Thomas C. Greenough

Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus

Spike (S) proteins of coronaviruses, including the coronavirus that causes severe acute respiratory syndrome (SARS), associate with cellular receptors to mediate infection of their target cells. Here we identify a metallopeptidase, angiotensin-converting enzyme 2 (ACE2), isolated from SARS coronavirus (SARS-CoV)-permissive Vero E6 cells, that efficiently binds the S1 domain of the SARS-CoV S protein. We found that a soluble form of ACE2, but not of the related enzyme ACE1, blocked association of the S1 domain with Vero E6 cells. 293T cells transfected with ACE2, but not those transfected with human immunodeficiency virus-1 receptors, formed multinucleated syncytia with cells expressing S protein. Furthermore, SARS-CoV replicated efficiently on ACE2-transfected but not mock-transfected 293T cells. Finally, anti-ACE2 but not anti-ACE1 antibody blocked viral replication on Vero E6 cells. Together our data indicate that ACE2 is a functional receptor for SARS-CoV.

Persistence of episomal HIV-1 infection intermediates in patients on highly active anti-retroviral therapy

Treatment of HIV-1-infected individuals with a combination of anti-retroviral agents results in sustained suppression of HIV-1 replication, as evidenced by a reduction in plasma viral RNA to levels below the limit of detection of available assays. However, even in patients whose plasma viral RNA levels have been suppressed to below detectable levels for up to 30 months, replication-competent virus can routinely be recovered from patient peripheral blood mononuclear cells and from semen. A reservoir of latently infected cells established early in infection may be involved in the maintenance of viral persistence despite highly active anti-retroviral therapy. However, whether virus replication persists in such patients is unknown. HIV-1 cDNA episomes are labile products of virus infection and indicative of recent infection events. Using episome-specific PCR, we demonstrate here ongoing virus replication in a large percentage of infected individuals on highly active anti-retroviral therapy, despite sustained undetectable levels of plasma viral RNA. The presence of a reservoir of ‘covert’ virus replication in patients on highly active anti-retroviral therapy has important implications for the clinical management of HIV-1-infected individuals and for the development of virus eradication strategies.

Unusual Polymorphisms in Human Immunodeficiency Virus Type 1 Associated with Nonprogressive Infection

ABSTRACT Factors accounting for long-term nonprogression may include infection with an attenuated strain of human immunodeficiency virus type 1 (HIV-1), genetic polymorphisms in the host, and virus-specific immune responses. In this study, we examined eight individuals with nonprogressing or slowly progressing HIV-1 infection, none of whom were homozygous for host-specific polymorphisms (CCR5-Δ32, CCR2-64I, andSDF-1-3′A) which have been associated with slower disease progression. HIV-1 was recovered from seven of the eight, and recovered virus was used for sequencing the full-length HIV-1 genome; full-length HIV-1 genome sequences from the eighth were determined following amplification of viral sequences directly from peripheral blood mononuclear cells (PBMC). Longitudinal studies of one individual with HIV-1 that consistently exhibited a slow/low growth phenotype revealed a single amino acid deletion in a conserved region of the gp41 transmembrane protein that was not seen in any of 131 envelope sequences in the Los Alamos HIV-1 sequence database. Genetic analysis also revealed that five of the eight individuals harbored HIV-1 with unusual 1- or 2-amino-acid deletions in the Gag sequence compared to subgroup B Gag consensus sequences. These deletions in Gag have either never been observed previously or are extremely rare in the database. Three individuals had deletions in Nef, and one had a 4-amino-acid insertion in Vpu. The unusual polymorphisms in Gag, Env, and Nef described here were also found in stored PBMC samples taken 3 to 11 years prior to, or in one case 4 years subsequent to, the time of sampling for the original sequencing. In all, seven of the eight individuals exhibited one or more unusual polymorphisms; a total of 13 unusual polymorphisms were documented in these seven individuals. These polymorphisms may have been present from the time of initial infection or may have appeared in response to immune surveillance or other selective pressures. Our results indicate that unusual, difficult-to-revert polymorphisms in HIV-1 can be found associated with slow progression or nonprogression in a majority of such cases.

Down-modulation of mature major histocompatibility complex class II and up-regulation of invariant chain cell surface expression are well-conserved functions of human and simian immunodeficiency virus nef alleles.

