Longitudinal analysis of T cell receptor (TCR) gene usage by human immunodeficiency virus 1 envelope-specific cytotoxic T lymphocyte clones reveals a limited TCR repertoire.

Human immunodeficiency virus 1 (HIV-1) infection is associated with a vigorous cellular immune response that allows detection of cytotoxic T lymphocyte (CTL) activity using freshly isolated peripheral blood mononuclear cells (PBMC). Although restricting class I antigens and epitopes recognized by HIV-1-specific CTL have been defined, the effector cells mediating this vigorous response have been characterized less well. Specifically, no studies have addressed the breadth and duration of response to a defined epitope. In the present study, a longitudinal analysis of T cell receptor (TCR) gene usage by CTL clones was performed in a seropositive person using TCR gene sequences as a means of tracking responses to a well-defined epitope in the glycoprotein 41 transmembrane protein. 10 CTL clones specific for this human histocompatibility leukocyte antigen-B14-restricted epitope were isolated at multiple time points over a 31-mo period. All clones were derived from a single asymptomatic HIV-1-infected individual with a vigorous response to this epitope that was detectable using unstimulated PBMC. Polymerase chain reaction amplification using V alpha and V beta family-specific primers was performed on each clone, followed by DNA sequencing of the V-D-J regions. All 10 clones utilized V alpha 14 and V beta 4 genes. Sequence analysis of the TCR revealed the first nine clones isolated to also be identical at the nucleotide level. The TCR-alpha junctional region sequence of the tenth clone was identical to the junctional region sequences of the other nine, but this clone utilized distinct D beta and J beta gene segments. This study provides evidence that the observed high degree of HIV-1-specific CTL activity may be due to monoclonal or oligoclonal expansion of specific effector cells, and that progeny of a particular CTL clone may persist for prolonged periods in vivo in the presence of a chronic productive viral infection. The observed limited TCR diversity against an immunodominant epitope may limit recognition of virus variants with mutations in regions interacting with the TCR, thereby facilitating immune escape.

Multiple sites in HIV-1 reverse transcriptase associated with virological response to combination therapy.

OBJECTIVE: To determine whether analysis of sequence variation in reverse transcriptase at baseline can explain differences in response to combination antiretroviral therapy. METHODS: Amino acid sequences of reverse transcriptase obtained from baseline isolates from 55 patients included in a trial of zidovudine and didanosine versus zidovudine/didanosine/nevirapine (ACTG241) were analysed. Simple and multiple linear regression were used to determine the relationship between numbers and identity of mutations at baseline and virological response after 8 and 48 weeks. RESULTS: Numbers of baseline zidovudine resistance mutations were predictive of short-term response (week 8). Amino acid identity at position 215 explained > 20% of the variation in response at week 8, but less at week 48. Multiple regression identified the combinations: 215 + 44 and 41 + 202, each of which explained about 30% of the variation in week 8 response. A model incorporating amino acids 214 + 215 + 60 + 202 + baseline viral load explained > 40% of the variation in response at week 48. Unexpectedly, the mutant combination 601 + 215Y/F responded threefold better than 60V + 215Y/F over 48 weeks. CONCLUSIONS: Use of clinical data to analyse virological response to combination therapy has revealed effects of baseline amino acid mutations at sites not previously identified as being important in antiretroviral resistance. Predictors of long-term responses were different from those involved in the short term and may require more complex analysis.

Human immunodeficiency virus type 1 hypersusceptibility to amprenavir in vitro can be associated with virus load response to treatment in vivo.

The human immunodeficiency virus type 1 protease mutation N88S, which is occasionally selected by treatment with nelfinavir or indinavir, confers hypersusceptibility to amprenavir in vitro. The clinical relevance of this observation is unclear. We report a case of N88S developing after virologic failure of both indinavir- and nelfinavir-containing regimens that was managed successfully with a regimen that contained amprenavir.

HIV-1 reverse transcriptase/ribonuclease H: high level expression in Escherichia coli from a plasmid constructed using the polymerase chain reaction.

