JoAnn M. Mican

New Real-Time Reverse Transcriptase-Initiated PCR Assay with Single-Copy Sensitivity for Human Immunodeficiency Virus Type 1 RNA in Plasma

ABSTRACT More sensitive assays for human immunodeficiency virus type 1 (HIV-1) RNA are needed to detect, quantify, and characterize persistent viremia in patients who are receiving antiretroviral therapy and whose plasma HIV-1 RNA levels are suppressed to less than 50 to 75 copies/ml. We therefore developed an internally controlled real-time reverse transcriptase-initiated PCR assay that quantifies HIV-1 RNA concentrations down to 1 copy per ml of plasma. This assay with single-copy sensitivity (the single-copy assay) generates a reproducible linear regression plot of input copy number versus threshold cycle by using HIV-1 RNA transcripts at copy numbers ranging from 1 to 106 per reaction mixture. The single-copy assay was compared to the ultrasensitive AMPLICOR HIV-1 MONITOR assay and a more sensitive modification of the ultrasensitive assay by repeatedly testing a low-copy-number panel containing 200 to 0.781 copies of HIV-1 RNA per ml of plasma. This comparison showed that the single-copy assay had a greater sensitivity than the other assays and was the only assay that detected HIV-1 RNA at levels as low as 0.781 copies/ml. Testing of plasma samples from 15 patients who were receiving antiretroviral therapy and who had <75 HIV-1 RNA copies/ml revealed persistent viremia in all 15 patients, with HIV-1 RNA levels ranging from 1 to 32 copies/ml (median, 13 copies/ml). The greater sensitivity of the single-copy assay should allow better characterization of persistent viremia in patients who are receiving antiretroviral therapy and whose HIV-1 RNA levels are suppressed to below the detection limits of present assays.

Lytic Granule Loading of CD8+ T Cells Is Required for HIV-Infected Cell Elimination Associated with Immune Control

Virus-specific CD8+ T cells probably mediate control over HIV replication in rare individuals, termed long-term nonprogressors (LTNPs) or elite controllers. Despite extensive investigation, the mechanisms responsible for this control remain incompletely understood. We observed that HIV-specific CD8+ T cells of LTNPs persisted at higher frequencies than those of treated progressors with equally low amounts of HIV. Measured on a per-cell basis, HIV-specific CD8+ T cells of LTNPs efficiently eliminated primary autologous HIV-infected CD4+ T cells. This function required lytic granule loading of effectors and delivery of granzyme B to target cells. Defective cytotoxicity of progressor effectors could be restored after treatment with phorbol ester and calcium ionophore. These results establish an effector function and mechanism that clearly segregate with immunologic control of HIV. They also demonstrate that lytic granule contents of memory cells are a critical determinant of cytotoxicity that must be induced for maximal per-cell killing capacity.

Effect of interleukin-2 on the pool of latently infected, resting CD4+ T cells in HIV-1-infected patients receiving highly active anti-retroviral therapy

The size of the pool of resting CD4+ T cells containing replication-competent HIV in the blood of patients receiving intermittent interleukin (IL)-2 plus highly active anti-retroviral therapy (HAART) was significantly lower than that of patients receiving HAART alone. Virus could not be isolated from the peripheral blood CD4+ T cells in three patients receiving IL-2 plus HAART, despite the fact that large numbers of resting CD4+ T cells were cultured. Lymph node biopsies were done in two of these three patients and virus could not be isolated. These results indicate that the intermittent administration of IL-2 with continuous HAART may lead to a substantial reduction in the pool of resting CD4+ T cells that contain replication-competent HIV.

