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Physician-scientists played many important roles in controlling human immunodeficiency virus infection (HIV). They worked in academia, government, and industry, and their research was critical in the following areas: discovering the first active drugs; identifying the virologic, immunologic, and clinical perimeters of efficacy; and conceiving of and executing the subsequent clinical trials that led to the multiple drug regimens that we now have to treat the infection and its complications in all the populations now involved. It is now true that over half of the infected people in the world are under treatment. Because the overall number of physician-scientists is decreasing, we must consider the possibility that our ability to deal with future challenges to human health issues such as HIV may be compromised.

Fourteen chimpanzees were inoculated with pre- and posttreatment sera from seven patients with persistent hepatitis B virus infection and chronic hepatitis who had permanent responses of their infection to treatment with interferon and/or adenine arabinoside. Inoculation of pretreatment serum at a dilution of 10(-8) from a patient with a Type I response to treatment [disappearance of Dane particle DNA polymerase (DNAP) activity, HBeAg, and HBsAg from serum] resulted in infection, while undiluted posttreatment serum (all markers negative) failed to infect another animal. Pretreatment sera (DNAP, HBeAg, and HBsAg positive) from all six patients with a Type II response to treatment (disappearance of DNAP activity and HBeAg but not HBsAg from serum) led to infection in six chimpanzees after inoculation of serum dilutions varying between 10(-2) and 10(-7). Inoculation of undiluted posttreatment sera (HBsAg positive and DNAP and HBeAg negative) from the same six patients produced no evidence of hepatitis B virus infection in another six animals. These results indicate that a Type I or II response to treatment with these antiviral agents reduces the infectivity in the serum of patients with chronic hepatitis B to below the level of detection by this assay. Such changes should be useful in interrupting spread of the infection between individuals. Our findings suggest that the serum of some patients who, without treatment are HBsAg positive and DNAP and HBeAg negative, may also be free of detectable infectious hepatitis B virus.

For the HIV-1 reverse transcriptase inhibitor efavirenz, variant drug transporter gene ABCB1 may predict virologic response but not plasma efavirenz exposure. Conversely, variant drug metabolizing enzyme gene CYP2B6 predicts greater plasma efavirenz exposure but not virologic response. We examined whether long-term responses to efavirenz, and/or plasma efavirenz exposure, are better predicted by multilocus genetic interactions than by individual polymorphisms.We studied antiretroviral-naïve study participants randomized to receive efavirenz (with or without nelfinavir) plus two nucleoside analogues in study ACTG 384, and who had DNA available for analysis. Participants were followed up for up to 3 years. Nine single nucleotide polymorphisms in ABCB1, CYP2B6, CYP3A4, CYP3A5 and CYP2C19 were identified. Gene-gene interactions were identified using multifactor dimensionality reduction.Among 340 efavirenz recipients, higher efavirenz AUC24 h values were associated with a single locus model involving CYP2B6 516GT (73% accuracy; P0.001). This was also the best model among blacks (69% accuracy; P0.001), whereas among whites the best model involved a gene-gene interaction between CYP2B6 516GT and ABCB1 2677GT (82% accuracy, P0.001). Among 155 participants who received efavirenz without nelfinavir, virologic failure was associated with a two-locus interaction between ABCB1 2677GT and CYP2B6 516GT (65% accuracy, P0.001). Toxicity failure was best predicted by an interaction between ABCB1 2677GT and ABCB1 3435CT (71% accuracy, P0.001).Multilocus genetic interactions between variant drug metabolism and transporter genes may predict efavirenz pharmacokinetics and treatment responses. This finding may have implications for better individualizing antiretroviral therapy.

