Timothy W. Schacker

Microbial translocation is a cause of systemic immune activation in chronic HIV infection

Chronic activation of the immune system is a hallmark of progressive HIV infection and better predicts disease outcome than plasma viral load, yet its etiology remains obscure. Here we show that circulating microbial products, probably derived from the gastrointestinal tract, are a cause of HIV-related systemic immune activation. Circulating lipopolysaccharide, which we used as an indicator of microbial translocation, was significantly increased in chronically HIV-infected individuals and in simian immunodeficiency virus (SIV)-infected rhesus macaques (P ≤ 0.002). We show that increased lipopolysaccharide is bioactive in vivo and correlates with measures of innate and adaptive immune activation. Effective antiretroviral therapy seemed to reduce microbial translocation partially. Furthermore, in nonpathogenic SIV infection of sooty mangabeys, microbial translocation did not seem to occur. These data establish a mechanism for chronic immune activation in the context of a compromised gastrointestinal mucosal surface and provide new directions for therapeutic interventions that modify the consequences of acute HIV infection.

CD4+ T Cell Depletion during all Stages of HIV Disease Occurs Predominantly in the Gastrointestinal Tract

The mechanisms underlying CD4+ T cell depletion in human immunodeficiency virus (HIV) infection are not well understood. Comparative studies of lymphoid tissues, where the vast majority of T cells reside, and peripheral blood can potentially illuminate the pathogenesis of HIV-associated disease. Here, we studied the effect of HIV infection on the activation and depletion of defined subsets of CD4+ and CD8+ T cells in the blood, gastrointestinal (GI) tract, and lymph node (LN). We also measured HIV-specific T cell frequencies in LNs and blood, and LN collagen deposition to define architectural changes associated with chronic inflammation. The major findings to emerge are the following: the GI tract has the most substantial CD4+ T cell depletion at all stages of HIV disease; this depletion occurs preferentially within CCR5+ CD4+ T cells; HIV-associated immune activation results in abnormal accumulation of effector-type T cells within LNs; HIV-specific T cells in LNs do not account for all effector T cells; and T cell activation in LNs is associated with abnormal collagen deposition. Taken together, these findings define the nature and extent of CD4+ T cell depletion in lymphoid tissue and point to mechanisms of profound depletion of specific T cell subsets related to elimination of CCR5+ CD4+ T cell targets and disruption of T cell homeostasis that accompanies chronic immune activation.

HIV reservoir size and persistence are driven by T cell survival and homeostatic proliferation

HIV persists in a reservoir of latently infected CD4+ T cells in individuals treated with highly active antiretroviral therapy (HAART). Here we identify central memory (TCM) and transitional memory (TTM) CD4+ T cells as the major cellular reservoirs for HIV and find that viral persistence is ensured by two different mechanisms. HIV primarily persists in TCM cells in subjects showing reconstitution of the CD4+ compartment upon HAART. This reservoir is maintained through T cell survival and low-level antigen-driven proliferation and is slowly depleted with time. In contrast, proviral DNA is preferentially detected in TTM cells from aviremic individuals with low CD4+ counts and higher amounts of interleukin-7–mediated homeostatic proliferation, a mechanism that ensures the persistence of these cells. Our results suggest that viral eradication might be achieved through the combined use of strategic interventions targeting viral replication and, as in cancer, drugs that interfere with the self renewal and persistence of proliferating memory T cells.

