Immunization of macaques with live simian human immunodeficiency virus (SHIV) vaccines conferred protection against AIDS induced by homologous and heterologous SHIVs and simian immunodeficiency virus

To evaluate the vaccine potential of SHIVs attenuated by deletion of viral accessory genes, seven rhesus macaques were sequentially immunized with Delta vpu Delta nefSHIV-4 (vaccine-I) followed by Delta vpuSHIV(PPC) (vaccine-II). Despite the absence of virological evidence of productive infection with the vaccine strains, based on analysis of infectivity among peripheral blood mononuclear cells (PBMC) of the vaccinated animals, all seven animals developed binding as well as neutralizing antibodies against both vaccine-I and -II. The animals also developed vaccine virus-specific CTLs that recognized homologous as well as heterologous pathogenic SHIVs and SIV, and also soluble inhibitory factors that blocked the in vitro replication of the vaccine strains and different challenge viruses. Virus-specific cellular and humoral responses were sustained throughout a 58-week prechallenge period. To model aspects of natural transmission, the animals received a mucosal (rectal) challenge, with a mixture of three challenge viruses, SHIV(KU), SHIV(89.6)P, and SIV(mac)R71/17E. Two mock-vaccinated control animals inoculated with the same mixture of challenge viruses developed large numbers of infectious PBMC, high plasma viremia, and precipitous loss of CD4(+) T cells. The control animals did not develop any immune responses and succumbed to AIDS between 6 and 7 weeks postchallenge. All seven vaccinated animals became infected with challenge viruses as indicated by the presence of infectious cells in the PBMC and/or viral RNA in plasma. However, peak plasma viremia in vaccinates was two to nearly five logs lower than in the control animals and later plasma viral RNA became undetectable in all vaccinates. Vaccinated animals maintained normal CD4(+) T cell levels throughout the study. Challenge with pathogenic viruses caused massive anamnestic responses as determined by quantitation of virus-specific CD4(+) and CD8(+) T cells by intracellular IFN-gamma staining, and these cells persisted for at least 74 weeks. The study is still in progress and at this time DNA of SIV has become undetectable in lymph nodes of six of the seven vaccinates, SHIV(89.6)P in five of the seven, and SHIV(KU) in three of the seven animals.     

Development of virus-specific immune responses in SHIV(KU)-infected macaques treated with PMPA

Therapeutic intervention with highly active antiretroviral therapy (HAART) can lead to the suppression of HIV viremia below the threshold of detection for several years. However, impact of HAART on reconstitution of virus-specific immune responses remains poorly understood. In this study, four macaques were infected with pathogenic SHIV(KU). One week postinoculation two of the four animals were treated with PMPA [9-R-(2-phosphophomethoxypropyl)adenine] daily for 83 days. Two other macaques, that did not receive treatment, exhibited explosive virus replication accompanied by a near total loss of CD4(+) T cells and succumbed to AIDS-related complications within 6 months of infection. These animals did not develop any virus-specific immune responses. On the contrary, the animals that received PMPA showed transient loss of CD4(+) T cells that recovered during the treatment period. The virus burden declined below the level of detection that rebounded soon after cessation of PMPA therapy. The virus replicated productively for several weeks before both animals controlled the productive replication of virus. This control of virus replication was found to be associated with the development of virus-specific neutralizing antibodies, T-helper cells, and CTLs. Although PMPA did not eliminate virus from the animals, it provided them with enough time to mount virus-specific immune responses that eventually controlled the virus replication in the blood. Our results suggest that antiretroviral therapy, if initiated early during infection, would help the host in mounting virus-specific immune responses that might control productive replication of the virus.     