Background Efavirenz and nelfinavir are metabolized by cytochrome P-450 (CYP) 2B6 and CYP2C19, respectively, with some involvement by CYP3A. Nelfinavir is a substrate for P-glycoprotein, which is encoded by MDR1. The present study examined associations between genetic variants and long-term responses to treatment. Methods Adult AIDS Clinical Trials Group study 384 randomized antiretroviral-naive subjects to receive efavirenz and/or nelfinavir plus 2 nucleoside analogues, with follow-up lasting up to 3 years. Population pharmacokinetics were estimated from a nonlinear mixed-effects model. Polymorphisms in CYP2B6, CYP2C19, CYP3A4, CYP3A5, and MDR1 were characterized. Results The 504 participants in the genetic study included 340 efavirenz recipients and 348 nelfinavir recipients (184 of the 504 participants received both efavirenz and nelfinavir). Of the participants, 49% were white, 31% were black, and 19% were Hispanic. Plasma exposure to efavirenz and nelfinavir in each population was significantly associated with the polymorphisms CYP2B6 516G-->T and CYP2C19 681G-->A, respectively. Among efavirenz recipients, the MDR1 position 3435 TT genotype was associated with decreased likelihood of virologic failure and decreased emergence of efavirenz-resistant virus but not with plasma efavirenz exposure. Among nelfinavir recipients, a trend toward decreased virologic failure was associated with the polymorphism CYP2C19 681G-->A. Conclusions Genetic variants predict plasma exposure to efavirenz and nelfinavir, and they may predict virologic failure and/or emergence of drug-resistant virus. These associations with treatment responses must be validated in other studies.

Pharmacokinetic studies were conducted with human immunodeficiency virus-infected patients receiving efavirenz, nelfinavir, or both agents at weeks 4 and 32. Reductions of 25% and 45% were observed in the mean nelfinavir area under the concentration-time curve and minimum concentration of the drug in serum, and there was a 31% more rapid half-life for patients receiving both drugs compared to patients receiving nelfinavir alone. There were no significant differences in efavirenz pharmacokinetics.

Background. Maximum suppression of virus replication is often not achievable for persons infected with multidrug-resistant human immunodeficiency virus type 1 (HIV-1). Available data suggest that lamivudine contributes to partial viral suppression, despite the presence of M 184V mutations and high-level phenotypic lamivudine resistance. Methods. Selective lamivudine withdrawal was studied in 6 subjects who had incomplete viral suppression during antiretroviral treatment for multidrug-resistant HIV-1 infection. Results. Plasma levels of HIV-1 RNA increased to 0.5 log 10 copies/mL above baseline 6 weeks after the withdrawal of lamivudine treatment (P = .04), even though reversion of lamivudine resistance was not yet detected. Early increases in plasma levels of HIV-1 RNA after lamivudine withdrawal were associated with the presence of the T215Y/F mutation and broad phenotypic resistance to nucleoside reverse-transcriptase inhibitors at baseline. Genotypic and phenotypic reversion of lamivudine resistance was detected in 4 subjects 8-14 weeks after withdrawal of lamivudine therapy. The duration of lamivudine withdrawal ranged from 8 to 22 weeks; all subjects resumed lamivudine treatment. Plasma levels of HIV-1 RNA were 0.6 log 10 copies/mL above baseline (P = .03) when lamivudine therapy was resumed. After the resumption of lamivudine treatment, plasma HIV RNA levels decreased to baseline levels in 3 subjects but remained elevated in 3 subjects who had evolution of increased antiretroviral drug resistance during the period of lamivudine withdrawal. Safety concerns raised by this latter finding led to permanent closure of the study. Conclusions. In select cases of multidrug-resistant HIV-1 infection, lamivudine contributes to suppression of HIV-1 replication, despite the presence of M184V mutations and lamivudine resistance.

