Susan J. Little

Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections

The viruses HIV-1, Epstein–Barr virus (EBV), cytomegalovirus (CMV) and hepatitis C virus (HCV) are characterized by the establishment of lifelong infection in the human host, where their replication is thought to be tightly controlled by virus-specific CD8+ T cells. Here we present detailed studies of the differentiation phenotype of these cells, which can be separated into three distinct subsets based on expression of the costimulatory receptors CD28 and CD27. Whereas CD8+ T cells specific for HIV, EBV and HCV exhibit similar characteristics during primary infection, there are significant enrichments at different stages of cellular differentiation in the chronic phase of persistent infection according to the viral specificity, which suggests that distinct memory T-cell populations are established in different virus infections. These findings challenge the current definitions of memory and effector subsets in humans, and suggest that ascribing effector and memory functions to subsets with different differentiation phenotypes is no longer appropriate.

Rapid evolution of the neutralizing antibody response to HIV type 1 infection

A recombinant virus assay was used to characterize in detail neutralizing antibody responses directed at circulating autologous HIV in plasma. Examining serial plasma specimens in a matrix format, most patients with primary HIV infection rapidly generated significant neutralizing antibody responses to early (0–39 months) autologous viruses, whereas responses to laboratory and heterologous primary strains were often lower and delayed. Plasma virus continually and rapidly evolved to escape neutralization, indicating that neutralizing antibody exerts a level of selective pressure that has been underappreciated based on earlier, less comprehensive characterizations. These data argue that neutralizing antibody responses account for the extensive variation in the envelope gene that is observed in the early months after primary HIV infection.

Immune Activation and CD8+ T-Cell Differentiation towards Senescence in HIV-1 Infection

Progress in the fight against the HIV/AIDS epidemic is hindered by our failure to elucidate the precise reasons for the onset of immunodeficiency in HIV-1 infection. Increasing evidence suggests that elevated immune activation is associated with poor outcome in HIV-1 pathogenesis. However, the basis of this association remains unclear. Through ex vivo analysis of virus-specific CD8+ T-cells and the use of an in vitro model of naïve CD8+ T-cell priming, we show that the activation level and the differentiation state of T-cells are closely related. Acute HIV-1 infection induces massive activation of CD8+ T-cells, affecting many cell populations, not only those specific for HIV-1, which results in further differentiation of these cells. HIV disease progression correlates with increased proportions of highly differentiated CD8+ T-cells, which exhibit characteristics of replicative senescence and probably indicate a decline in T-cell competence of the infected person. The differentiation of CD8+ and CD4+ T-cells towards a state of replicative senescence is a natural process. It can be driven by excessive levels of immune stimulation. This may be part of the mechanism through which HIV-1-mediated immune activation exhausts the capacity of the immune system.

Evidence for increased T cell turnover and decreased thymic output in HIV infection.

The effects of HIV infection upon the thymus and peripheral T cell turnover have been implicated in the pathogenesis of AIDS. In this study, we investigated whether decreased thymic output, increased T cell proliferation, or both can occur in HIV infection. We measured peripheral blood levels of TCR rearrangement excision circles (TREC) and parameters of cell proliferation, including Ki67 expression and ex vivo bromodeoxyuridine incorporation in 22 individuals with early untreated HIV disease and in 15 HIV-infected individuals undergoing temporary interruption of therapy. We found an inverse association between increased T cell proliferation with rapid viral recrudescence and a decrease in TREC levels. However, during early HIV infection, we found that CD45RO−CD27high (naive) CD4+ T cell proliferation did not increase, despite a loss of TREC within naive CD4+ T cells. A possible explanation for this is that decreased thymic output occurs in HIV-infected humans. This suggests that the loss of TREC during HIV infection can arise from a combination of increased T cell proliferation and decreased thymic output, and that both mechanisms can contribute to the perturbations in T cell homeostasis that underlie the pathogenesis of AIDS.