PD-1 is a regulator of virus-specific CD8+ T cell survival in HIV infection

Here, we report on the expression of programmed death (PD)-1 on human virus-specific CD8+ T cells and the effect of manipulating signaling through PD-1 on the survival, proliferation, and cytokine function of these cells. PD-1 expression was found to be low on naive CD8+ T cells and increased on memory CD8+ T cells according to antigen specificity. Memory CD8+ T cells specific for poorly controlled chronic persistent virus (HIV) more frequently expressed PD-1 than memory CD8+ T cells specific for well-controlled persistent virus (cytomegalovirus) or acute (vaccinia) viruses. PD-1 expression was independent of maturational markers on memory CD8+ T cells and was not directly associated with an inability to produce cytokines. Importantly, the level of PD-1 surface expression was the primary determinant of apoptosis sensitivity of virus-specific CD8+ T cells. Manipulation of PD-1 led to changes in the ability of the cells to survive and expand, which, over several days, affected the number of cells expressing cytokines. Therefore, PD-1 is a major regulator of apoptosis that can impact the frequency of antiviral T cells in chronic infections such as HIV, and could be manipulated to improve HIV-specific CD8+ T cell numbers, but possibly not all functions in vivo.

Clinical and Epidemiologic Features of Primary HIV Infection

Primary infection with the human immunodeficiency virus (HIV) ranges from asymptomatic seroconversion to a severe symptomatic illness resembling infectious mononucleosis that can result in hospitalization [1]. However, most reports of primary HIV infection have characterized the events surrounding acquisition of HIV in only a few patients, and most investigators have obtained this information many months after HIV has been acquired. In this report, we summarize the events leading to the acquisition of HIV and the initial clinical and diagnostic evaluation of 46 patients with primary HIV infection. Methods Study Design Patients were recruited into an institutional review board-approved protocol for the study of primary HIV infection. Eligible participants were documented to be 1) simultaneously positive for HIV p24 antigen (or plasma HIV RNA) and negative for HIV antibody (measured by an enzyme immunoassay [EIA] for HIV) at recruitment; 2) serologically (that is, measured by HIV EIA plus Western blot assay) positive at recruitment; or 3) serologically positive at recruitment and serologically negative within the 12 months before study entry, with a documented retroviral syndrome developing 3 months before study entry. We placed no restrictions on sex, age, or race. All enrolled patients who had HIV seroconversion provided written documentation of a previously negative HIV EIA test result from a laboratory proficient in HIV antibody testing. Between September 1993 and January 1995, 60 persons were referred to the study clinic to be screened for primary HIV infection; 46 met the inclusion criteria. The 14 excluded persons did not provide written documentation of previous negative HIV antibody test results or of recent seroconversion. At the enrollment visit, a standardized 160-item questionnaire was administered in private to each patient to collect demographic information, recent and earlier sexual history, and details of all recent medical illnesses. History of sexual contact was corroborated on subsequent visits. Recent medical records were also reviewed, and each patient had a complete physical examination. Any stored serum or plasma samples available from previous HIV antibody testing or a recent illness were retrieved and tested for p24 antigen, plasma HIV RNA, and antibodies against HIV to determine whether the illness associated with the serum sample had been caused by HIV seroconversion. No patient received antiretroviral therapy before or during the initial evaluation period. All patients were asked to have lumbar puncture to obtain cerebrospinal fluid specimens. Twenty-four of the 46 patients agreed to have lumbar puncture. Laboratory Analysis All laboratory studies were done at the University of Washington. Laboratories at the university are certified by the American College of Pathology and the AIDS (acquired immunodeficiency syndrome) Clinical Trials Group for the HIV antibody test, the p24 antigen test, and HIV RNA polymerase chain reaction assays. Enzyme immunoassays for HIV were done according to the manufacturers instructions (Genetic Systems Corp., Seattle, Washington). All positive EIA results were confirmed by Western blot assay (Epitope, Inc., Beaverton, Oregon). Levels of HIV p24 antigen were assayed by EIA according to the manufacturers directions (Abbott Laboratories, Chicago, Illinois); p24 antigen levels less than 20 pg/mL were considered to represent a negative result. Plasma HIV RNA was detected using the Chiron (Emeryville, California) branched-chain DNA assay. The lower limit of detection for this assay is 10 103 DNA copies/mL of plasma [2, 3]. Quantitative co-cultures of peripheral blood mononuclear cells and standard HIV type 1 (HIV-1) cultures of cerebrospinal fluid and plasma were done at the University of Washington Retrovirology Laboratory using previously published methods [4, 5]. T-cell subsets were measured by the Hematopathology Laboratory of the University of Washington using flow cytometry. Statistical Analysis Differences in demographic characteristics and sexual contact history were evaluated using the Fisher exact test or the Mann-Whitney test, as specified. For patients with symptomatic illness, we used the date of symptom onset as the date of HIV acquisition. For