Background: HIV-1 isolates harboring multiple nucleoside reverse transcriptase inhibitor (NRTI) resistance mutations are more susceptible (hypersusceptible) to the nonnucleoside reverse transcriptase inhibitors (NNRTIs) than isolates lacking NRTI resistance mutations, but this has only been reported with a single-cycle replication phenotypic assay. In fact, there was a report that a commercial multicycle assay did not readily detect hypersusceptibility. Objective: To see whether NNRTI hypersusceptibility can be demonstrated in other types of phenotypic assays, including multicycle assays and enzyme inhibition assays. Methods: The susceptibility of HIV-1 clones derived from different patients in multicycle assays was tested in peripheral blood mononuclear cells (PBMCs) and in an established cell line. In addition, the reverse transcriptase (RT) of many of these clones was expressed and their susceptibility tested in an RT inhibition assay. Nevirapine and efavirenz susceptibilities were tested and compared with a control wild-type virus or RT. Results: Hypersusceptibility to nevirapine and efavirenz was detected using each of the methods described above. R 2 values correlating the other methods with single-cycle assay values were between 0.66 and 0.96. In addition to the high correlations, the different methods gave similar numeric results. Conclusions: NNRTI hypersusceptibility is readily seen in multicycle susceptibility assays and in enzyme inhibition assays.

HIV-1 strains that possess a one or two amino acid insert between codons 102 and 103 of the reverse transcriptase (RT) gene were identified in three HIV-1-infected individuals. Each strain also had one or more known mutations associated with nucleoside RT inhibitors (NRTIs) and non-nucleoside RT inhibitors (NNRTIs). Recombinant viruses from these strains had reduced susceptibility to efavirenz and nevirapine, and homology modelling predicted a loss of binding contacts with efavirenz. Mutagenesis studies indicated that replication of insert-containing strains was dependent on RT gene mutations and polymorphisms that co-evolved with the insert. These results suggest that inserts in the NNRTI-binding pocket contribute to NNRTI resistance, but are tolerated only under specific genetic conditions.

We observed a previously uncharacterized mutation in the protease substrate cleft, L23I, in 31 of 4,303 persons undergoing human immunodeficiency virus type 1 genotypic resistance testing. In combination with V82I, L23I was associated with a sevenfold reduction in nelfinavir susceptibility and a decrease in replication capacity. In combination with other drug resistance mutations, L23I was associated with multidrug resistance and a compensatory increase in replication capacity.

Highly active antiretroviral therapy (HAART), consisting mainly of two nucleoside reverse transcriptase inhibitors (NRTIs) and one protease inhibitor (PI), is offered to10% of HIV-infected subjects in Oman. The aims of the present study were to determine the frequency of resistance-associated mutations in these patients, and to assess the contribution of drug resistance to treatment outcome. Among 29 patients on HAART foror =6 months, virological, failure was observed in 27 (93%). Genotypic analysis indicated that in five of these 27 patients, there were no mutations that confer resistance to reverse transcriptase inhibitors (RTIs). The genotypes of 17 other patients carried one or two RTI mutations, mainly the lamivudine-associated resistance mutation M184V. Three or more RTI mutations were found in only five (14.7%) patients with virological failure, including three patients on the nonnucleoside RTI efavirenz. Major PI mutations were infrequent, and were detected in seven (26%) of 27 patients failing HAART, mainly as single mutation at codons 82 or 90. In contrast, accessory mutations in the protease gene were present in all patients. However, there were significant differences in the prevalence of accessory mutations at codons 36 and 77 among clade B and non-B viruses. When genotypic data of this study were used to change therapy of seven patients whose isolates had multiple resistance mutations, adequate viral suppression was observed in five. Our results indicate that the high rate of treatment failure among patients in Oman is mainly due to factors other than resistance to antiretroviral drugs. These factors, which may include nonadherence to therapy and treatment interruptions, need to be investigated.

T-1249 is a 39-aa synthetic peptide that inhibits fusion of human immunodeficiency virus (HIV) to the host target cell. A 14-day open-label, phase 1/2 dose-escalation monotherapy study of the safety and antiretroviral activity of T-1249 was performed on 115 HIV-1-infected adults. At baseline, the majority of the patients had advanced HIV disease (baseline median CD4(+) cell count, 57 cells/microL) and had extensive pretreatment (i.e., pre-T-1249) experience with antiretroviral medications (median, 11 antiretroviral drugs). Patients received T-1249 monotherapy by subcutaneous injection, for 14 days, at doses ranging from 6.25 to 192 mg/day. T-1249 was generally well tolerated, and no dose-limiting toxicity was identified. Injection-site reactions were the most commonly reported adverse event (57%). Dose-dependent decreases in plasma HIV-1 RNA load were observed; the median maximum change from baseline across dose groups ranged from -0.29 log(10) copies/mL (95% confidence interval [CI], -0.43 to -0.05 log(10) copies/mL) for the lowest dose to -1.96 log(10) copies/mL (95% CI, -2.02 to -1.37 copies/mL) for the highest dose. These results indicate that T-1249 is a potent new therapeutic agent for HIV-1 infection.

