Antiviral CD8+ T cells in the genital tract control viral replication and delay progression to AIDS after vaginal SIV challenge in rhesus macaques immunized with virulence attenuated SHIV 89.6

The recently failed clinical efficacy trial of an acquired immunodeficiency syndrome (AIDS) vaccine that elicits antiviral CD8⁺ T‐cell responses has emphasized the challenge of producing an effective vaccine against human immunodeficiency virus (HIV). In the simian immunodeficiency virus (SIV)/ rhesus monkey model of AIDS, live‐attenuated lentivirus ‘vaccines’ provide the best protection from uncontrolled viral replication and clinical disease after pathogenic SIV challenge. This review summarizes a recent series of studies in which we show that after vaginal SIV challenge of rhesus macaques immunized with an attenuated lentivirus protection from uncontrolled viral replication is primarily mediated by CD8⁺ T cells in the vaginal mucosa. Immunization with a chimeric simian/human immunodeficiency virus (SHIV) results in a systemic infection that induces a moderate population of SIV‐specific CD8⁺ and CD4⁺ T cells with cytolytic potential in the vaginal mucosa. Depletion of CD8⁺ T cells at the time of SIV challenge completely abrogates the protection mediated by prior infection with attenuated SHIV. Further after vaginal SIV challenge, the only significant expansion of SIV‐specific T cells occurs in the vagina in these animals. No significant expansion of T‐cell responses was observed in systemic lymphoid tissues. Thus, the presence of SIV‐specific CD8⁺ T cells in the vagina on the day of vaginal SIV challenge and a modest expansion of local effector T cells is sufficient to stop uncontrolled SIV replication. It seems that T‐cell based vaccine strategies that can elicit mucosal effector CD8⁺ T‐cell populations and avoid inducing systemic T‐cell proliferation upon exposure to HIV have the greatest potential for mimicking the success of live‐attenuated lentiviral vaccines.     

Simian immunodeficiency virus-specific T-cell-mediated proliferative response of infected rhesus macaques

While cell-mediated immunity is known to play an important role in controlling viral infections, its role in human and experimental animal models of human AIDS has not been established. To address this issue, four juvenile rhesus macaques were infected with simian immunodeficiency virus SIVMAC. Freshly isolated peripheral blood mononuclear cells from these SIVMAC-infected macaques and four uninfected control macaques were assessed for T-cell proliferative activity to SIV, monthly, for 10 consecutive months. T cells from SIV-infected monkeys failed to proliferate in response to SIV added directly to the culture. However, when SIV was processed by autologous antigen-presenting cells prior to culture with purified T cells, proliferative responses were uniformly demonstrated in SIV-infected monkeys, but not in uninfected controls. Proliferation in response to heat-inactivated SIV was mediated by CD4+ T cells and was shown to be MHC class II-restricted. However, the proliferative response to infectious SIV was mediated by both CD4+ and CD8+ T cells and was MHC class-restricted. As disease progressed, a decline in the T-cell proliferative response was observed.     

Enhanced SIV replication and accelerated progression to AIDS in macaques primed to mount a CD4 T cell response to the SIV envelope protein

Given the dual role of CD4 T cells as both immune effectors and targets for HIV infection, the balance of CD4 versus CD8 T cell-mediated responses induced by candidate AIDS vaccines may be critical in determining postvaccination infection outcomes. An attenuated recombinant varicella-zoster virus vaccine expressing the simian immunodeficiency virus (SIV) envelope (Env) elicited nonneutralizing Env-binding antibodies and little if any cytotoxic T lymphocyte responses in rhesus macaques (Macaca mulatta). After challenge with SIV, Env vaccinees manifested increased levels of SIV replication, more rapid CD4 depletion, and accelerated progression to AIDS compared with controls. Enhanced SIV replication correlated with increased CD4 T cell proliferation soon after SIV challenge, apparently the result of an anamnestic response to SIV antigens. Thus activation of virus-specific CD4 T cells at the time of exposure to a CD4 T cell-tropic lentivirus, in the absence of an effective CD8 response, may enhance virus replication and disease. These data suggest suggest that candidate AIDS vaccines may not simply be either efficacious or neutral; they may also have the potential to be harmful.     