patients who were HIV negative on EIA within the 6 months before study entry and who had no history of symptoms, the estimated acquisition date was established as the date midway between the most recent negative and the subsequent positive HIV antibody test result. One author assigned the acquisition date for all patients. Results Patients We enrolled 46 patients during a 28-month period. Patients were referred because they were at risk for HIV infection and developed symptoms of a retroviral illness or because they were found to be HIV-positive on routine testing and had documented evidence of a recent negative test. Patients were recruited from the University of Washington HIV clinics (43%), regional AIDS prevention programs (15%), King County Jail (12%), and local community physicians (30%). Forty-three of the 46 patients (93%) were men. The median age was 30 years, and 13 of 46 patients (28%) were younger than 25 years of age. Forty-two of the 43 men had had sex with men as a risk factor for HIV acquisition; the other male patient reported only heterosexual contact. Four patients (3 men and 1 woman) reported having used injection drugs. Our cohort was similar to persons reported to have HIV-1 infection in King County, Washington (Table 1). Table 1. Demographic Characteristics of Patients with Primary HIV Infection Compared with the 1995 Demographic Characteristics of Persons with Reported Cases of AIDS in King County, Washington* In 17 of 46 patients (37%), either HIV-1 p24 antigen or HIV RNA was detected when the HIV EIA result was negative. The remaining 29 patients (63%) were identified with a new positive result on an HIV Western blot assay and a previous negative result on HIV EIA paired serologic assays. These 29 patients included persons who were tested because they were at high risk and thus had routinely obtained HIV serologic testing, persons who were tested for recent symptoms consistent with primary HIV infection, and persons who were entering a new personal relationship and wanted to know their HIV serologic status. In these 29 patients, the median time from the last documented negative test result was 5 months (range, 1 to 12 months), and the median time from symptom onset to study entry was 80 days (range 2 to 178 days). No differences were noted between the demographic characteristics of the 17 patients identified as positive for HIV p24 antigen or for HIV RNA at enrollment and the demographic characteristics of the 29 patients identified through paired serologic testing. Clinical Presentation Forty-one patients (89%) reported symptoms associated with HIV seroconversion. Sixteen of 17 patients who were positive for HIV p24 antigen or HIV RNA or were negative for HIV EIA antibody at study entry had signs or symptoms suggestive of an acute retroviral syndrome; 15 of these 16 patients (94%) sought medical assistance, and 12 of these 15 were eventually tested for HIV infection during their illness. Of the remaining 29 patients, 25 (86%) also reported symptoms consistent with an acute retroviral syndrome during the interval between their previously negative antibody test result and study entry (Table 2). Twenty-two of these 25 (88%) sought medical care for these symptoms: Nine had HIV testing at the initial medical encounter, and 5 others referred themselves for testing shortly after the illness developed. Three of 46 patients (6%) reported that they had intentionally engaged in high-risk sexual behavior in attempts to become infected with HIV. Table 2. Clinical Presentation of HIV Seroconversion* Twenty-three patients were participating in routine surveillance programs in which HIV testing was done every 6 months. Twenty of these 23 (87%) reported symptoms consistent with an acute retroviral syndrome, and 19 of the 20 (95%) had medical evaluation for these symptoms (48% went to their own primary care provider, 31% went to an emergency department, and 21% went to a walk-in clinic). A diagnosis of primary HIV infection was considered at these medical encounters in only 5 of these 19 patients (26%). The five most common symptoms reported during primary HIV infection were fever, sore throat, fatigue, weight loss, and myalgia (Figure 1, top). The median maximal temperature reported was 38.9 C (range, 39 C to 40.5 C), and the median weight loss was 5 kg (range, 1.4 to 10 kg). Abnormalities on physical examination for symptoms of primary HIV infection were found in 23 of 35 patients (66%) seeking medical help. These abnormalities included postural hypotension, oral ulceration, exudative pharyngitis, thrush, genital or rectal ulceration, adenopathy, and signs of neuropathy. Signs and symptoms of aseptic meningitis (fever, headache, photophobia, and stiff neck) were noted in 10 of the 41 patients (24%) presenting for medical consultation. The median duration of symptoms of the acute retroviral illness was 14 days (Figure 1, bottom). Seven of the 41 patients (17%) with symptomatic illness were hospitalized. Frequency of hospitalization was the only significant difference noted in the clinical presentation of patients who were positive for p24 antigen (or for HIV RNA) and had negative EIA results compared with patients who were identified by serial testing: 29% compared with 8%. Figure 1. Top. Bottom. Virologic Studies at Study Entry The time from seroconversion to study entry was a significant factor in the ability to detect HIV-1 RNA in plasma (Table 3). Of 16 patients enrolled within 60 days of HIV acquisition (median, 28 days), 14 had HIV RNA detected in plasma (median titer, 116 103 RNA copies/mL [range, <10 to 553 103 RNA copies/mL]). Only 1 of these 14 patients had HIV p24 antigenemia. In the