Virus-specific T cell responses in macaques acutely infected with SHIV(sf162p3)

CD4(+) T helper and CD8(+) cytotoxic T lymphocyte responses are believed to play an important role in the control of primary HIV and SIV infection. However, the role of these cells in macaques acutely infected with SHIV(sf162p3) has not been well characterized. In this study, ten adult rhesus macaques were intravaginally infected with SHIV(sf162p3), and antigen-specific cytokine responses to SHIV-Tat, Nef, Gag and Env peptide pools were examined through 70 days post inoculation (p.i.) using ELISPOT and/or cytokine flow cytometry (CFC). Peak plasma viral replication occurred between 14 and 21 days p.i. followed by low to undetectable plasma viremia by 70 days of infection in most macaques. Although some animals had strong virus-specific cellular immune responses, many had weak or minimal responses that did not correlate with the post peak decline in plasma viremia.     

Viral burden and disease progression in rhesus monkeys infected with chimeric simian-human immunodeficiency viruses

To determine the role of viral burden in simian-human immunodeficiency virus (SHIV)-induced disease, cellular provirus and plasma viral RNA levels were measured after inoculation of rhesus monkeys with four different SHIVs. These SHIVs included SHIV-HXBc2 and SHIV-89.6, constructed with env, tat, rev, and vpu derived from either cell line-passaged or primary patient isolates of human immunodeficiency virus type 1; the viral quasispecies SHIV-89.6P derived after in vivo passage of SHIV-89.6; and a molecular clone, SHIV-KB9, derived from SHIV-89.6P. SHIV-HXBc2 and SHIV-89.6 are nonpathogenic in rhesus monkeys; SHIV-89.6P and SHIV-KB9 cause rapid CD4(+) T cell depletion and an immunodeficiency syndrome. Relative SHIV provirus levels were highest during primary infection in monkeys infected with SHIV-89.6P, the virus that caused the most rapid and dramatic CD4(+) T cell depletion. However, by 10 weeks postinoculation, provirus levels were similar in monkeys infected with the pathogenic and nonpathogenic chimeric viruses. The virus infections that resulted in the highest peak and chronic viral RNA levels were the pathogenic viruses SHIV-89.6P and SHIV-KB9. SHIV-89. 6P uniformly caused rapid and profound CD4(+) T cell depletion and immunodeficiency. Infection with the SHIV-KB9 resulted in very low CD4(+) T cell counts without seroconversion in some monkeys and a substantial but less profound CD4(+) T cell depletion and rapid seroconversion in others. Surprisingly, the level of plasma viremia did not differ between SHIV-KB9-infected animals exhibiting these contrasting outcomes, suggesting that host factors may play an important role in AIDS virus pathogenesis.     

Influence of peak viral load on the extent of CD4+ T-cell depletion in simian HIV infection

Simian HIV (SHIV) infection of macaques with CXCR4 tropic viruses results in early and profound CD4 T-cell depletion in the first few weeks of infection. Analyzing data from a large study of vaccination and SHIV-89.6P challenge, we observe a strong correlation between peak viral load and the extent of CD4 T-cell depletion in acute infection, consistent with a simple kinetic model of viral infection of CD4 T cells. We have modeled the dynamics of the interaction of virus and CD4 T cells over time to investigate the rate of CD4 T-cell infection and death. This analysis indicates that up to 80% of CD4 T cells are infected at peak viremia and that the proportion of CD4 T cells destroyed is correlated with the peak viral load. The simple relation between viral load and CD4 T-cell depletion allows prediction of the level of viral control required to prevent CD4 T-cell depletion in acute SHIV infection. Whether such a simple relation also holds for HIV or simian immunodeficiency virus infections remains to be determined, particularly in the gut and other anatomic sites in which most early T-cell depletion occurs.     