The goal of this study was to use X-ray crystallography to investigate the structural basis of resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors. We overexpressed, purified, and crystallized a multidrug-resistant (MDR) HIV-1 protease enzyme derived from a patient failing on several protease inhibitor-containing regimens. This HIV-1 variant contained codon mutations at positions 10, 36, 46, 54, 63, 71, 82, 84, and 90 that confer drug resistance to protease inhibitors. The 1.8-angstrom (Å) crystal structure of this MDR patient isolate reveals an expanded active-site cavity. The active-site expansion includes position 82 and 84 mutations due to the alterations in the amino acid side chains from longer to shorter (e.g., V82A and I84V). The MDR isolate 769 protease “flaps” stay open wider, and the difference in the flap tip distances in the MDR 769 variant is 12 Å. The MDR 769 protease crystal complexes with lopinavir and DMP450 reveal completely different binding modes. The network of interactions between the ligands and the MDR 769 protease is completely different from that seen with the wild-type protease-ligand complexes. The water molecule-forming hydrogen bonds bridging between the two flaps and either the substrate or the peptide-based inhibitor are lacking in the MDR 769 clinical isolate. The S1, S1′, S3, and S3′ pockets show expansion and conformational change. Surface plasmon resonance measurements with the MDR 769 protease indicate higher k off rates, resulting in a change of binding affinity. Surface plasmon resonance measurements provide k on and k off data ( K d = k off / k on ) to measure binding of the multidrug-resistant protease to various ligands. This MDR 769 protease represents a new antiviral target, presenting the possibility of designing novel inhibitors with activity against the open and expanded protease forms.

We established a database of HIV-1 reverse transcriptase (RT) and protease (PR) sequences and mutations to monitor the prevalence of antiretroviral drug resistance and mutational patterns in clinical samples submitted for testing to a major U.S. reference laboratory. At the end of 1998, 80% of the clinical samples tested harbored HIV strains with genotypically predicted resistance to at least one antiretroviral (ARV) drug. By the third quarter of 2002, the frequency of genotypically predicted resistance declined to 65% of samples tested. The prevalence of both PR and nucleoside RT inhibitor resistance declined over this period, while an increase in resistance to nonnucleoside RT inhibitors was found. These genotypic results strongly correlated with a nationwide decrease in the prescription of PR and nucleoside RT inhibitors, and an increase in the prescription of nonnucleoside RT inhibitors over the time period. The increased number of strains that were genotypically sensitive to all classes of ARV probably indicates an increase in genotypic assay use in ARV-naive individuals, however, the trends and correlations in this data set were similar when evaluated after removal of genotypically sensitive strains. Continued monitoring of ARV resistance prevalence, patterns, and utilization trends in clinical databases provides insight into the evolving relationship between clinical practice and ARV resistance.