ABSTRACT Recently, it has been demonstrated that the human immunodeficiency virus type 1 (HIV-1) Nef from laboratory strains down-modulates cell surface expression of mature major histocompatibility complex class II (MHC-II) molecules, while up-regulating surface expression of the invariant chain (Ii) associated with immature MHC-II (P. Stumptner-Cuvelette, S. Morchoisne, M. Dugast, S. Le Gall, G. Raposo, O. Schwartz, and P. Benaroch, Proc. Natl. Acad. Sci. USA 98:12144-12149, 2001). These Nef functions could contribute to impaired CD4+-T-helper-cell responses found in HIV-1-infected patients with progressive disease. However, it is currently unknown whether nef alleles derived from HIV-1-infected individuals or from other primate lentiviruses also modulate MHC-II and Ii. In the present study, we demonstrate that both activities are conserved among primary HIV-1 nef alleles, as well as among HIV-2 and simian immunodeficiency virus (SIV) nef alleles. Down-modulation of mature MHC-II required high levels of Nef expression. In contrast, surface expression of Ii was already strongly increased at low to medium levels of Nef expression. Notably, nef genes derived from two of four HIV-1-infected long-term nonprogressors did not up-regulate Ii, whereas nef alleles derived from 10 individuals with progressive disease were active in this assay. Unlike other in vitro Nef functions, the average activity of Nef in modulating MHC-II and Ii surface expression did not change significantly during the course of infection. Mutational analysis confirmed that MHC-II down- and Ii up-regulation are functionally separable from each other and from other Nef functions and identified acidic residues, located at the base of the flexible C-proximal loop of Nef, that are critical for increased Ii expression. Overall, our results suggest that the ability of Nef to interfere with MHC-II antigen presentation might play a role in AIDS pathogenesis.

Misdiagnosis of HIV infection by HIV-1 plasma viral load testing: a case series.