Persistence of HIV in Gut-Associated Lymphoid Tissue despite Long-Term Antiretroviral Therapy

Human immunodeficiency virus (HIV) persists in peripheral blood mononuclear cells despite sustained, undetectable plasma viremia resulting from long-term antiretroviral therapy. However, the source of persistent HIV in such infected individuals remains unclear. Given recent data suggesting high levels of viral replication and profound depletion of CD4(+) T cells in gut-associated lymphoid tissue (GALT) of animals infected with simian immunodeficiency virus and HIV-infected humans, we sought to determine the level of CD4(+) T cell depletion as well as the degree and extent of HIV persistence in the GALT of infected individuals who had been receiving effective antiviral therapy for prolonged periods of time. We demonstrate incomplete recoveries of CD4(+) T cells in the GALT of aviremic, HIV-infected individuals who had received up to 9.9 years of effective antiretroviral therapy. In addition, we demonstrate higher frequencies of HIV infection in GALT, compared with PBMCs, in these aviremic individuals and provide evidence for cross-infection between these 2 cellular compartments. Together, these data provide a possible mechanism for the maintenance of viral reservoirs revolving around the GALT of HIV-infected individuals despite long-term viral suppression and suggest that the GALT may play a major role in the persistence of HIV in such individuals.

Treatment intensification does not reduce residual HIV-1 viremia in patients on highly active antiretroviral therapy

In HIV-1-infected individuals on currently recommended antiretroviral therapy (ART), viremia is reduced to <50 copies of HIV-1 RNA per milliliter, but low-level residual viremia appears to persist over the lifetimes of most infected individuals. There is controversy over whether the residual viremia results from ongoing cycles of viral replication. To address this question, we conducted 2 prospective studies to assess the effect of ART intensification with an additional potent drug on residual viremia in 9 HIV-1-infected individuals on successful ART. By using an HIV-1 RNA assay with single-copy sensitivity, we found that levels of viremia were not reduced by ART intensification with any of 3 different antiretroviral drugs (efavirenz, lopinavir/ritonavir, or atazanavir/ritonavir). The lack of response was not associated with the presence of drug-resistant virus or suboptimal drug concentrations. Our results suggest that residual viremia is not the product of ongoing, complete cycles of viral replication, but rather of virus output from stable reservoirs of infection.

Decreased Survival of B Cells of HIV-viremic Patients Mediated by Altered Expression of Receptors of the TNF Superfamily

Human immunodeficiency virus (HIV) infection leads to numerous perturbations of B cells through mechanisms that remain elusive. We performed DNA microarray, phenotypic, and functional analyses in an effort to elucidate mechanisms of B cell perturbation associated with ongoing HIV replication. 42 genes were up-regulated in B cells of HIV-viremic patients when compared with HIV-aviremic and HIV-negative patients, the majority of which were interferon (IFN)-stimulated or associated with terminal differentiation. Flow cytometry confirmed these increases and indicated that CD21low B cells, enhanced in HIV-viremic patients, were largely responsible for the changes. Increased expression of the tumor necrosis factor (TNF) superfamily (TNFSF) receptor CD95 correlated with increased susceptibility to CD95-mediated apoptosis of CD21low B cells, which, in turn, correlated with HIV plasma viremia. Increased expression of BCMA, a weak TNFSF receptor for B lymphocyte stimulator (BLyS), on CD21low B cells was associated with a concomitant reduction in the expression of the more potent BLyS receptor, BAFF-R, that resulted in reduced BLyS binding and BLyS-mediated survival. These findings demonstrate that altered expression of genes associated with IFN stimulation and terminal differentiation in B cells of HIV-viremic patients lead to an increased propensity to cell death, which may have substantial deleterious effects on B cell responsiveness to antigenic stimulation.