The human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT)/ribonuclease H has been expressed to high levels in Escherichia coli from a recombinant plasmid constructed using the polymerase chain reaction (PCR) for in vitro mutagenesis. Translational initiation and termination codons were introduced by the PCR at points corresponding to sites of cleavage of the RT from the gag-pol precursor polyprotein by the HIV-1 protease; the HIV-1 protease is not expressed from this construct. Most of the RT coding sequences derived from PCR were exchanged for a DNA fragment cloned by standard methods to minimize the possibility that an unwanted mutation was introduced during the in vitro amplification. The RT is expressed in bacteria from this plasmid as 66 and 51 kDa proteins, has both RNA-dependent DNA polymerase and ribonuclease H (RNase H) activities, and is indistinguishable from native HIV-1 RT in electrophoretic mobility and immunoreactivity. Peptide sequencing of the amino terminus of the HIV-1 RT purified from bacterial lysates is also presented. A novel activity gel assay was used to confirm that only the 66 kd protein catalyzes the RNase H reaction; this assay will simplify analysis of this catalytic activity. This HIV-1 RT expression plasmid is of interest because of the high level of expression in bacteria and the demonstrated RNase H activity of the enzyme. This plasmid will be distributed for research purposes through the NIH AIDS Repository and will facilitate enzymologic, structural, and immunologic evaluation of reverse transcription and its chemotherapeutic inhibition.

Race/ethnicity as a risk factor for HIV-1 infection among Connecticut intravenous drug users.

This cross-sectional study of 341 entrants to drug abuse treatment in four Connecticut cities in 1986-1987 evaluated whether demographic, behavioral, viral serologic, or economic differences explained the disproportionate risk of human immunodeficiency virus type 1 (HIV-1) infection among black and Hispanic intravenous drug users (IVDUs), relative to non-Hispanic white IVDUs. Blacks [odds ratio (OR) = 9.0, 95% confidence interval (CI) = 5.1-15.9] and Hispanics (OR = 4.1, 95% CI = 1.9-8.8) were at increased risk of HIV-1 infection, relative to non-Hispanic whites. Those who lived closer to New York City, injected drugs more frequently, used intravenous drugs for a longer duration, used shooting galleries, had greater numbers of sexual partners, had human cytomegalovirus (CMV) or hepatitis B virus (HBV) antibodies, and had the lowest annual incomes were also at increased risk. However, none of these other factors accounted for the black and Hispanic HIV-1 risk in stratified analysis. Black race, Hispanic ethnicity, proximity to New York City, and number of drug injections in the past year each also remained significant, independent risk factors in a multivariate analysis. The increased HIV-1 risk of nonwhite IVDUs remained unexplained. Behavioral, sociologic, and/or biologic factors not identified in this study may modulate HIV-1 transmission dynamics in IVDUs.

Limits of resistance testing.

Interpretation of antiretroviral testing is complicated by many factors, including those related to HIV biology and genetics, as well as the intricacies of drug selection pressure in vivo. These complex factors can limit the usefulness of resistance testing. However, knowledge about these issues can help to avoid misinterpreting resistance test results and thereby help clinicians to use resistance testing to individualize antiretroviral drug choices.

Management of antiretroviral therapy for HIV infection: modelling when to change therapy.

OBJECTIVE: To evaluate four strategies for monitoring plasma HIV RNA levels and/or resistance genotypes to decide when to change antiretroviral therapy. The strategies include: (i) 1997 guidelines recommending a therapy switch when plasma RNA exceeds a threshold level; (ii) a viral load policy, using a fixed increase in viral load as the trigger; (iii) a genotype policy, requiring a smaller viral rebound than (ii) and detection of genotypic resistance before switching; and (iv) a proactive policy, switching drug regimens at a predetermined time if viral load has not rebounded. DESIGN AND SETTING: A Monte Carlo simulation tracks patients' viral loads and presence of opportunistic infection during therapy. The model uses clinical and virological data and statistical variation in patient parameters for the evaluation of therapeutic strategies. MAIN OUTCOME MEASURES: To determine which strategies minimize viral rebound detection delay while maintaining a low (prespecified) probability of switching therapy before rebound. RESULTS: 1997 Guidelines and the viral load policy create lengthy delays in detection of rebound, particularly when patients are drug-naive and the detection limit of the viral load assay is 500 copies/ml. A detection limit of 20 copies/ml decreases this delay substantially. Genotyping achieves only minor additional delay reductions. Of the strategies tested, the proactive policy leads to the shortest delays. CONCLUSIONS: This model indicates that prolonged periods may be required for viral load to rebound to detectable levels following prolonged suppression. Proactive switching produces the best outcome in our model because it may reduce the duration of viral replication under pressure of a failing regimen before detection of viral rebound. This strategy should be evaluated in clinical trials.