The current standard of care for patients infected with HIV includes plasma viral load tests to monitor the effectiveness of antiretroviral regimens (1, 2). The assays approved for this use detect cell-free plasma viral RNA by using various amplification techniques (2). Access to these sensitive techniques and the trend toward earlier and more aggressive treatment approaches have led to the use of these assays as primary tools for the diagnosis of acute HIV infection (3, 4). Plasma viral load tests for HIV-1 were neither developed nor evaluated for the diagnosis of HIV infection; therefore, their diagnostic specificity is not well delineated when applied to persons who are negative for HIV antibody (5). We report two cases of false-positive results obtained by using branched-chain DNA assay (Chiron Quantiplex, Emeryville, California) and one case of a false-positive result obtained by using HIV reverse transcriptase polymerase chain reaction (RT-PCR) (Roche Amplicor Monitor, Basel, Switzerland) plasma viral load assay. These three cases demonstrate the potential problems of using HIV-1 plasma viral load tests for diagnosis of HIV infection. Case One A previously healthy 12-year-old boy, whose HIV-infected mother is cared for at one of our institutions, presented for evaluation of a positive plasma viral load of 1254 copies/mL determined by using the branched-chain DNA assay (Chiron Quantiplex) for HIV-1 RNA. The patients mother had received a diagnosis of HIV infection around the time of his birth, and the patient had tested negative for HIV-1 by enzyme-linked immunosorbent assay (ELISA) several times in the years after his birth. Although the patient reported no risk factors for HIV infection, he underwent plasma viral load testing after his primary care physician noted a skin lesion that was interpreted as herpes zoster. At our institution, the patient subsequently had a negative result on HIV-1 ELISA, a normal CD4 cell count and CD4:CD8 ratio, and a negative plasma viral load (also determined by using the branched-chain DNA assay). His skin lesion was diagnosed as impetigo, and he remains in excellent health 3 months after his initial presentation. Case Two A previously healthy, pregnant 40-year-old woman presented for an HIV test. Her male sexual partner, with whom she had recently had repeated unprotected vaginal intercourse, had been given a diagnosis of HIV infection 1 week before her office visit. The patient tested negative for HIV-1 antibody on the oral mucosal transudate (OraSure, Epitope, Inc., Beaverton, Oregon) HIV-1 oral specimen collection device but continued to be concerned about her HIV status. One week after her initial presentation, she underwent a plasma viral load test (Chiron Quantiplex) for HIV-1 RNA that yielded a positive value of 1574 copies/mL. The patient was told that she was probably infected with HIV. During the next 3 months, she had a negative result on an HIV-1 ELISA, a normal CD4 cell count and CD4:CD8 ratio, and three HIV-1 plasma viral load tests (all done by using branched-chain DNA assay) that showed an undetectable viral load. When the patient delivered a healthy baby 7 months after her initial presentation, another HIV-1 ELISA yielded negative results. Case Three A 20-year-old healthy woman was referred for further evaluation by her primary care physician when she had a positive result on HIV-1 ELISA and an indeterminate result on a Western blot test. The patients only risk factor was heterosexual intercourse, but she stated that her partner had used condoms consistently during the previous year. During a 4-month period after her indeterminate result on the Western blot test, she had a positive result on ELISA and an indeterminate result on a Western blot test on separate occasions. Five months later, both ELISA and a Western blot test yielded negative results, but the patient had a plasma viral load of 1300 copies/mL (determined by using RT-PCR assay [Roche Amplicor Monitor]). She was subsequently counseled that she was probably infected with HIV. Nearly 6 months after her initial indeterminate HIV test result, she was tested by a third laboratory and was negative for HIV-1 antibodies on both ELISA and Western blot test. She had a normal CD4 cell count and CD4:CD8 ratio and a plasma viral load that was undetectable on RT-PCR assay (Roche Amplicor Monitor). She remains healthy 8 months after her initial presentation. Discussion These three cases, which were observed in one region during a 2-month period, are probably examples of false-positive results on HIV-1 plasma viral load tests. Only one other case of a false-positive HIV-1 plasma viral load has been fully documented; that test had been performed by using RT-PCR, and the result was thought to be related to the administration of an HIV-1 vaccine (6). The patients described here had normal CD4 cell counts and CD4:CD8 ratios, low plasma viral loads, and subsequent negative results on HIV-1 ELISA and plasma viral load tests. To our knowledge, the lowest reported plasma viral load during seroconversion is more than 17 times higher than the highest viral load detected in our three patients (7). Although transient HIV infection has been reported in infants, it is unlikely in two of our patients because they had not recently been exposed to HIV (8, 9). Other potential explanations of false-positive HIV-1 plasma viral load include laboratory error, cross-contamination, and mix-up of specimens. From the patients perspective, false-positive results on an HIV test are potentially devastating, regardless of the cause. Further clinical experience is required to determine whether specific clinical circumstances correlate with an increased incidence of false-positive HIV-1 plasma viral load results. The current standard diagnostic protocol for HIV infection is based on detection of HIV-1-specific antibodies. The combination of screening ELISA followed by a confirmatory Western blot assay has been more than 99% accurate in detecting HIV infection (10, 11). This protocol has a relatively low rate of false-positive results (approximately 0.0006%) but can have negative or indeterminate results during the 3 to 4 weeks before seroconversion (12-14). Although host antibody responses may be undetectable during this acute infection period, the viral load in plasma is usually very high and initial viremia usually occurs in 4 to 11 days (4, 7, 15, 16). The occurrence of high levels of viremia during primary HIV infection has led some physicians to use plasma viral load assays as diagnostic tests to detect early HIV infection. However, plasma viral load assays are designed for monitoring the effectiveness of antiretroviral therapies and for measuring the quantity of virus in patients with confirmed HIV infection, not for the diagnosis of HIV infection. Their performance in patients who are not infected with HIV is unknown (1, 2). The first case illustrates the importance of following the most recent testing protocol for the diagnosis of HIV infection. The patients pediatrician requested a plasma viral load assay because the patient, whose mother has asymptomatic HIV infection, presented with a skin rash thought to be consistent with herpes zoster. In this case, because primary HIV infection was not suspected, an HIV-1 ELISA should have been ordered and, if reactive, followed by a Western blot assay. In the second case, a plasma viral load assay was ordered despite a negative result on an oral mucosal transudate test (OraSure) because the patient was pregnant and was at substantial risk for recent exposure to HIV. However, on the basis of current knowledge about viral replication during primary HIV infection, the patients plasma viral load would probably have been much higher if she had been infected with HIV in the previous 2 weeks (15). In the third case and in other cases described in the literature, plasma viral load testing was used to further analyze an indeterminate result on an HIV-1 Western blot assay (3). To minimize the occurrence of false-positive results, testing protocols for the diagnosis of HIV infection should include tests that complement each other. The HIV-1 ELISA assay, which has excellent diagnostic sensitivity, remains an important, inexpensive screening tool. Because of its high specificity, the HIV-1 Western blot assay is a reliable confirmatory test after reactive ELISA. Only patients who have a high pretest probability of a positive result should be evaluated for primary HIV infection by using plasma viral load testing. Such patients include those who are at high risk for recent exposure to HIV and present with indeterminate or negative results on Western blot tests and especially those with an appropriate accompanying clinical syndrome (4). A patient with a high HIV-1 plasma viral load is most likely in the process of seroconversion; although it is theoretically possible that a patient with an undetectable or low plasma viral load may have recently been infected with HIV, that possibility is much less likely. It is important to consider the pretest likelihood of acute infection when counseling patients with negative results on serologic testing and a low plasma viral load. Physicians should explain that the patient may not be infected with HIV-1 but should take precautions to avoid infecting others until follow-up testing provides a definite result. If HIV-1 plasma viral load testing is used in the diagnosis of primary HIV infection before the development of serum antibodies, low positive plasma viral load results should be interpreted with caution and the patients true disease status should be confirmed with repeated plasma viral load testing and follow-up serologic testing.