Coinfection with Schistosoma mansoni reactivates viremia in rhesus macaques with chronic simian-human immunodeficiency virus clade C infection

We tested the hypothesis that helminth parasite coinfection would intensify viremia and accelerate disease progression in monkeys chronically infected with an R5 simian-human immunodeficiency virus (SHIV) encoding a human immunodeficiency virus type 1 (HIV-1) clade C envelope. Fifteen rhesus monkeys with stable SHIV-1157ip infection were enrolled into a prospective, randomized trial. These seropositive animals had undetectable viral RNA and no signs of immunodeficiency. Seven animals served as virus-only controls; eight animals were exposed to Schistosoma mansoni cercariae. From week 5 after parasite exposure onward, coinfected animals shed eggs in their feces, developed eosinophilia, and had significantly higher mRNA expression of the T-helper type 2 cytokine interleukin-4 (P = 0.001) than animals without schistosomiasis. Compared to virus-only controls, viral replication was significantly increased in coinfected monkeys (P = 0.012), and the percentage of their CD4(+) CD29(+) memory cells decreased over time (P = 0.05). Thus, S. mansoni coinfection significantly increased viral replication and induced T-cell subset alterations in monkeys with chronic SHIV clade C infection.     

Influence of MHC class I and II haplotypes on the experimental infection of Mauritian cynomolgus macaques with SHIVSF162P4cy

Mauritian cynomolgus macaques (MCM) are widely used in human immunodeficiency virus research because of their restricted major histocompatibility complex (MHC) diversity which provides the opportunity to address the influence of host factors on vaccine studies. We herein report the impact of MHC haplotype on the outcome of 21 MCM infections with the CCR5-tropic simian/human immunodeficiency virus (SHIV)(SF162P4cy). MCM were susceptible to SHIV(SF162P4cy) infection as shown by viremia and loss of CD4+ T cells. A significant association between haplotype M7 (class IA, IB, II) and persistent viremia was observed in chronic phase, whereas recombinant class IA haplotype was associated with a reduction of viral RNA during acute infection. Class IB M4 haplotype displayed significantly lower acute phase provirus copy numbers. In addition, statistical analysis indicated a detrimental effect of haplotype M4 (class IA, IB) on the course of infection as indicated by lower CD4+ T-cell levels during chronic infection. A decrease in post-acute phase CD4+ T-cell numbers was also observed in haplotype M2 animals. This is the first report that documents the effects of host MHC class I and II molecules on the SHIV(SF162P4cy) infection in MCM, particularly with regard to the association between recombinant class IA, M4, and M7 haplotypes and the dynamic of viral replication and level of CD4+ T cells.     

Selective induction of cell-mediated immunity and protection of rhesus macaques from chronic SHIV(KU2) infection by prophylactic vaccination with a conserved HIV-1 envelope peptide-cocktail

Infection of Indian-origin rhesus macaques by the simian human immunodeficiency virus (SHIV) is considered to be a suitable preclinical model for directly testing efficacy of vaccine candidates based on the HIV-1 envelope. We used this model for prophylactic vaccination with a peptide-cocktail comprised of highly conserved HIV-1 envelope sequences immunogenic/antigenic in macaques and humans. Separate groups of macaques were immunized with the peptide-cocktail by intravenous and subcutaneous routes using autologous dendritic cells (DC) and Freund's adjuvant, respectively. The vaccine elicited antigen specific IFN-gamma-producing cells and T-cell proliferation, but not HIV-neutralizing antibodies. The vaccinated animals also exhibited efficient cross-clade cytolytic activity against target cells expressing envelope proteins corresponding to HIV-1 strains representative of multiple clades that increased after intravenous challenge with pathogenic SHIV(KU2). Virus-neutralizing antibodies were either undetectable or present only transiently at low levels in the control as well as vaccinated monkeys after infection. Significant control of plasma viremia leading to undetectable levels was achieved in majority of vaccinated monkeys compared to mock-vaccinated controls. Monkeys vaccinated with the peptide-cocktail using autologous DC, compared to Freund's adjuvant, and the mock-vaccinated animals, showed significantly higher IFN-gamma production, higher levels of vaccine-specific IFN-gamma producing CD4(+) cells and significant control of plasma viremia. These results support DC-based vaccine delivery and the utility of the conserved HIV-1 envelope peptide-cocktail, capable of priming strong cell-mediated immunity, for potential inclusion in HIV vaccination strategies.     