BACKGROUND The optimal sequencing ofantiretroviral regimens for the treatment of infection with human immunodeficiency virus type 1 (HIV-1) is unknown. We compared several different antiretroviral treatment strategies. METHODS This multicenter, randomized, partially double-blind trial used a factorial design to compare pairs of sequential three-drug regimens, starting with a regimen including zidovudine and lamivudine or a regimen including didanosine and stavudine in combination with either nelfinavir or efavirenz. The primary end point was the length of time to the failure ofthe second three-drug regimen. RESULTS A total of 620 subjects who had not previously received antiretroviral therapy were followed for a median of 2. 3 years. Starting with a three-drug regimen containing efavirenz combined with zidovudine and lamivudine (but not efavirenz combined with didanosine and stavudine) appeared to delay the failure ofthe second regimen, as compared with starting with a regimen containing nelfinavir (hazard ratio for failure ofthe second regimen, 0.71; 95 percent confidence interval, 0.48 to 1.06), as well as to delay the second virologic failure (hazard ratio, 0.56; 95 percent confidence interval, 0.29 to 1.09), and significantly delayed the failure ofthe first regimen (hazard ratio, 0.39) and the firstvirologic failure (hazard ratio, 0.34). Starting with zidovudine and lamivudine combined with efavirenz (but not zidovudine and lamivudine combined with nelfinavir) appeared to delay the failure of the second regimen, as compared with starting with didanosine and stavudine (hazard ratio, 0.68), and significantly delayed both the first and the second virologic failures (hazard ratio for the firstvirologic failure, 0.39; hazard ratio for the second virologic failure, 0.47), as well as the failure ofthe first regimen (hazard ratio, 0.35). The initial use of zidovudine, lamivudine, and efavirenz resulted in a shorter time to viral suppression. CONCLUSIONS The efficacy ofantiretroviral drugs depends on how they are combined. The combination of zidovudine, lamivudine, and efavirenz is superior to the other antiretroviral regimens used as initial therapy in this study.

The 24-week extension of AIDS Clinical Trials Group Protocol 359, a study of human immunodeficiency virus (HIV)‐infected, indinavir-experienced patients, was designed to study the durability of “salvage” treatment regimens. Patients received saquinavir in combination with either ritonavir or nelfinavir and, in addition, delavirdine, adefovir, or both. Patients who demonstrated a virologic response at weeks 12‐16 were eligible to continue therapy in the extension through week 48. Of the 105 eligible subjects who were enrolled in the extension, 86 (82%) completed 48 weeks, and 49 (57%) of those 86 had HIV RNA levels 500 copies/ mL at week 48. For these 86 subjects who completed 48 weeks, the median change in CD4 cell count from baseline was +72 cells/mm 3 . Greater body weight, higher CD4 cell count, and greater degree of phenotypic susceptibility to indinavir and saquinavir at baseline were significantly associated with durable virologic suppression. These results show that some patients who experience treatment failure can demonstrate durable virologic and immunologic responses with salvage antiretroviral regimens. Current treatment guidelines recommend starting therapy for human immunodeficiency virus (HIV) infection with 2 nucleoside analogue reverse-transcriptase inhibitors in combination with 1 or 2 protease inhibitors or a nonnucleoside analogue reversetranscriptase inhibitor [1, 2]. However, 20%‐63% of patients from clinical cohorts experience virologic treatment failure while receiving combination antiretroviral therapy [3‐7]. Recent prospective studies have attempted to identify strategies for treatment of the treatment-experienced patient [8‐13], but these studies have focused primarily on 8‐24-week virologic responses to treatment. The durability of virologic and immunologic re

To determine the impact of HIV-1 drug resistance at baseline and antiretroviral drug levels (DL) during follow-up on virologic response to the next antiretroviral regimen.Baseline genotypic and phenotypic susceptibility was obtained for plasma virus from patients failing a protease inhibitor-containing regimen. Untimed plasma antiretroviral DL were performed and the distribution of DL after 12 weeks of follow-up was classified as above (DLHigh) or below (DLLow) the median. Inhibitory quotients [IQ = (DL at week 12)/(fold change in IC50 to wild-type)] were determined for each drug in the regimen. Primary outcome was change in log10 plasma HIV-1 RNA viral load (DeltaVL) from baseline to 12 weeks.There were 137 patients who had baseline resistance data available for the antiretroviral drugs used in the salvage regimen, and DL at week 12. Each drug with DLHigh was associated with DeltaVL = -0.40 (P = 0.0002) while each drug with DLLow had DeltaVL = -0.16 (P = 0.11). In multivariate models DeltaVL associated with each active drug (defined by genotype) with DLHigh was -0.48 log10 (P0.0001), and with each active drug with DLLow was -0.22 (P = 0.03). The DeltaVL was -0.18 if no drugs in the regimen had an IQmedian, compared to -0.58 for one drug, -1.06 for two drugs, -0.86 for three drugs, and -1.44 for four or five drugs with IQmedian (P0.0001 for trend).In salvage therapy, both the number of active drugs and the DL for each drug in the new regimen determine the antiviral response.