Differential Th17 CD4 T-cell depletion in pathogenic and nonpathogenic lentiviral infections

Acute HIV infection is characterized by massive loss of CD4 T cells from the gastrointestinal (GI) tract. Th17 cells are critical in the defense against microbes, particularly at mucosal surfaces. Here we analyzed Th17 cells in the blood, GI tract, and broncheoalveolar lavage of HIV-infected and uninfected humans, and SIV-infected and uninfected sooty mangabeys. We found that (1) human Th17 cells are specific for extracellular bacterial and fungal antigens, but not common viral antigens; (2) Th17 cells are infected by HIV in vivo, but not preferentially so; (3) CD4 T cells in blood of HIV-infected patients are skewed away from a Th17 phenotype toward a Th1 phenotype with cellular maturation; (4) there is significant loss of Th17 cells in the GI tract of HIV-infected patients; (5) Th17 cells are not preferentially lost from the broncheoalveolar lavage of HIV-infected patients; and (6) SIV-infected sooty mangabeys maintain healthy frequencies of Th17 cells in the blood and GI tract. These observations further elucidate the immunodeficiency of HIV disease and may provide a mechanistic basis for the mucosal barrier breakdown that characterizes HIV infection. Finally, these data may help account for the nonprogressive nature of nonpathogenic SIV infection in sooty mangabeys.

Persistent HIV-1 replication is associated with lower antiretroviral drug concentrations in lymphatic tissues

Significance We show that HIV continues to replicate in the lymphatic tissues of some individuals taking antiretroviral regimens considered fully suppressive, based on undetectable viral loads in peripheral blood, and that one mechanism for persistent replication in lymphatic tissues is the lower concentrations of the antiretroviral drugs in those tissues compared with peripheral blood. These findings are significant because they provide a rationale and framework for testing the efficacy of new agents and combinations of drugs that will fully suppress replication in lymphatic tissues. More suppressive regimens could improve immune reconstitution, as well as provide the effective regimens needed for functional cure and eradication of infection. Antiretroviral therapy can reduce HIV-1 to undetectable levels in peripheral blood, but the effectiveness of treatment in suppressing replication in lymphoid tissue reservoirs has not been determined. Here we show in lymph node samples obtained before and during 6 mo of treatment that the tissue concentrations of five of the most frequently used antiretroviral drugs are much lower than in peripheral blood. These lower concentrations correlated with continued virus replication measured by the slower decay or increases in the follicular dendritic cell network pool of virions and with detection of viral RNA in productively infected cells. The evidence of persistent replication associated with apparently suboptimal drug concentrations argues for development and evaluation of novel therapeutic strategies that will fully suppress viral replication in lymphatic tissues. These strategies could avert the long-term clinical consequences of chronic immune activation driven directly or indirectly by low-level viral replication to thereby improve immune reconstitution.