Virus-specific T-cell responses associated with hepatitis C virus (HCV) persistence in the liver after apparent recovery from HCV infection

Hepatitis C virus (HCV) RNA persistence in the liver has been described even after apparent resolution of HCV infection. Because T-cell reactivity plays a role in recovery from HCV infection, virus-specific T-cell responses were investigated in apparently recovered individuals in whom hepatic HCV RNA persistence was documented: 15 sustained virological responders to interferon (IFN)-treatment and 9 asymptomatic aviremic anti-HCV carriers. HCV-specific CD4(+) T-cell proliferative responses were detected significantly more often in apparently recovered individuals (sustained virological responders: 60%; asymptomatic anti-HCV carriers: 66%) compared with 50 chronic hepatitis C patients (28%; P < 0.05). However, T-cell frequencies and numbers tended to decline over time and the number of HCV proteins targeted by CD4(+) T-cell proliferative responses was limited. Interestingly, liver viral load correlated inversely with virus-specific immune responses. Thus, CD4(+) T-cell responders showed significantly lower hepatic HCV RNA levels (P < 0.05). HCV-specific IFN-gamma-secreting CD4(+) T-cells were not detected in all the apparently recovered patients although they were found significantly more often compared with chronic hepatitis C patients (P < 0.05). Also, HCV NS3-specific CD8(+) T-cells were detected in 11 HLA-A2-positive apparently recovered individuals (8 sustained virological responders and 3 asymptomatic anti-HCV carriers); T-cell frequencies tended to be greater in those patients who had lower hepatic viral levels. In conclusion, HCV-specific T-cells are detectable in apparently recovered individuals in whom HCV RNA can persist in the liver indicating that HCV replication may be prolonged in the face of an insufficient or inadequate virus-specific CD4(+) and CD8(+) T-cell response.     

Vaccine-elicited immune responses prevent clinical AIDS in SHIV(89.6P)-infected rhesus monkeys

Accumulating evidence has demonstrated the importance of cytotoxic T lymphocytes (CTLs) and helper T lymphocytes in controlling HIV-1 replication. We have elicited immune responses in rhesus monkeys utilizing DNA vaccines augmented by the administration of IL-2/Ig, a fusion protein consisting of interleukin-2 and the Fc portion of IgG2. These vaccine-elicited immune responses did not prevent infection following a high-dose intravenous challenge with SHIV(89.6P) but did control viremia to nearly undetectable levels and prevented immunodeficiency and clinical disease. In contrast, control monkeys developed high levels of viremia and exhibited a rapid loss of CD4(+) T cells, significant clinical disease progression, and death in half of the animals by day 140 following challenge. Vaccine approaches that elicit immune responses capable of reducing plasma viral loads, but not capable of inducing sterilizing immunity, may still provide substantial clinical benefits.     

The impact of early immune destruction on the kinetics of postacute viral replication in rhesus monkey infected with the simian-human immunodeficiency virus 89.6P

Set-point viral load is positively correlated with the extent of initial viral replication in pathogenic simian-human immunodeficiency virus (SHIV) infection. To elucidate the mechanisms underlying the correlation, we conducted a systematic investigation in rhesus monkeys infected with the highly pathogenic SHIV 89.6P. This model is widely used in the preclinical evaluation of AIDS vaccine candidates and a thorough understanding of the model's biology is important to the proper interpretation of these evaluations. We found that the levels of peak viremia were positively correlated not only with the levels of set-point viremia but, importantly, with the extent of initial overall immune destruction as indicated by the degree of CD4+ T cell depletion and lymph node germinal center (GC) formation. The extent of initial overall immune destruction was inversely correlated with subsequent development and maintenance of virus-specific cellular and humoral immune responses. Thus, these data suggest that the extent of early immune damage determines the development and durability of virus-specific immunity, thereby playing a critical role in establishing the levels of set-point viral replication in SHIV infection. Vaccines that limit both the initial viral replication and the extent of early immune damage will therefore mediate long-term virus replication control and mitigation of long-term immune destruction in this model of immunodeficiency virus infection.     