Zidovudine and other nucleoside analogue reverse transcriptase inhibitors (NRTIs), like zalcitabine and didanosine used for treatment of individuals infected with HIV-1, can select for viruses with Q151M and other associated mutations (for example, A62V, S68G, V75I, F77L, F116Y) in the reverse transcriptase (RT) enzyme. These mutations confer resistance to multiple nucleoside analogues, and thereby compromise the efficacy of this class of drugs. Presently available phenotypic assays for detection of multiple nucleoside analogue resistant (MNR) HIV-1 require testing for each NRTI individually. Here we report an enzymatic RT assay that uses resistance to zidovudine triphosphate (zidovudine-TP) as a diagnostic biochemical marker of MNR HIV-1. This assay exploits the different biochemical mechanisms for zidovudine-resistance conferred by either Q151M or T215Y/F mutations and the inability of conventional RT assays to detect T215Y/F-associated zidovudine resistance. The assay detects RT activity directly in plasma by using Amp-RT, an ultra-sensitive PCR-based RT assay. We show that enzymatic resistance to zidovudine-TP is specific to MNR RT and is distinguishable from both wild-type (WT) and RT containing classical zidovudine-resistant mutations (D67N, K70R, T215Y/F, K219Q). Compared to WT, MNR HIV-1 RT had 5- to 36-fold increases in the concentration of drug required to inhibit 50% (IC50) of RT activity, depending on the presence of Q151M alone or with additional MNR mutations. A screening assay utilizing 1 μM zidovudine-TP was developed and validated on 14 reference isolates, 37 plasma specimens, and seven patient-derived viruses. Twenty-three specimens were found to have reduced susceptibility to zidovudine-TP, and all had Q151M. In contrast, 21 specimens were sensitive to zidovudine-TP, of which 12 had WT genotypes, four had T215Y/F, and five had T69S-insertions along with T215Y/F mutations. This RT-based phenotypic assay provides a specific and rapid tool for the direct identification and monitoring of Q151M-associated MNR HIV-1 in plasma.

Twenty-four of over 24,000 patients genotyped over the past 3 years were found to have human immunodeficiency virus (HIV) isolates that possess an insert in the protease gene. In this report, we evaluated the spectrum of protease gene insertion mutations in patient isolates and analyzed the effect of these various insertion mutations on viral phenotypes. The inserts were composed of 1, 2, 5, or 6 amino acids that mapped at or between codons 35 and 38, 17 and 18, 21 and 25, or 95 and 96. Reduced susceptibility to protease inhibitors was found in isolates which possess previously reported drug resistance mutations. Fitness assays, including replication and competition experiments, showed that most of the isolates with inserts grew somewhat better than their counterparts with a deletion of the insert. These experiments demonstrate that, rarely, insertion mutations can develop in the HIV type 1 protease gene, are no more resistant than any other sequences which have similar associated resistance mutations, and can provide a borderline advantage in replication.

The T69D mutation in the human immunodeficiency virus type 1 reverse transcriptase (RT) gene has been associated with reduced susceptibility to dideoxycytosine (ddC); however, several other mutations at codon 69 have been observed in antiretroviral drug-treated patients. The Stanford HIV RT and Protease Sequence Database was interrogated and showed that 23% of patients treated with nucleoside RT inhibitors (NRTI) had mutations at codon 69. These variants included T69N, -S, -A, -G, -E, -I, and -K mutations that were present in patients treated with NRTI but not in drug-naive patients. Treatment history information showed that a substantial percentage of these codon 69 changes occurred in patients administered non-ddC-containing regimens. Different and specific patterns of other RT gene mutations were associated with the various codon 69 mutations. Drug susceptibility assays showed that viral constructs containing codon 69 variants could have reduced susceptibility to ddC and other RT inhibitors. These results suggest that the T69D mutation is not the only codon 69 variant associated with drug resistance and that ddC is not the only drug affected.