High Prevalence of Osteonecrosis of the Femoral Head in HIV-Infected Adults

Context Osteonecrosis (avascular necrosis) of the hip is an uncommon but painful and disabling condition that sometimes requires total hip replacement. Contribution This large survey showed that an unusually high percentage of HIV-infected adults (4.4% [95% CI, 2.5% to 7.2%]) had osteonecrosis of the hip, as detected by magnetic resonance imaging. Implications Although screening asymptomatic HIV-infected patients is not warranted, osteonecrosis should be considered in HIV-infected patients with persistent groin or hip pain. The Editors The natural history of HIV infection has changed substantially with the wide use of highly active antiretroviral regimens that include potent protease inhibitors or non-nucleoside reverse transcriptase inhibitors (1). The incidence of HIV-related deaths and HIV-associated opportunistic infections has decreased dramatically. As survival and quality of life have improved, the focus of health care providers has shifted to management of the increasingly complex drug regimens and their associated drug interactions and toxicities. Previously unrecognized or underrecognized complications related to HIV infection or therapies for HIV infection, such as lactic acidosis, hyperlipidemia, and fat redistribution, are increasingly being recognized (2). Osteonecrosis of the hip (also known as avascular necrosis) is a disabling condition that frequently requires total hip replacement (3-5). Osteonecrosis in HIV-infected patients was first reported in 1990 (6). Subsequent anecdotal reports have suggested that osteonecrosis is being diagnosed with increasing frequency in the setting of HIV infection (7-31). Between May 1999 and November 2000, six HIV-infected patients followed at the Warren Grant Magnuson Clinical Center of the U.S. National Institutes of Health (NIH) presented with groin or hip pain and received a diagnosis of osteonecrosis. The first two of these patients received their diagnosis within a 4-day period and were the first HIV-infected patients ever to receive such a diagnosis at the NIH Clinical Center. We also learned that an increasing number of cases of osteonecrosis were being reported to the U.S. Food and Drug Administrations Adverse Event Reporting System. We were concerned that osteonecrosis might be becoming a major HIV-related complication. We hypothesized that, if this were true, additional patients may have developed clinically silent osteonecrosis. Because magnetic resonance imaging (MRI) has been used to study asymptomatic osteonecrosis of the hip in other high-risk populations (32-37), we undertook a prospective MRI-based study to determine the prevalence of and evaluate possible risk factors for asymptomatic osteonecrosis of the hip in a sample of HIV-infected patients. Methods Participant Recruitment and Questionnaire Between 1 June and 15 December 1999, adults who were enrolled in studies of the treatment or natural history of HIV infection at the NIH Clinical Center or who were receiving health care services at the National Naval Medical Center in Bethesda, Maryland, were invited to participate in an MRI-based screening study of osteonecrosis of the hip. All patients seen in the outpatient clinics of the National Institute of Allergy and Infectious DiseasesNIH Clinical Center HIV program during the study period were informed of the protocol and encouraged to participate. Because this study focused on asymptomatic patients, we excluded persons with current groin or hip pain. Because the incidence of osteonecrosis was unknown, we considered several possible sample sizes (100 to 300 patients) and estimations of the 95% CI for low prevalence rates of asymptomatic osteonecrosis (0.1% to 3.5%). We determined that a sample size of at least 300 HIV-infected participants would be reasonable and attainable. Before undergoing MRI, participants completed a standard questionnaire on joint symptoms; medical history; medication use; and personal habits, including ethanol use, cigarette smoking, and routine exercise regimens. Questions related to medication use made a clear distinction between corticosteroids and other types of steroids, such as androgenic and anabolic steroids. Patients were asked why corticosteroids were prescribed and the route, frequency, and duration of use. The Institutional Review Board of the National Institute of Allergy and Infectious Diseases approved the protocol. All participants gave written informed consent. Data Collection We retrieved laboratory and clinical data from patient databases. Laboratory values obtained within 4 months of the MRI date were used in the analyses; for certain variables, we also analyzed the highest or lowest values recorded in a laboratory database that contained data back to 1984. Physiatrist Evaluation After approximately 150 patients were scanned and asymptomatic osteonecrosis was detected in 4 patients, we prospectively determined whether subtle physical findings might identify