CD8(+) T cells in preventing HIV infection and disease

The complex interplay between the host immune response and HIV has been the subject of intense research over the last 25 years. HIV and simian immunodeficiency virus (SIV) CD8 T cells have been of particular interest since they were demonstrated to be temporally associated with reduction in virus load shortly following transmission. Here, we briefly review the phenotypic and functional properties of HIV-specific and SIV-specific CD8 T-cell subsets during HIV infection and consider the influence of viral variation with specific responses that are associated with disease progression or control. The development of an effective HIV/AIDS vaccine combined with existing successful prevention and treatment strategies is essential for preventing new infections. In the context of previous clinical HIV/AIDS vaccine trials, we consider the challenges faced by therapeutic and vaccine strategies designed to elicit effective HIV-specific CD8 T cells.     

Infection of human monocytes with HIV-1Ba-L. Effect on accessory cell function for T-cell proliferation in vitro

Major laboratory manifestations of human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS) include altered levels of circulating CD4+ lymphocytes and decreased in vitro T-cell mitogenic responses. Since T-cell proliferation is regulated by monocytes (M phi), studies were undertaken to determine whether defective M phi function contributes to these poor mitogenic responses. M phi were isolated from peripheral blood mononuclear cells (PBMC) of normal donors by adherence to plastic. After 5 days in culture, the adherent cells were inoculated with the HIV-1 M phi-tropic strain, Ba-L. Under these conditions HIV infection in M phi can be detected 5-7 days after inoculation. Ten to fourteen days postinoculation, the adherent cells were harvested with lidocaine and cocultured with fresh autologous T cells and T-cell mitogens in a 3-day assay. We found decreased proliferative anti-CD3 responses to Leu4 and OKT3 and variable responses to concanavalin A (Con A) by T cells cultured with HIV-infected monocytes compared with T cells cultured with uninfected M phi. Supernatants from HIV-infected M phi cultures decreased proliferative responses of normal PBMC to anti-CD3 monoclonal antibodies. Heat-activated supernatants had the same effect. Inhibitors of HIV binding did not restore proliferative responses of HIV-infected cultures to normal levels. These results indicate that HIV infection of M phi causes the release of soluble factor(s) that suppress anti-CD3-induced T-cell proliferative responses.     

Spontaneous and antigen-induced production of HIV-inhibitory beta-chemokines are associated with AIDS-free status.

The beta-chemokines RANTES, macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta suppress infection by macrophage-tropic strains of HIV and simian immunodeficiency virus (SIV) by binding and down-regulating the viral coreceptor, CCR5. Accordingly, we have examined whether higher levels of CCR5 ligands are associated with a more favorable clinical status in AIDS. A cross-sectional study of 100 subjects enrolled in the Multicenter AIDS Cohort Study at the Baltimore site was conducted to measure chemokine production and lymphocyte proliferation by peripheral blood mononuclear cells (PBMC). Statistical analyses of the data revealed that the production of HIV-suppressive beta-chemokines by HIV antigen-stimulated PBMC was significantly higher in HIV-positive subjects without AIDS compared with subjects with clinical AIDS. Increased chemokine production was also correlated with higher proliferative responses to HIV antigens. Both parameters were significantly lower in the AIDS versus non-AIDS group. Notably, significantly higher levels of MIP-1alpha were also observed with unstimulated PBMC from seronegative subjects at risk for HIV infection released as compared with seropositive and non-Multicenter AIDS Cohort Study seronegative subjects. The association of chemokine production with antigen-induced proliferative responses, more favorable clinical status in HIV infection, as well as with an uninfected status in subjects at risk for infection suggests a positive role for these molecules in controlling the natural course of HIV infection.     