Effect of Treatment, during Primary Infection, on Establishment and Clearance of Cellular Reservoirs of HIV-1

Patients in whom virologic suppression is achieved with highly active antiretroviral therapy (HAART) retain long-lived cellular reservoirs of human immunodeficiency virus type 1 (HIV-1); this retention is an obstacle to sustained control of infection. To assess the impact that initiating treatment during primary HIV-1 infection has on this cell population, we analyzed the decay kinetics of HIV-1 DNA and of infectivity associated with cells activated ex vivo in 27 patients who initiated therapy before or <6 months after seroconversion and in whom viremia was suppressed to <50 copies/mL. The clearance rates of cellular reservoirs could not be distinguished by these techniques (median half-life, 20 weeks) during the first year of HAART. The clearance of HIV-1 DNA slowed significantly during the subsequent 3 years of treatment (median half-life, 70 weeks), consistent with heterogeneous cellular reservoirs being present. Total cell-associated infectivity (CAI) after 1 year of treatment was undetectable (<0.07 infectious units/million cells [IUPM]) in most patients initiating treatment during primary infection either before (9/9) or <6 months after (6/8) seroconversion. In contrast, all 17 control patients who initiated HAART during chronic infection retained detectable CAI after 3-6 years of treatment (median reservoir size, 1.1 IUPM; P<.0005). These results suggest that treatment <6 months after seroconversion may facilitate long-term control of cellular reservoirs that maintain HIV-1 infection during treatment.

Enhanced CD4+ T-Cell Recovery with Earlier HIV-1 Antiretroviral Therapy

BACKGROUND The relationship between the timing of the initiation of antiretroviral therapy (ART) after infection with human immunodeficiency virus type 1 (HIV-1) and the recovery of CD4+ T-cell counts is unknown. METHODS In a prospective, observational cohort of persons with acute or early HIV-1 infection, we determined the trajectory of CD4+ counts over a 48-month period in partially overlapping study sets: study set 1 included 384 participants during the time window in which they were not receiving ART and study set 2 included 213 participants who received ART soon after study entry or sometime thereafter and had a suppressed plasma HIV viral load. We investigated the likelihood and rate of CD4+ T-cell recovery to 900 or more cells per cubic millimeter within 48 months while the participants were receiving viral-load-suppressive ART. RESULTS Among the participants who were not receiving ART, CD4+ counts increased spontaneously, soon after HIV-1 infection, from the level at study entry (median, 495 cells per cubic millimeter; interquartile range, 383 to 622), reached a peak value (median, 763 cells per cubic millimeter; interquartile range, 573 to 987) within approximately 4 months after the estimated date of infection, and declined progressively thereafter. Recovery of CD4+ counts to 900 or more cells per cubic millimeter was seen in approximately 64% of the participants who initiated ART earlier (≤4 months after the estimated date of HIV infection) as compared with approximately 34% of participants who initiated ART later (>4 months) (P<0.001). After adjustment for whether ART was initiated when the CD4+ count was 500 or more cells per cubic millimeter or less than 500 cells per cubic millimeter, the likelihood that the count would increase to 900 or more cells per cubic millimeter was lower by 65% (odds ratio, 0.35), and the rate of recovery was slower by 56% (rate ratio, 0.44), if ART was initiated later rather than earlier. There was no association between the plasma HIV RNA level at the time of initiation of ART and CD4+ T-cell recovery. CONCLUSIONS A transient, spontaneous restoration of CD4+ T-cell counts occurs in the 4-month time window after HIV-1 infection. Initiation of ART during this period is associated with an enhanced likelihood of recovery of CD4+ counts. (Funded by the National Institute of Allergy and Infectious Diseases and others.).