Human Immunodeficiency Virus Type 1-Specific Cytotoxic T Lymphocytes (CTL), Virus Load, and CD4 T Cell Loss: Evidence Supporting a Protective Role for CTL In Vivo

The relationships between primary human immunodeficiency virus type 1 (HIV-1) Gag-specific cytotoxic T lymphocyte (CTL) frequency, virus load, and CD4 T cell loss were evaluated in a group of 46 HIV-1-infected persons with hemophilia. Freshly isolated peripheral blood mononuclear cells in limiting dilution assays were used to measure HIV-1 Gag-specific CTL frequencies. Concurrent measurements of virus load and lymphocyte surface markers were obtained. No correlation between Gag-specific CTL frequency and concurrent CD4 cell count was observed. A significant inverse relationship was observed between HIV-1 Gag-specific CTL frequency and provirus load as measured by polymerase chain reaction. Subjects with higher CTL frequencies were found to have more stable CD4 cell counts over time. These results provide additional evidence to support the concept that the predominant role of this virus-specific cellular immune response is to limit viral replication and CD4 cell loss in HIV-1 infection.

Declining CD4 T-Cell Counts in a Person Infected with nef-Deleted HIV-1

To the Editor: We have recently seen declining CD4 T-lymphocyte counts in a man with long-term, nonprogressive infection with only nef-deleted forms of human immunodeficiency virus type 1 (HIV-1). ...

Development and Characterization of a Severe Acute Respiratory Syndrome—Associated Coronavirus—Neutralizing Human Monoclonal Antibody That Provides Effective Immunoprophylaxis in Mice

Abstract Background. Severe acute respiratory syndrome (SARS) remains a significant public health concern after the epidemic in 2003. Human monoclonal antibodies (MAbs) that neutralize SARS-associated coronavirus (SARSCoV) could provide protection for exposed individuals. Methods. Transgenic mice with human immunoglobulin genes were immunized with the recombinant major surface (S) glycoprotein ectodomain of SARS-CoV. Epitopes of 2 neutralizing MAbs derived from these mice were mapped and evaluated in a murine model of SARS-CoV infection. Results. Both MAbs bound to S glycoprotein expressed on transfected cells but differed in their ability to block binding of S glycoprotein to Vero E6 cells. Immunoprecipitation analysis revealed 2 antibody-binding epitopes: one MAb (201) bound within the receptor-binding domain at aa 490–510, and the other MAb (68) bound externally to the domain at aa 130–150. Mice that received 40 mg/kg of either MAb prior to challenge with SARS-CoV were completely protected from virus replication in the lungs, and doses as low as 1.6 mg/kg offered significant protection. Conclusions. Two neutralizing epitopes were defined for MAbs to SARS-CoV S glycoprotein. Antibodies to both epitopes protected mice against SARS-CoV challenge. Clinical trials are planned to test MAb 201, a fully human MAb specific for the epitope within the receptor-binding region.

CD4 Down-Modulation by Human Immunodeficiency Virus Type 1 Nef Correlates with the Efficiency of Viral Replication and with CD4+ T-Cell Depletion in Human Lymphoid Tissue Ex Vivo

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Nef protein is an important virulence factor. Nef has several functions, including down-modulation of CD4 and class I major histocompatibility complex cell surface expression, enhancement of virion infectivity, and stimulation of viral replication in peripheral blood mononuclear cells. Nef also increases HIV-1 replication in human lymphoid tissue (HLT) ex vivo. We analyzed recombinant and primarynef alleles with highly divergent activity in different in vitro assays to clarify which of these Nef activities are functionally linked. Our results demonstrate that Nef activity in CD4 down-regulation correlates significantly with the efficiency of HIV-1 replication and with the severity of CD4+ T-cell depletion in HLT. In conclusion, HIV-1 Nef variants with increased activity in CD4 down-modulation would cause severe depletion of CD4+ T cells in lymphoid tissues and accelerate AIDS progression.

Phase I/II evaluation of nevirapine alone and in combination with zidovudine for infection with human immunodeficiency virus

In these Phase I/II open-label clinical trials, 62 persons with human immunodeficiency virus type 1 (HIV-1) infection and CD4+ cell counts < 400/mm3 received nevirapine at doses of 12.5, 50, and 200 mg/day, alone or in combination with zidovudine, 200 mg q8h. Nevirapine was well tolerated in the doses tested. Mean steady-state trough levels were 0.23, 1.1, and 1.9 micrograms/ml for the 12.5, 50, and 200 mg/day doses, respectively. Early suppression of p24 antigen levels and increase in CD4+ cell count were reversed following rapid emergence of virus less susceptible to nevirapine. Resistant strains were isolated from all participants by 8 weeks. Nevertheless, reduction of p24 antigen levels to < 50% of baseline values persisted for 12 weeks or more in four of seven persons who received 200 mg nevirapine/day in combination with zidovudine: these individuals had been antigenemic on long-term zidovudine therapy. This study demonstrates a direct relationship between drug resistance and effects on surrogate markers in HIV-1 infection.