Human herpesvirus 6A accelerates AIDS progression in macaques

Although HIV is the necessary and sufficient causative agent of AIDS, genetic and environmental factors markedly influence the pace of disease progression. Clinical and experimental evidence suggests that human herpesvirus 6A (HHV-6A), a cytopathic T-lymphotropic DNA virus, fosters the progression to AIDS in synergy with HIV-1. In this study, we investigated the effect of coinfection with HHV-6A on the progression of simian immunodeficiency virus (SIV) disease in pig-tailed macaques (Macaca nemestrina). Inoculation of HHV-6A resulted in a rapid appearance of plasma viremia associated with transient clinical manifestations and followed by antibody seroconversion, indicating that this primate species is susceptible to HHV-6A infection. Whereas animals infected with HHV-6A alone did not show any long-term clinical and immunological sequelae, a progressive loss of CD4(+) T cells was observed in all of the macaques inoculated with SIV. However, progression to full-blown AIDS was dramatically accelerated by coinfection with HHV-6A. Rapid disease development in dually infected animals was heralded by an early depletion of both CD4(+) and CD8(+) T cells. These results provide in vivo evidence that HHV-6A may act as a promoting factor in AIDS progression.     

Immunity in natural SIV infections

In stark contrast to human immunodeficiency virus (HIV)‐infected individuals who, if left untreated, almost invariably progress to acquired immunodeficiency syndrome (AIDS), natural hosts for the simian immunodeficiency viruses (SIV) remain asymptomatic throughout the course of infection. This observation represents one of the main unresolved puzzles of AIDS research, particularly if one considers that natural SIV infections are characterized by chronically high levels of viraemia as well as intrinsic virus cytopathicity comparable with that of HIV. In this review, I discuss the basic immunological features of natural, nonpathogenic SIV infections, the evidence suggesting that attenuated, rather than extraordinarily strong, immune responses to the virus may favour their benign course, and the implications of these findings in terms of HIV therapy and vaccines.     

Human herpesvirus 8 cellular immune responses in homosexual men.

Little is known about cellular immunity to human herpesvirus 8 (HHV-8), the virus associated with Kaposi's sarcoma (KS). T cell proliferative responses to purified HHV-8 were measured in homosexual men, a group with elevated HHV-8 seroprevalence and high risk of KS. None of 20 blood donor controls had T cell responses to HHV-8. Among human immunodeficiency virus (HIV)-negative homosexual men, 8 (42%) of 19 HHV-8 seropositive men responded as did 4 (16%) of 25 HHV-8 seronegative men. Among HIV-positive homosexual men, however, none of 21 HHV-8 seropositives had T cell responses to HHV-8, even though most responded to common recall antigens, and 10 had >/=400 CD4 cells/mm3. The results suggest that HHV-8 T cell proliferative responses are common in HIV-negative homosexual men and that HIV infection may be associated with diminished HHV-8 cellular immunity, possibly before there is substantial depletion of CD4 cells. If correct, this could explain why KS occurs relatively early in HIV infection/AIDS.     

Developing an HIV cytotoxic T‐lymphocyte vaccine: issues of CD8 T‐cell quantity,quality and location

Issues of quantity, quality and location impact the ability of CD8 T cells to mediate protection from infection. These issues are considered in light of human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) vaccination. Methods are reviewed that result in 100‐ to 1000‐fold higher frequencies of vaccine‐specific memory CD8 T cells than that achieved by current HIV/SIV vaccine approaches. Data demonstrating that location within mucosal tissues has a direct impact on memory CD8 T‐cell function are discussed. Arguments are made that establishing memory CD8 T cells within mucosal sites of transmission, a priori to natural infection, may be essential for conferring optimal and rapid protection. Lastly, it is proposed that heterologous prime‐boost vaccination with recombinant live replicating vectors, which has the potential to induce tremendous numbers of cytolytic memory CD8 T cells within mucosal tissues, would provide a far more stringent test of the hypothesis that memory CD8 T cells could, in principal, form the basis for a preventative HIV vaccine.     