Premature Induction of an Immunosuppressive Regulatory T Cell Response during Acute Simian Immunodeficiency Virus Infection

Here we report the results of an investigation into the possibility that one mechanism responsible for the establishment of persistent human immunodeficiency virus infection is an early regulatory T (Treg) cell response that blunts virus-specific responses. Using the simian immunodeficiency virus (SIV)-infected rhesus macaque model, we show that, indeed, viral replication and immune activation in lymphatic tissue drive a premature immunosuppressive response, with dramatic increases in the frequencies of CD4+CD25+FOXP3+ Treg cells, transforming growth factor- beta 1+ cells, interleukin-10+ cells, and indoleamine 2,3-dioxygenase+CD3+ cells. When we compared SIV infection with rhesus cytomegalovirus (RhCMV) infection, we found that the frequency of Treg cells paralleled the magnitude of immune activation during both infections but that the magnitude of immune activation and of the Treg cell response were lower and peaked much later during RhCMV infection. Importantly, the frequency of Treg cells inversely correlated with the magnitude of the SIV-specific cytotoxic T lymphocyte response. We conclude that an early Treg cell response during acute SIV infection may contribute to viral persistence by prematurely limiting the antiviral immune response before infection is cleared.

Persistent HIV-1 replication maintains the tissue reservoir during therapy

Lymphoid tissue is a key reservoir established by HIV-1 during acute infection. It is a site associated with viral production, storage of viral particles in immune complexes, and viral persistence. Although combinations of antiretroviral drugs usually suppress viral replication and reduce viral RNA to undetectable levels in blood, it is unclear whether treatment fully suppresses viral replication in lymphoid tissue reservoirs. Here we show that virus evolution and trafficking between tissue compartments continues in patients with undetectable levels of virus in their bloodstream. We present a spatial and dynamic model of persistent viral replication and spread that indicates why the development of drug resistance is not a foregone conclusion under conditions in which drug concentrations are insufficient to completely block virus replication. These data provide new insights into the evolutionary and infection dynamics of the virus population within the host, revealing that HIV-1 can continue to replicate and replenish the viral reservoir despite potent antiretroviral therapy.

Collagen deposition in HIV-1 infected lymphatic tissues and T cell homeostasis.

Lymphatic tissues (LTs) are structurally organized to promote interaction between antigens, chemokines, growth factors, and lymphocytes to generate an immunologic response and maintain normal-sized populations of CD4(+) and CD8(+) T cells. Inflammation and tissue remodeling that accompany local innate and adaptive immune responses to HIV-1 replication cause damage to the LT architecture. As a result, normal populations of CD4(+) and CD8(+) T cells cannot be supported and antigen-lymphocyte interactions are impaired. This conclusion is supported herein following LT sampling before and during anti-HIV therapy in persons with acute, chronic, and late-stage HIV-1 infection. Among seven individuals treated with anti-retroviral therapy (ART) and four individuals deferring therapy we found evidence of significant paracortical T cell zone damage associated with deposition of collagen, the extent of which was inversely correlated with both the size of the LT CD4(+) T cell population and the change in peripheral CD4(+) T cell count with anti-HIV therapy. The HIV-1-associated inflammatory changes and scarring in LT both limit the ability of the tissue to support and reestablish normal-sized populations of CD4(+) T cells and suggest a novel mechanism of T cell depletion that may explain the failure of ART to significantly increase CD4(+) T cell populations in some HIV-1-infected persons.