Comparative analysis of HIV type 1 genotypic resistance across antiretroviral trial treatment regimens.

From data on HIV-1 genotypes collected from antiretroviral trial participants who fail virologically, we describe methods for comparing distributions of acquired HIV-1 mutations across different treatment regimens. Given a definition of a "mutational distance" that summarizes the genetic change of a subject's virus in a way that captures the resistance cost of exposure to an antiretroviral regimen, these comparative analyses inform about the relative treatability of emergent virus by next-line therapy directed to the same viral target. The utility of the methods is illustrated by application to data from AIDS Clinical Trials Group (ACTG) Study 241. We find that patients failing zidovudine/didanosine/nevirapine accumulated a 2.41-fold greater nonnucleoside reverse transcriptase inhibitor (RTI) mutational distance than patients failing zidovudine/didanosine [95% confidence interval (1.55, 5.26), p < 0.000001], quantitating expectations that adding a nonnucleoside RTI to a double nucleoside regimen may attenuate future effectiveness of nonnucleoside RTI therapy for nucleoside-experienced patients if viremia is not suppressed. We also find that persons with extensive prior experience with suboptimal nucleoside therapy who were virologically failing zidovudine/didanosine/nevirapine or zidovudine/didanosine accumulated a similar nucleoside RTI mutational distance, implying that the addition of the nonnucleoside RTI did not preserve future nucleoside options.

Interactions between HIV-1 and cytomegalovirus in human osteosarcoma cells carrying both viruses.

Cytomegalovirus (CMV) and the human immunodeficiency virus type 1 (HIV-1) may interact in the pathogenesis of AIDS. We compared CMV replication in human osteosarcoma (HOS) cells to that in HOS cells genetically engineered to contain an envelope-deficient HIV-1 proviral construct (designated HOS-HXG). Following acute CMV infection of each cell line, HOS-HXG cells contained higher numbers of intranuclear CMV nucleocapsids than did HOS cells. Infectious CMV could be persistently detected in culture supernatant fluids of the CMV-infected HOS-HXG cells, whereas CMV was lost over several weeks from HOS cells infected with CMV in parallel. HIV-1 CMV pseudotypes were not detected in supernatant fluids from CMV-infected HOS-HXG cells. On day 119 after CMV infection, these cultures were superinfected with HIV-1. These dually infected HOS-HXG cells produced infectious HIV-1 and exhibited markedly enhanced CMV replication compared to parental CMV-infected HOS-HXG cells. Two different HIV-1 tat gene function antagonists, Ro24-7429 and chemically modified antibodies to the Tat protein, did not inhibit the replication of CMV in either acute or persistent infections of HOS-HXG cells at concentrations that inhibited HIV-1 replication.

Clinical monitoring of HIV-1 infection in the ERA of antiretroviral resistance testing.

Viral replication of HIV-1 in the human body is a dynamic process. Incomplete suppression of replication during antiretroviral therapy ultimately selects for resistance that imparts an adaptive advantage to HIV-1. Therefore, the goal of antiretroviral therapy is complete suppression of viral replication. Viral suppression to below the lowest possible limits of detection has been associated with an optimal clinical response and delay of drug resistance. An ultrasensitive viral load assay with a very low threshold of detection remains our best laboratory tool to monitor the response to therapy. Patients may fail HAART for many reasons. Only when other potential causes of treatment failure are excluded should antiretroviral resistance testing be considered. Genotypic and phenotypic assays for assessing resistance are now available, and recent retrospective and prospective data support their use in clinical management as an adjunct to helping to choose among different antiretroviral drugs. Despite the growing enthusiasm for these tests, improvements in sensitivity, turnaround time, and quality control are still needed. A practitioner's decision about when to initiate or change therapy in an HIV-infected patient should depend primarily on viral load results, and not on antiretroviral resistance test results. Moreover, resistance testing is no substitute for a thorough clinical and drug history. As we approach the third decade of the HIV epidemic, we will learn how to use antiretroviral resistance tests in conjunction with (not in lieu of) proven clinical and laboratory tools.