Malignant lymphoma associated with human AIDS and with SIV-induced immunodeficiency in macaques.

Malignant lymphomas associated with human (HIV) and simian (SIV) immunodeficiency virus infections are reviewed and compared. Recent observation of a high frequency of lymphomas in a series of cynomolgus macaques, highly immunodeficient after infection with SIVsm(smm3) are described. In addition to the increased frequency in human and monkey AIDS, SIV and HIV lymphomas share several important features. Clinically and by histology they present as aggressive high-grade malignant tumors with a predilection for extranodal growth in viscera, skin, central nervous system, testis, and retroorbitally. Most malignant lymphomas are of B-cell origin. AIDS lymphomas in humans are heterogeneous with regard to Epstein-Barr virus (EBV) association. Similarly, most lymphomas in monkeys experimentally infected with SIV tested to date were shown to be associated with an EBV-like simian herpes virus. These observations point to the possibility of using SIV-immunodeficient macaques for study of EBV and other oncogenic and immunosuppressive factors in AIDS-associated lymphomagenesis.     

Modulation of intracellular restriction factors contributes to methamphetamine- mediated enhancement of acquired immune deficiency syndrome virus infection of macrophages

Epidemiological studies have demonstrated that the use of methamphetamine (METH), a sympathomimetic stimulant, is particularly common among patients infected with HIV. In vitro studies have determined that METH enhances HIV infection of CD4+ T cells, monocyte-derived dendritic cells, and macrophages. In addition, animal studies have also showed that METH treatment increases brain viral load of SIV-infected monkeys and promotes HIV replication and viremia in HIV/hu-CycT1 transgenic mice. However, the mechanisms (s) of METH actions on HIV remain to be determined. In this study, we investigated the impact of METH on intracellular restriction factors against HIV and SIV. We demonstrated that METH treatment of human blood mononuclear phagocytes significantly affected the expression of anti-HIV microRNAs and several key elements (RIG-I, IRF-3/5, SOCS-2, 3 and PIAS-1, 3, X, Y) in the type I IFN pathway. The suppression of these innate restriction factors was associated with a reduced production of type I IFNs and the enhancement of HIV or SIV infection of macrophages. These findings indicate that METH use impairs intracellular innate antiviral mechanism(s) in macrophages, contributing to cell susceptibility to the acquired immune deficiency syndrome (AIDS) virus infection.     

Massive infection and loss of CD4+ T cells occurs in the intestinal tract of neonatal rhesus macaques in acute SIV infection

Rapid, profound, and selective depletion of memory CD4+ T cells has now been confirmed to occur in simian immunodeficiency virus (SIV)-infected adult macaques and human immunodeficiency virus (HIV)-infected humans. Within days of infection, marked depletion of memory CD4+ T cells occurs primarily in mucosal tissues, the major reservoir for memory CD4+ T cells in adults. However, HIV infection in neonates often results in higher viral loads and rapid disease progression, despite the paucity of memory CD4+ T cells in the peripheral blood. Here, we examined the immunophenotype of CD4+ T cells in normal and SIV-infected neonatal macaques to determine the distribution of naive and memory T-cell subsets in tissues. We demonstrate that, similar to adults, neonates have abundant memory CD4+ T cells in the intestinal tract and spleen and that these are selectively infected and depleted in primary SIV infection. Within 12 days of SIV infection, activated (CD69+), central memory (CD95+CD28+) CD4+ T cells are marked and persistently depleted in the intestine and other tissues of neonates compared with controls. The results in dicate that "activated" central memory CD4+ T cells are the major target for early SIV infection and CD4+ T cell depletion in neonatal macaques.     

HGP-30, a synthetic analogue of human immunodeficiency virus (HIV) p17, is a target for cytotoxic lymphocytes in HIV-infected individuals.