patients with MRI evidence of osteonecrosis of the hip. Patients enrolled after 12 July 1999 were asked (but not required) to be examined by a physiatrist who was unaware of the MRI results. The examination included evaluation of range of motion in the hip, which involved testing in all planes to end range with and without resistance and joint-loading maneuvers; pain was assessed in each test (38). MRI Scanning HIV-Infected Participants Magnetic resonance imaging was performed by using an LX Horizon 1.5-T MRI system (General Electric Medical Systems, Milwaukee, Wisconsin). We modeled the protocol for screening MRI on a previously described method for bilateral screening of the hips for osteonecrosis (37). Coronal T1-weighted spin-echo sequences were done with a repetition time of 400 ms and an echo time of 8 ms (using a 256 192 matrix). These were followed by coronal fatsuppressed T2-weighted fast spin-echo inversion recovery sequences with a repetition time of 3266 ms, an echo time of 38 ms, and an inversion time of 150 ms (256 224 matrix). Other variables used for both screening sequences included three excitations, a 40.0 40.0cm field of view, and 5-mm contiguous sections. All MRI scans were initially interpreted by one of the investigators. Positive scans were later intermixed with 35 randomly selected negative scans of HIV-infected participants and were reviewed by a second radiologist, who was unaware of the previous interpretation. Patients with osteonecrosis on the screening MRI underwent dedicated high-resolution MRI of the affected hip or the more abnormal hip. HIV-Negative Participants We assumed that asymptomatic osteonecrosis of the hip detectable only by MRI must occur infrequently in healthy adult humans with no known risk factors. To document this, we recruited persons without hip or groin pain who had no known risk factors for osteonecrosis. Exclusion criteria for this comparison group were a history of alcohol abuse, hip or leg fracture, pancreatitis, diabetes mellitus, hypertriglyceridemia (requiring therapy), systemic lupus erythematosus, blood-clotting disorders, or systemic corticosteroid use during the previous year or for greater than 1 month ever (total lifetime use). We recruited these participants from an NIH healthy volunteer database and by posting flyers at the NIH Clinical Center. The participants in the comparison group were approximately matched for age and sex to participants in the screening study HIV-infected patients; the comparison group underwent the MRI screening and consented to participate using the same protocol and procedures that were used in the HIV group. Hematologic Evaluation Participants with MRI evidence of osteonecrosis were evaluated for a possible hypercoagulable state by using the following assays: two assays for the presence of a lupus anticoagulant (DRVVT, American Diagnostica, Inc., Greenwich, Connecticut, and Staclot, Diagnostica Stago, Asnieres-Sur-Seine, France); assays for immunoglobulin (Ig)G and IgM anticardiolipin antibodies (Quantilite, Innova, San Diego, California); functional assays for protein C, protein S, and antithrombin III levels (Diagnostica Stago; an assay for thrombinantithrombin complex levels [Dade Behring Marburg, Marburg, Germany]; an assay for functional plasma activator inhibitor-1 levels (Biopool, Umea, Sweden); and routine laboratory assays for serum homocysteine and serum lipoprotein(a) levels. These patients were also evaluated for genetic predisposition to hypercoagulability by using restriction enzyme-based genetic tests for factor V Leiden, a prothrombin gene abnormality (G20210A), and for the heat-labile folate reductase (5,10 methylene tetrahydrofolate reductase) gene abnormality, which may lead to hyperhomocystinemia (39-41). For a comparison group, we measured anticardiolipin antibodies, lupus anticoagulant, and protein S levels in a sample of 50 controls with HIV infection who had no evidence of osteonecrosis on MRI. These controls were randomly selected from patients who had been previously scanned as part of the study and who were seen in our clinics for routine visits over a 3-week period (19 November to 9 December 1999). Statistical Analysis We used the method of Clopper and Pearson (42) to calculate exact CIs for proportions. Associations of osteonecrosis with categorical variables were evaluated by using a two-sided Fisher exact test or, for variables with more than two categories, the FisherFreemanHalton test (43). Associations with continuous variables were evaluated by using a two-sided Wilcoxon rank-sum test with continuity correction. Confidence intervals for relative risks for osteonecrosis were estimated by using the likelihood score method (44). For CIs of odds ratios, we estimated variance according to the method of Robins and colleagues (45). For these calculations, we used the NCSS 2000 software package (Number Cruncher Statistical Systems, Kaysville, Utah) and the StatXact 4 software package (Cytel Software Corp., Cambridge, Mass