HIV-1 drug resistance. Molecular pathogenesis and laboratory monitoring.

Molecular and clinical aspects of HIV-1 pathogenesis are described, including the turnover of the HIV-1 population in vivo, the clinical significance of resistance to HIV-1 reverse transcriptase (RT) inhibitors, and the inter-relationship between virus replication and RT inhibitor resistance. The molecular genetics of RT inhibitor resistance, including interactive effects of different RT mutations and the implications of those effects for combination chemotherapy, are summarized. Structural studies of RT and the biochemical bases of drug resistance are discussed.

HIV-1 chemotherapy and drug resistance.

BACKGROUND: Therapy with inhibitors of human immunodeficiency virus type 1 (HIV-1) replication has had only transient clinical benefit to date. It has been speculated that drug-resistant virus mutants may contribute to therapeutic failure. OBJECTIVE: To extrapolate from the biology of drug-resistant HIV-1 to improve antiretroviral chemotherapeutic strategies. STUDY DESIGN: The literature was reviewed in regard to clinical and virologic correlates of HIV-1 drug resistance, methodology for detection of resistant virus, and chemotherapeutic strategies for prolonging suppression of virus replication. RESULTS: HIV-1 isolates resistant to different nucleoside reverse transcriptase (RT) inhibitors, nonnucleoside RT inhibitors, and protease inhibitors have been implicated to different extents with virologic or clinical failure of therapeutic effectiveness. The in vivo antiviral effect of certain nonnucleoside RT inhibitors as monotherapy is lost coincident with emergence of a dominant population of resistant virus. Disease progression is more rapid among patients at advanced stages of HIV-1 disease with highly zidovudine (AZT)-resistant virus (50% inhibitory concentration 1.0 muM AZT) and is not attributable to effects of other baseline predictors of progression studied to date. However, there is no definitive evidence that high level AZT resistance causes the loss of therapeutic benefit of AZT. Several of the research methods used for detection of drug-resistant mutants could be developed for future use as screening assays in clinical virology laboratories. CONCLUSIONS: Strategies for individualizing therapy based on switching, or adding, drugs at first detection of drug-resistant HIV-1 aim to minimize replication of viruses that are drug-resistant, as well as those that remain susceptible to the drug. If clinical investigation reveals that such approaches extend duration of antiretroviral therapeutic benefit, HIV-1 drug-resistance assessment may be increasingly requested from clinical virology laboratories. Monitoring antiviral suppression by quantifying plasma HIV-1 RNA appears more practical at present, however. Different combination regimens are also being studied to see if some regimens delay emergence of resistant virus longer than others. In the future, sequential empiric changes to new combination regimens every few months may also bear investigation to attempt to "pre-empt" HIV-1 resistance development.

Epidemic human immunodeficiency virus (HIV) infection among intravenous drug users (IVDU).

Human immunodeficiency virus (HIV) infection is epidemic among intravenous drug users (IVDU), particularly in the northeastern United States. IVDU are playing a critical role in the spread of HIV by infecting their heterosexual partners and children, as well as their needle-sharing partners. The epidemiology of HIV infection among IVDU is reviewed here, including a compilation of seroprevalence data. Relevant determinants of the future spread of HIV among IVDU are discussed, including the major risk factors for HIV seropositivity, the modes of HIV transmission, and aspects of the natural history of HIV infection in IVDU. The public health policy implications of these issues include the need for education of adolescents and the general public about the risks of drug injection and heterosexual intercourse with IVDU, as well as motivation of IVDU to stop injecting, never share injection paraphernalia, or, at least, clean needles effectively.

Isolation and characterization of phosphonoacetic acid-resistant mutants of human cytomegalovirus.

Mutants of the human cytomegalovirus (HCMV) that were 6- to 13-fold more resistant to phosphonoacetic acid than the wild-type HCMV (Towne) were isolated. Extracts from mycoplasma-free, mutant-infected cells had phosphonoacetate-resistant DNA polymerase activity in vitro. This strongly suggests that the selected mutations are in the HCMV DNA polymerase genes of these viruses.

Clinical use of genotypic and phenotypic drug resistance testing to monitor antiretroviral chemotherapy.