Sequential immunization of macaques with two differentially attenuated vaccines induced long-term virus-specific immune responses and conferred protection against AIDS caused by heterologous simian human immunodeficiency Virus (SHIV(89.6)P)

Four rhesus macaques were sequentially immunized with live vaccines DeltavpuDeltanefSHIV-4 (vaccine-I) and Deltavpu SHIV(PPC) (vaccine-II). The vaccine viruses did not replicate productively in the peripheral blood mononuclear cells (PBMCs) of the vaccinated animals. All four animals developed binding antibodies against both the vaccine-I and -II envelope glycoproteins but neutralizing antibodies only against vaccine-I. They developed vaccine virus-specific CTLs that also recognized homologous as well as heterologous pathogenic SHIVs. Thirty weeks after the last immunization, the vaccinated animals and three unvaccinated control animals were challenged iv with a highly virulent heterologous SHIV(89.6)P. As expected, the three unvaccinated control animals developed large numbers of infectious PBMCs, high plasma viremia, and precipitous loss of CD4(+) T cells. Two controls did not develop any immune response and succumbed to AIDS in about 6 months. The third control animal developed neutralizing antibodies and had a more chronic disease course, but eventually succumbed to AIDS-related complications 81 weeks after inoculation. The four vaccinated animals became infected with challenge virus as indicated by the presence of challenge virus-specific DNA in the PBMCs and RNA in plasma. However, virus in these animals replicated approximately 200- to 60,000-fold less efficiently than in control animals and eventually, plasma viral RNA became undetectable in three of the four vaccinates. The animals maintained normal CD4(+) T-cell levels throughout the observation period of 85 weeks after a transient drop at Week 3 postchallenge. They also maintained CTL responses throughout the observation period. These studies thus showed that the graded immunization schedule resulted in a safe and highly effective long-lasting immune response that was associated with protection against AIDS by highly pathogenic heterologous SHIV(89.6)P.     

CD4+ lymphocytopenia in acute infection of Asian macaques by a vaginally transmissible subtype-C,CCR5-tropic Simian/Human Immunodeficiency Virus (SHIV)

An R5-tropic SHIV(CHN19P4) was previously generated using a primary HIV-1 subtype-C envelope. We have further characterized this SHIV in two species of macaques. To determine whether this isolate is transmissible vaginally, female pig-tailed macaques were inoculated with 2 x 10(3) TCID50 of SHIV(CHN19P4) by the vaginal route. Animals became infected with a high peak plasma viremia (>10(7) viral copies/mL) and rapid seroconversion. The viremia was accompanied by CD4+ lymphocytopenia in the gut lamina propria lymphocyte (LPL) population. Comparable CD4+ T-cell loss was not seen in peripheral blood and colonic lymph nodes. These findings demonstrate a unique R5-tropic SHIV that can be used to study envelope-related issues in vaginal transmission of the most prevalent subtype of HIV-1. We also found that rhesus macaques intravenously inoculated with 1 x 10(3) TCID50 of SHIV(CHN19P4) became infected and showed CD4+ lymphocytopenia in the gut LPL population. Despite inactivation of the vpu gene in SHIV(CHN19P4), the virus appears to target mainly gut-associated lymphoid tissues during the initial stage of infection as has been described for SHIV(SF162P), another R5-tropic (subtype B) recombinant virus. Our data indicate that the R5-mediated CD4+ lymphocytopenia in the gut is likely independent of HIV-1 genotypes and of the function of vpu at the acute phase of viral infection.     