Compromised B cell responses to influenza vaccination in HIV-infected individuals.

BACKGROUND Yearly influenza vaccination, although recommended for human immunodeficiency virus (HIV)-infected individuals, has not received thorough evaluation in the era of antiretroviral therapy. We assessed the impact of HIV disease on B cell responses to influenza vaccination. METHODS Sixty-four HIV-infected and 17 HIV-negative individuals received the 2003-2004 trivalent inactivated influenza vaccine. Frequencies of influenza-specific antibody-secreting cells (ASCs) were measured by enzyme-linked immunospot (ELISPOT) assay, and antibody responses were measured by hemagglutination-inhibition (HI) assay. Memory responses to influenza were measured by ELISPOT assay after polyclonal activation of B cells in vitro. RESULTS Prevaccination HI titers were significantly higher in HIV-negative than in HIV-infected individuals. Peak HI titers and influenza-specific ASC frequencies were directly correlated with CD4+ T cell counts in HIV-infected individuals. Influenza-specific memory B cell responses were significantly lower in HIV-infected than in HIV-negative individuals and were directly correlated with CD4+ T cell counts. CONCLUSIONS HIV infection is associated with a weak antibody response to influenza vaccination that is compounded by a poor memory B cell response. CD4+ T cell count is a critical determinant of responsiveness to influenza vaccination, and the contribution of plasma HIV RNA level is suggestive and warrants further investigation.

Defective Human Immunodeficiency Virus-Specific CD8+ T-Cell Polyfunctionality, Proliferation, and Cytotoxicity Are Not Restored by Antiretroviral Therapy

ABSTRACT Identifying the functions of human immunodeficiency virus (HIV)-specific CD8+ T cells that are not merely modulated by the level of virus but clearly distinguish patients with immune control from those without such control is of paramount importance. Features of the HIV-specific CD8+ T-cell response in antiretroviral-treated patients (designated Rx <50) and untreated patients (long-term nonprogressors [LTNP]) matched for very low HIV RNA levels were comprehensively examined. The proliferative capacity of HIV-specific CD8+ T cells was not restored in Rx <50 to the level observed in LTNP, even though HIV-specific CD4+ T-cell proliferation in the two patient groups was comparable. This diminished HIV-specific CD8+ T-cell proliferation in Rx <50 was primarily due to a smaller fraction of antigen-specific cells recruited to divide and not to the numbers of divisions that proliferating cells had undergone. Exogenous interleukin-2 (IL-2) induced proliferating cells to divide further but did not rescue the majority of antigen-specific cells with defective proliferation. In addition, differences in HIV-specific CD8+ T-cell proliferation could not be attributed to differences in cellular subsets bearing a memory phenotype, IL-2 production, or PD-1 expression. Although polyfunctionality of HIV-specific CD8+ T cells in Rx <50 was not restored to the levels observed in LTNP despite prolonged suppression of HIV RNA levels, per-cell cytotoxic capacity was the functional feature that most clearly distinguished the cells of LTNP from those of Rx <50. Taken together, these data suggest that there are selective qualitative abnormalities within the HIV-specific CD8+ T-cell compartment that persist under conditions of low levels of antigen.

Normalization of B Cell Counts and Subpopulations after Antiretroviral Therapy in Chronic HIV Disease

BACKGROUND Untreated human immunodeficiency virus (HIV) disease leads to abnormalities in all major lymphocyte populations, including CD4(+) T cells, CD8(+) T cells, and B cells. However, little is known regarding the effect of antiretroviral therapy (ART)-induced decrease in HIV viremia on B cell numbers and subpopulations. METHODS We conducted a longitudinal study to evaluate changes in B cell numbers and subpopulations that occur during the course of 12 months of effective ART in a group of individuals with chronic HIV infection. RESULTS ART-induced decrease in HIV viremia was associated with a significant increase in B cell counts, similar to increases in CD4(+) T cell counts yet distinct from the lack of increase in CD8(+) T cells. The increase in B cell counts was accompanied by a significant decrease in the frequency of apoptosis-prone B cell subpopulations, namely mature activated and immature transitional B cells, which are overrepresented in untreated HIV disease. The increase in B cell counts was reflected by a significant increase in naive and resting memory B cells, both of which represent populations that are essential for generating adequate humoral immunity. CONCLUSIONS Normalization of B cell counts and subpopulations may help to explain the improvement in humoral immunity reported to occur after an ART-induced decrease in HIV viremia.