Evaluation of the immune response of individuals exposed to human immunodeficiency virus (HIV) is an important component of any plan designed to lead toward the development of an AIDS vaccine. Since the levels of antibodies to HIV p17 and the synthetic p17 peptide HGP-30 correlate with stages of progression to AIDS, studies were initiated to determine whether cytotoxic lymphocytes directed toward target cells pulsed with HGP-30 and radioactive chromium were present in seropositive individuals. The significance of such cells in controlling HIV viral infection has recently been enhanced by reports that HIV p17 is on the surface of infected cells and that an inactivated virus vaccine depleted of viral envelope appears to be effective in controlling expression. The selection of HGP-30 as the p17 peptide to be evaluated in early studies is based on the presence of both T-cell and B-cell epitopes as predicted by computer modeling and mouse studies and the demonstration of in vitro neutralization activity by antibodies to the epitope. By using B-lymphoblastoid cells pulsed with HGP-30 and radioactive chromium as autologous targets and mixed leukocyte culture-expanded peripheral blood lymphocytes as effectors, CD8+ cytotoxic T lymphocytes against HGP-30-coated targets were identified in seropositive individuals. In this report we demonstrate that a synthetic p17 epitope can be a target for major histocompatibility complex-restricted cytotoxic T lymphocytes in HIV-infected individuals.     

Isolation of human immune deficiency virus from African AIDS patients and from persons without AIDS or IgG antibody to human immune deficiency virus

We previously reported a high incidence of acquired immune deficiency syndrome (AIDS) in Kinshasa, Zaire, as well as a high frequency of antibody to human immunodeficiency virus (HIV), which includes HTLV-III and LAV viruses, in persons without AIDS. In this report we assessed the frequency of HIV virus infection in persons with and without clinical AIDS and the association of virus isolation to presence of antibody. We isolated HIV from 27 (77%) of 35 patients with AIDS, and 5 of 9 patients with AIDS-related complex (ARC). Virus was also isolated from plasma and cerebrospinal fluid of patients in the study. The presence of antibody was a reliable marker for virus infection in African patients with AIDS. HIV was isolated from 5 of 27 control patients without AIDS, 3 of whom had normal T helper to T suppressor ratios and normal numbers of T helper cells. Two of these patients had no detectable antibody to HIV by ELISA or Western blot methods. In a population, such as the general heterosexual population of Kinshasa, with frequent infection by HIV and with few clearly definable risk groups, screening for antibodies to HIV may not be sufficient to identify some virus infected persons.     

Mononuclear phagocytes of blood and bone marrow: comparative roles as viral reservoirs in human immunodeficiency virus type 1 infections.

We examined human immunodeficiency virus (HIV) production in cultured mononuclear cells from 36 seropositive homosexual males. Production was detected by using an HIV p24 antigen ELISA assay in blood mononuclear cells in 54% of asymptomatic, 71% of acquired immunodeficiency syndrome (AIDS)-related complex, and 100% of AIDS patients. When the peripheral blood mononuclear cells were separated into monocytes and CD4+ T cells, we found that the CD4+ T-cell fraction was preferentially infected in the three clinical stages. The ability to isolate HIV from blood monocyte-derived macrophages was similar in the three stages (24-33%) and required coculture with phytohemagglutinin-stimulated lymphoblasts. Bone marrow and blood mononuclear cells cultured simultaneously yielded virus from both sources in 13 individuals. Again the prime source of virus was the nonadherent bone marrow mononuclear cells, which contained CD4+ T cells, and not the adherent monocyte-enriched fraction. We conclude that blood mononuclear cells yield productive virus more readily as disease progresses and that infection is detected in association with CD4+ T-cell-enriched fractions. In our large sample of patients, monocyte infection was detected in only a small fraction, suggesting that this cell type is neither a primary nor an exclusive reservoir of HIV infection.     