Higher levels of IL-18 circulate during primary infection of monkeys with a pathogenic SHIV than with a nonpathogenic SHIV

We have monitored kinetics of peripheral blood Interleukin (IL)-18 level, viral RNA load, and CD4(+) T cell counts in cynomolgus and rhesus macaques following infections of various simian/human immunodeficiency viruses (SHIVs) causing differential pathogenicity. Infections of cynomolgus and rhesus macaques with pathogenic SHIVs-C2/1 and -89.6PD, respectively, induced high levels of plasma IL-18 (0.1-1 ng/ml) and enhanced apoptosis of peripheral blood T cells during primary viremia, along with a rapid decline of CD4(+) T cells and a high level of set point viral load after primary viremia (six of six cases). In contrast, infections of cynomolgus macaques with nonpathogenic SHIVs-TH09V3 and -MD14 did not cause such IL-18 elevation, showing no decline of CD4(+) T cells and no or low viral set point level following primary viremia (three of three cases). Thus, the elevation of circulating IL-18 level during primary viral infection can be a good indicator of an active pathogenic viral infection. However, the role of increased IL-18 remains to be elucidated and needs further investigation.     

Immunodominance of HIV-1-specific CD8(+) T-cell responses in acute HIV-1 infection: at the crossroads of viral and host genetics

The development of HIV-1-specific CD8(+) T-cell responses during acute HIV-1 infection is associated with a dramatic decline in HIV-1 replication and the resolution of the acute retroviral syndrome. These HIV-1-specific CD8(+) T cells typically target a small number of viral epitopes in a distinct hierarchical order, and high-level viremia in chronic progressive infection leads to broadly diversified HIV-1-specific CD8(+) T-cell responses with a less clear immunodominance pattern. It is argued here that the specific hierarchical pattern of immune responses in acute HIV-1 infection is the result of a tightly regulated process that, among other factors, is critically impacted by the kinetics of viral protein expression, the HLA class I background of the infected individual and the autologous sequence of the infecting virus.     

The clinical benefits of tenofovir for simian immunodeficiency virus-infected macaques are larger than predicted by its effects on standard viral and immunologic parameters

Previous studies have demonstrated that tenofovir (9-[2-(phosphonomethoxy)propyl]adenine; PMPA) treatment is usually very effective in suppressing viremia in macaques infected with simian immunodeficiency virus (SIV). The present study focuses on a subset of infant macaques that were chronically infected with highly virulent SIVmac251, and for which prolonged tenofovir treatment failed to significantly suppress viral RNA levels in plasma despite the presence of tenofovirsusceptible virus at the onset of therapy. While untreated animals with similarly high viremia developed fatal immunodeficiency within 3-6 months, these tenofovir-treated animals had significantly improved survival (up to 3.5 years). This clinical benefit occurred even in animals for which tenofovir had little or no effect on CD4 and CD8 lymphocyte counts and antibody responses to SIV and test antigens. Thus, the clinical benefits of tenofovir were larger than predicted by plasma viral RNA levels and other routine laboratory parameters.     

Presence of Intact vpu and nef genes in nonpathogenic SHIV is essential for acquisition of pathogenicity of this virus by serial passage in macaques

Use of the macaque model of human immunodeficiency virus (HIV) pathogenesis has shown that the accessory genes nef and vpu are important in the pathogenicity of simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus (SHIV). We examined the ability of two nonpathogenic SHIVs, SHIV(PPC) and DeltavpuDeltanefSHIV(PPC), to gain pathogenicity by rapid serial passage in macaques. In this study, each virus was passaged by blood intravenously four times at 4-week intervals in macaques. Animals were monitored for 40 weeks for levels of CD4 T cells and quantitative measures of virus infection. DeltavpuDeltanefSHIV(PPC) maintained a limited phase of productive replication in the four animals, with no loss of CD4(+) T cells, whereas SHIV(PPC) became more pathogenic in later passages, judging by plasma viral load and viral mRNA in lymph nodes, infectious peripheral blood mononuclear cells and CD4(+) T cell loss. The nef, LTR, and env of the SHIV(PPC) viruses underwent numerous mutations, compared to DeltavpuDeltanefSHIV(PPC). This study confirms the seminal role that nef, LTR, and vpu could play in regulation of pathogenesis of HIV infection.     