Assays that detect antiretroviral drug resistance in human immunodeficiency virus have recently become available to clinicians. Phenotypic assays measure the drug susceptibility of the virus by determining the concentration of drug that inhibits viral replication in tissue culture. Genotypic assays determine the presence of mutations that are known to confer decreased drug susceptibility. Although each type of assay has specific advantages, limitations associated with these tests often complicate the interpretation of results. Several retrospective clinical trials have suggested that resistance testing may be useful in the assessment of the success of salvage antiretroviral therapy. Prospective, controlled trials have demonstrated that resistance testing improves short-term virological response. Resistance testing is currently recommended to help guide the choice of new drugs for patients after treatment has failed and for pregnant women. Resistance testing should also be considered for treatment-naïve patients, to detect transmission of resistant virus.

Fitness of human immunodeficiency virus type 1 protease inhibitor-selected single mutants.

Human immunodeficiency virus type 1 (HIV-1) evolution under chemotherapeutic selection pressure in vivo involves a complex interplay between an increasing magnitude of drug resistance and changes in viral replicative capacity. To examine the replicative fitness of HIV-1 mutants with single, drug-selected substitutions in protease (PR), we constructed virus that contained the most common mutations in indinavir-selected clinical isolates, PR M46I and V82T, and the most common polymorphic change in drug-naïve patients, PR L63P. These mutants were competed in vitro in the absence of drug against the otherwise isogenic WT virus (NL4-3). Phenotypic drug susceptibility was determined with a recombinant virus assay using a single cycle of virus growth. PR M46I and L63P were as fit as WT. However, PR V82T was out-competed by WT. None of these mutants had appreciable phenotypic resistance to any of the protease inhibitors, including indinavir. The PRV82T mutant was hypersusceptible to saquinavir. Thus, the impaired fitness of the V82T single mutant is consistent with its low frequency in protease inhibitor-naïve patients. The similar fitness of WT (NL4-3), L63P, and M46I is consistent with the common occurrence of L63P in the absence of protease inhibitor-selection pressure, but not with the rare detection of M46I in drug-naïve patients.

Susceptibility testing by polymerase chain reaction DNA quantitation: a method to measure drug resistance of human immunodeficiency virus type 1 isolates.

Polymerase chain reaction (PCR) DNA quantitation (PDQ) susceptibility testing rapidly and directly measures nucleoside sensitivity of human immunodeficiency virus type 1 (HIV-1) isolates. PCR is used to quantitate the amount of HIV-1 DNA synthesized after in vitro infection of peripheral blood mononuclear cells. The relative amounts of HIV-1 DNA in cell lysates from cultures maintained at different drug concentrations reflect drug inhibition of virus replication. The results of PDQ susceptibility testing of 2- or 3-day cultures are supported by assays measuring HIV-1 p24 antigen production in supernatants of 7- or 10-day cultures. DNA sequence analyses to identify mutations in the reverse transcriptase gene that cause resistance to 3'-azido-3'-deoxythymidine also support the PDQ results. With the PDQ method, both infectivity titration and susceptibility testing can be performed on supernatants from primary cultures of peripheral blood mononuclear cells. PDQ susceptibility testing should facilitate epidemiologic studies of the clinical significance of drug-resistant HIV-1 isolates.

Physical mapping of the human cytomegalovirus (HCMV) (Towne) DNA polymerase gene: DNA-mediated transfer of a genetic marker for an HCMV gene.

DNA-mediated transfer of a drug resistance marker (phosphonoacetate resistance) for the HCMV (Towne) DNA polymerase (pol) gene has been used to genetically confirm the physical localization of the HCMV (Towne) DNA pol gene. The HCMV (Towne) genomic region homologous to the pol genes of other herpesviruses was first identified by moderate stringency Southern hybridization. Restriction fragments from this region were molecularly cloned from previously characterized phosphonoacetic acid resistant (PAAr) HCMV genomes (R. T. D'Aquilla and W. C. Summers, J. Virol., 61, 1291-1295, 1987). A high frequency of recombinant PAAr virus was found among the progeny of cotransfections of infectious, wild-type HCMV (Towne) DNA only with pol-homologous restriction fragments cloned from PAAr HCMV. The co-transfection technique described here may facilitate further gene mapping in HCMV. The results presented here provide functional proof that the HCMV pol gene is encoded by the sequences previously identified as homologous to other herpesvirus pool genes.