A dendritic cell-based vaccine for treating HIV infection: background and preliminary results

Antibody response against human immunodeficiency virus-1 (HIV) is ineffective and cellular immune response is not strong enough to achieve the complete suppression or at least a strong control of viral replication in HIV- infected patients. In 2001, we showed in vitro that dendritic cells (DCs) of HIV-infected patients loaded with autologous HIV chemically inactivated by aldrithiol-2 were capable of raising an HIV-specific cellular immune response powerful enough to allow the destruction of autologous HIV- infected CD4 T cells. In 2003, we showed that simian immunodeficiency virus (SIV)-infected macaques vaccinated with inactivated SIV-loaded autologous DCs raised a strong SIV-specific cellular response. Ten months after vaccination, plasma viral load of 7 out of the 10 vaccinated monkeys remained 1000-fold lower than initially. In December 2004, we published results observed in 18 untreated HIV-infected patients vaccinated with autologous monocyte-derived DCs loaded with autologous inactivated HIV. A year following vaccination, 8 patients had a plasma viral load decrease >90%; among them, 4 had viral load <1000 copies mL(-1). Moreover, by one year, the viral load decline of the 18 patients was significantly correlated with their percentage of HIV-1-gag-specific CD8(+) T cells expressing perforin and that of HIV-1-specific CD4(+) T(H)1 cells. This is the first demonstration of the capacity of a therapeutic vaccine to induce an effective HIV-specific T cell response associated with sustained viral suppression in untreated viremic patients. The manipulation of antigen presenting cells to elicit virus-specific cellular responses is a promising tool to control persistant viral infections.     

Immunization with live attenuated simian immunodeficiency virus induces strong type 1 T helper responses and beta-chemokine production

Immunization with live attenuated simian immunodeficiency virus (SIV) strains has proved to be one of the most effective strategies to induce protective immunity in the SIV/macaque model. To better understand the role that CD4(+) T helper responses may play in mediating protection in this model, we characterized SIV-specific proliferative and cytokine responses in macaques immunized with live attenuated SIV strains. Macaques chronically infected with live attenuated SIV had strong proliferative responses to SIV proteins, with stimulation indices of up to 74. The magnitude of the proliferative response to SIV Gag varied inversely with the degree of attenuation; Gag-specific but not envelope-specific responses were lower in animals infected with more highly attenuated SIV strains. SIV-specific stimulation of lymphocytes from vaccinated macaques resulted in secretion of interferon-gamma, IL-2, regulated-upon-activation, normal T cells expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta but not IL-4 or IL-10. Intracellular flow cytometric analysis documented that, in macaques vaccinated with SIVmac239Deltanef, up to 2% of all CD4(+)T cells were specific for SIV p55. The ability of live attenuated SIV to induce a strong, sustained type 1 T helper response may play a role in the success of this vaccination approach to generate protection against challenge with wild-type SIV.     

Human and simian immunodeficiency retroviruses: activation and differential transactivation of gene expression

New West African human immunodeficiency viruses (HIV-2s) and simian immunodeficiency virus (SIV) contain functional transactivator (tat) gene and tat response elements. Their long terminal repeats (LTR) and tat genes are more related among themselves than to HIV-1 LTR and tat gene. The viral gene expression of HIV-2 as well as SIV can be stimulated by T cell activators, such as mitogens and phorbol esters. HIV-2 and SIV display a much broader transactivation response specificity than does HIV-1. The LTR-directed gene expression of HIV-2/SIV is not only transactivated by their own tat gene and by HIV-1 tat gene but also by factors in human T cell leukemia/lymphoma virus type I (HTLV-I) and simian virus 40 (SV40) infected cells, involving HTLV-I tat gene and SV40 T antigens, respectively. HIV-1 LTR-directed gene expression is much less transactivated by HIV-2/SIV tat genes and by factors in HTLV-I- and SV40-infected cells. Immune activation and heterologous transactivation of the LTR-directed gene expression may be relevant to the latency of virus infection and progression toward the acquired immunodeficiency syndrome (AIDS).