Diagnosis of Primary HIV-1 Infection

Primary HIV infection is characterized by diverse clinical symptoms (1, 2). Since patients with primary infection are just developing HIV antibodies, recognition of the syndrome in at-risk persons should prompt antibody testing as well as a virologic assay (3, 4). Diagnosing primary infection may decrease HIV transmission (5) and allow consideration of early treatment (2, 6). To date, the optimal patients to screen and the best algorithm for use of diagnostic tests have not been determined. The objectives of this study were to determine the sensitivity and specificity of virologic tests and specific clinical symptoms for diagnosing primary HIV infection. Methods Patients Patients with potential exposure to HIV and compatible symptoms were referred from clinics, testing centers, emergency departments, and community physicians for primary infection screening at Cedars-Sinai Medical Center in Los Angeles, California, and the University of California, San Diego. In Los Angeles, 127 patients were screened between March 1993 and August 1995 (cohort 1) for enrollment in a placebo-controlled trial of zidovudine (7). A similar group of 255 patients was screened in Los Angeles between June 1996 and July 1999 (cohort 2), and 54 patients were screened between June 1996 and March 1999 in San Diego (cohort 3). Patients in cohort 2 received a standardized questionnaire regarding results of past HIV tests, presence of specific symptoms, and potential exposure to HIV during the preceding 2 months. Virologic and Serologic Assays The study was approved by local institutional review boards. All patients provided informed consent and received pre- and post-test counseling. Patients in cohorts 1 and 2 had real-time testing for HIV antibodies and p24 antigen by a polyclonal enzyme immunoassay (Abbott Laboratories, Abbott Park, Illinois); plasma was stored at 70 C. In cohort 2, real-time plasma HIV RNA measurements were performed by branched-chain DNA (bDNA) assay, version 2.0 (lower limit of detection, 500 copies/mL), between June 1996 and July 1998 and by version 3.0 (lower limit of detection, 50 copies/mL) between August 1998 and July 1999 (Chiron Diagnostics, Emeryville, California). Stored samples from cohort 1 were retrospectively tested by bDNA assay, version 3.0. After April 1998, p24 antigen was measured by using the monoclonal HIV AG-1 enzyme immunoassay (Abbott Laboratories), which replaced the previous assay. All samples with detectable p24 antigen were confirmed by neutralization (Abbott Laboratories). Samples positive for HIV antibody were confirmed by Western blot (Cambridge Biotech Corp., Rockville, Maryland). Samples that yielded indeterminate Western blots (<2 envelope bands, core bands, or both) were tested again in approximately 1 month to document seroconversion. Patients in cohort 3 were screened in San Diego by using HIV antibody enzyme immunoassay (Abbott Laboratories). Plasma HIV RNA level was determined by using Amplicor HIV Monitor (Roche Diagnostic Systems, Indianapolis, Indiana), which had a lower limit of detection of 400 copies/mL. Samples that were positive for HIV RNA but negative for HIV antibody were tested for p24 antigen (Beckman Coulter, Fullerton, California). Statistical Analysis Primary infection was defined as a confirmed positive virologic test result with either a negative HIV antibody assay result or an indeterminate Western blot. Because there is no virologic gold standard, we assumed that levels of plasma HIV RNA had a sensitivity of 100% for diagnosing primary infection. False-positive HIV RNA measurements were defined as those that were negative on repeated testing and those obtained in patients who did not undergo seroconversion. In cohort 1, frozen plasma samples that were negative for HIV antibody and p24 antigen were retrospectively tested for HIV RNA. Follow-up was not available for these patients; therefore, before testing any samples, we determined that an HIV RNA level greater than 10 000 copies/mL would be considered a true-positive result. This HIV RNA level, in our experience, has not been seen in false-positive samples. Sensitivity and specificity, along with corresponding 95% confidence intervals, were determined by using tabulated exact binomial limits for sample sizes less than or equal to 100 (8) and by using the normal approximation to the binomial for sample sizes greater than 100. Patients in cohort 2 were analyzed for predictors of primary infection on the basis of uniformly collected demographic characteristics, exposure history, and symptoms. All variables were compared by using a two-sample t-test. To assess the impact of several predictor variables, a stepwise discriminant analysis was performed by using variables that differed significantly between patients with and those without primary infection (significance was indicated by a Pvalue<0.05). Role of the Funding Sources The funding sources had no role in the collection, analysis, or interpretation of the data or in the decision to submit the paper for publication. Results Demographic characteristics were