ lymphocytes. Depletion of CD4+ cells from mononuclear cell populations of HIV-infected subjects decreased SP. Thus, in HIV-1-infected subjects, SP increases as HIV-1-related disease progresses and appears to be CD4 dependent.     

Regulatory T cells and chronic immune activation in human immunodeficiency virus 1 (HIV-1)-infected children

The function of CD4⁺ T cells with regulatory activity (T_regs) is the down-regulation of immune responses. This suppressive activity may limit the magnitude of effector responses, resulting in failure to control human immunodeficiency virus 1 (HIV-1) infection, but may also suppress chronic immune activation, a characteristic feature of HIV-1 disease. We evaluated the correlation between viral load, immune activation and T_regs in HIV-1-infected children. Eighty-nine HIV-1-infected children (aged 6–14 years) were included in the study and analysed for HIV-1 plasmaviraemia, HIV-1 DNA load, CD4 and CD8 cell subsets. T_reg cells [CD4⁺ CD25^highCD127^lowforkhead box P3 (FoxP3^high)] and CD8-activated T cells (CD8⁺CD38⁺) were determined by flow cytometry. Results showed that the number of activated CD8⁺CD38⁺ T cells increased in relation to HIV-1 RNA plasmaviraemia (r = 0·403, P < 0·0001). The proportion of T_regs also correlated positively with HIV-1 plasmaviraemia (r = 0·323, P = 0·002), but correlated inversely with CD4⁺ cells (r = −0·312, P = 0·004), thus suggesting a selective expansion along with increased viraemia and CD4⁺ depletion. Interestingly, a positive correlation was found between the levels of T_regs and CD8⁺CD38⁺ T cells (r = 0·305, P = 0·005), and the percentage of T_regs tended to correlate with HIV-1 DNA load (r = 0·224, P = 0·062). Overall, these findings suggest that immune activation contributes to the expansion of T_reg cells. In turn, the suppressive activity of T_regs may impair effector responses against HIV-1, but appears to be ineffective in limiting immune activation.     

Human immunodeficiency virus type 1 (HIV-1) and human hematopoietic progenitor cells

Summary Besides a progressive depletion of CD4+ T-lymphocytes, other peripheral blood cytopenias, (granulocytopenia, anemia and thrombocytopenia) are frequently observed in HIV-1 seropositive individuals, especially in patients with overt AIDS. Various experimental evidences suggest that HIV-1 could play a direct role in the pathogenesis of HIV-1 related peripheral blood cytopenias, affecting the survival/proliferation capacity of hematopoietic progenitors. CD34+ human hematopoietic progenitors, however, are substantially not susceptible to HIV-1 infection either in vitro and in vivo and their defects seem rather related to an alteration of bone marrow and peripheral blood microenvironments due to the presence of soluble HIV-1 specific products.