Frequent detection of cell-associated HIV-1 RNA in patients with plasma viral load <50 copies/ml

Despite prolonged undetectable plasma viral load some HIV-1 infected patients have been reported to develop resistance-associated mutations leading to treatment failure. The mechanisms for this phenomenon and the point of origin for residual viral evolution are still not elucidated. In order to quantify cell-associated HIV-1 RNA in patients with different levels of plasma viremia paired cell-associated HIV-1 RNA loads and plasma viral loads were determined. Weak inverse correlation between these parameters and the amounts of CD4(+) T cells was observed, whereas there was no correlation between viral loads and CD8(+) T cells or CD14(+) monocytes, respectively. In a subset of patients, cell-associated and plasma HIV-1 env V3 sequences were analyzed. Plasma viral load and the amount of cell-associated HIV-RNA correlated strongly. However, in 62.3% of patients with undetectable plasma viral load cell-associated HIV-RNA could be detected. Analyses of HIV-RNA in plasma and blood cells showed identical sequences in 4/19 patients, whereas the majority of patients had differing HIV-1 RNA sequences in plasma and cells, respectively. In summary, this study shows that residual viral replication in peripheral blood still occurs in the majority of patients with undetectable plasma viral load. Since these replication events could lead to ongoing viral evolution it should be considered to optimize antiretroviral therapy in order to minimize the development of drug resistance.     

Declining levels of rescued lymphoproliferative response to human cytomegalovirus (HCMV) in AIDS patients with or without HCMV disease following long-term HAART.

OBJECTIVE: To investigate the lymphoproliferative response (LPR) to human cytomegalovirus (HCMV) in two groups of AIDS patients undergoing long-term highly active antiretroviral therapy (HAART): group 1 ( n = 22) with nadir CD4(+) cell count <50/microl and no HCMV disease; group 2 ( n = 16) with <50/microl CD4(+) T-cell count and HCMV disease. All patients had previously undergone antiretroviral monotherapy or dual therapy before initiating HAART. STUDY DESIGN AND METHODS: The two groups of patients were tested prospectively for CD4(+) T cell count, HIV RNA load, HCMV viremia, and LPR to HCMV at baseline, and then after 3 and 4 years of HAART. A control group of 13 recently diagnosed treatment-naive AIDS patients with CD4(+) T-cell counts <100/microl was also investigated. RESULTS: No LPR to HCMV was found in any of the treatment-naive patients nor in any patient of the two groups examined at baseline, when HCMV viremia was 13.6% in the patient group without disease and 87.5% in the group with disease ( p <.0001). After 3 years of HAART, the frequency of patients who recovered an LPR to HCMV was not significantly different (81.8% in the group without HCMV disease, and 68.7% in the group with HCMV disease), whereas, compared with baseline, the HIV load decreased and the CD4(+) T-cell count increased significantly and to a comparable extent in the two groups of patients. In addition, the frequency of patients with HCMV viremia, although reduced, became comparable in both groups. After 4 years of HAART, the frequency of responders to HCMV without and with HCMV disease dropped to comparable levels (50.0 vs. 56.3%, respectively) in association with high median CD4(+) T-cell counts and low median HIV RNA plasma levels. In parallel, the frequency of patients with HCMV viremia did not change significantly. In addition, after between 3 and 4 years of HAART, although the frequency of stable responders and nonresponders remained unchanged (50%) in both groups, most of the remaining patients showed declining levels of responsiveness to HCMV. Although some patients from both groups were found to have CD4(+) T-cell counts >150/microl in the absence of LPR to HCMV, thus suggesting dissociation of specific and nonspecific immune reconstitution, a significant correlation was found between CD4(+) T-cell count and LPR to HCMV (r = 0.44; p <.001). From a clinical standpoint, anti-HCMV therapy could be safely discontinued in 8 patients with HCMV retinitis showing CD4(+) T-cell counts >150/microl, recovery of HCMV LPR, and no HCMV viremia. CONCLUSIONS: Declining levels of the previously recovered LPR to HCMV are often observed after long-term HAART. However, because the role of LPR in the evolution of HCMV infection and disease during HAART remains to be defined, the clinical impact of the declining LPR to HCMV must still be clarified in long-term prospective studies.