similar across cohorts. Overall, 89% of patients were male, 74% were white, 13% were Hispanic, and 9% were African American. Regarding risk factors, 77% of patients were homosexual men, 18% were heterosexual women, and 4% used intravenous drugs. Patients were categorized as having 1) primary infection with undetectable HIV antibodies or an indeterminate Western blot [12.4%], 2) chronic infection with a positive Western blot [18.1%], or 3) no infection (69.5%). Sensitivity and Specificity of Virologic Assays The results of screening tests are summarized in Table 1. Patients with primary infection had undetectable HIV antibodies or indeterminate Western blot but a confirmed positive virologic test result. Two patients in cohort 1 were negative for HIV antibody and p24 antigen but positive for HIV RNA, with levels greater than 100 000 copies/mL. For the purpose of this analysis, they were considered to be true positive for primary HIV infection. Overall, p24 antigen testing had a sensitivity of 88.7% (95% CI, 77.0% to 95.7%). The mean concentration of HIV RNA in the five patients who had primary infection but undetectable p24 antigen was 251 189 copies/mL (range, 100 000 to 630 957 copies/mL). The overall specificity of tests for HIV RNA and p24 antigen was 97.4% (CI, 94.9% to 98.9%) and 100% (CI, 99.3% to 100%), respectively. Eight of 303 uninfected patients (2.6%) had false-positive results on HIV RNA testing (mean concentration, 269 copies/mL [range, 52 to 1950 copies/mL]). Table 1. Serologic and Virologic Results in Patients Screened for Primary HIV Infection Predictors of Primary HIV Infection Cohort 2 was divided into patients with primary infection and those who were uninfected or had chronic HIV infection (Table 2). The primary infection group involved 25 patients who had a negative antibody test result or indeterminate Western blot and 15 patients who had had a negative antibody test result in the preceding 3 months. Of these 15, 4 had had a negative Western blot in the previous 3 weeks and 11 had had a documented negative antibody test result or evolving Western blot in the previous 3 months. Those with primary infection were more likely to be homosexual; to have been exposed to an HIV-infected person; and to report fever, myalgia, arthralgia, rash, or night sweats (P<0.05 for all comparisons) (Table 2). Combining fever, myalgia, and rash increased the predictive value of symptoms; however, no combination of symptoms identified more than 75% of patients with primary infection. Table 2. Clinical Predictors of Primary HIV Infection in Cohort 2 Discussion Our study shows that no clinical symptoms have sufficient sensitivity or specificity for primary infection to allow targeted screening of at-risk persons. In addition, we show that while both p24 antigen and plasma HIV RNA assays are useful virologic tests for diagnosing primary infection, each has limitations. Assays for HIV RNA are likely to be more sensitive but are associated with lower specificity and can therefore yield more false-positive results. Similar to our study, a study in India that screened patients in sexually transmitted disease clinics showed that certain symptoms occur with increased frequency in patients with primary HIV infection (9). Nevertheless, both this study and our study demonstrate that selective screening would miss a substantial number of patients with primary infection. Thus, to maximize the number of identified infected patients, many seronegative persons must be screened. Several groups have reported that assays for HIV RNA are more sensitive than those for p24 antigen in diagnosing primary infection. However, these studies often screened asymptomatic patients, such as those found to experience seroconversion during cohort studies (10) and those with indeterminate Western blots (11). This makes the findings less relevant for the prospective screening of symptomatic patients. Similar to our study, other studies have shown that the assay for p24 antigen is sensitive in symptomatic antibody-negative patients, in whom virologic testing is necessary for diagnosis (4, 7, 12). Overall, these studies and our own emphasize the importance of analyzing the sensitivity and specificity of virologic assays in the context of the patients clinical and serologic status. When selecting a virologic test, it is important to consider its cost in clinical practice. In our study, it cost approximately

Persistence of Transmitted Drug Resistance among Subjects with Primary Human Immunodeficiency Virus Infection

6000 (

Human Immunodeficiency Virus Type 1-Specific CD8+ T-Cell Responses during Primary Infection Are Major Determinants of the Viral Set Point and Loss of CD4+ T Cells

20 per test) to screen all seronegative patients in cohort 2 for p24 antigen. To identify a single patient missed by p24 antigen testing, we were required to perform HIV RNA assays on more than 100 patients at

Transmission Fitness of Drug-Resistant Human Immunodeficiency Virus and the Prevalence of Resistance in the Antiretroviral-Treated Population

100 per test, an additional cost of more than