Point mutations and inserts in the β3-β4 region of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) are associated with resistance to nucleoside analog inhibitors. This report describes HIV-1 strains from seven patients that were found to have a 3-bp deletion in the β3-β4 region of the RT gene. These patient strains also had a mean of 6.2 drug resistance-associated mutations in their RT genes (range, 3 to 10 mutations). The deletion was most frequently found in strains with the Q151M mutation. Nonnucleoside RT inhibitor mutations were found in six of seven strains. Culture-based drug sensitivity assays showed that deletion-containing isolates had reduced susceptibility to four to eight RT inhibitors. Site-directed mutagenesis experiments showed that the deletion alone conferred reduced susceptibility to nucleoside analogs. Changes in the three-dimensional models of the RT deletion mutants were consistently observed at the β3-β4 loop and at helices C and E in both the presence and the absence of dTTP. Loss of hydrogen bonds between the RT and dTTP were also observed in the RT deletion mutant. These results suggest that the deletion in the RT gene contributes to resistance to several nucleoside analogs through a complex interaction with other mutations in the RT gene.

This study examines sequential lymph nodes from 13 drug-naive patients before and after 24 weeks of highly active antiretroviral therapy (HAART). A multipronged approach was used to study changes in HIV-1 RNA in each paired lymph node in relation to tissue architecture and frequency of naive T cells. After 24 weeks, all patients showed significant suppression of plasma viral load and 12 of 13 showed concordant viral suppression in the lymph node (p = 0.001). Using in situ hybridization and quantitative image analysis, we showed that HIV-1 RNA was reduced to below detectable levels (two copies per cell) in follicular dendritic cell (FDC) and mononuclear cell pools. Independent immunohistochemical analysis of lymph node sections revealed that 5 of 13 patients displayed increased FDC networks and 6 of 13 showed no change and all patients showed increases in tissue-resident CD4+ cells. All lymph node biopsies at 24 weeks showed increased proportions of CD4+ and CD8+ cells coexpressing the naive markers CD45RA and CD62L when compared with baseline values. Significant correlations existed between viral load suppression and loss of activated CD8+ T cells after 24 weeks in both lymph node and blood, which was mirrored by significantly lowered frequencies of activated peripheral Gag peptide/MHC tetramer+ CD8+ cells. Overall, these data show that a potent and successful treatment strategy that significantly suppresses and removes FDC-resident HIV-1 results in improvements in lymphoid architecture and by so doing provides the structures available for increased numbers of naive cells to interact with cognate antigen. In addition, our article shows that suppression of HIV-1 replication results in diminished frequencies of peripherally activated antigen-specific CD8+ cells.

Objective To determine the short-term effects of using genotypic antiretroviral resistance testing (GART) with expert advice in the management of patients failing on a protease inhibitor and two nucleoside reverse transcriptase inhibitors. Design Prospective randomized controlled trial. Setting Multicenter community-based clinical trials network. Patients One-hundred and fifty-three HIV-infected adults with a threefold or greater rise in plasma HIV-1 RNA on at least 16 weeks of combination antiretroviral therapy. Interventions Randomization was either to a GART group, where genotype interpretation and suggested regimens were provided to clinicians, or to a no-GART group, where treatment choices were made without such input. Main outcomes measures Plasma HIV-1 RNA levels and CD4 cell counts were measured at 4, 8, and 12 weeks following randomization. The primary endpoint was change in HIV-1 RNA levels from baseline to the average of the 4 and 8 week levels. Results The average baseline CD4 cell count was 230 x 10(6) cells/l and the median HIV-1 RNA was 28,085 copies/ml. At entry, 82 patients were failing on regimens containing indinavir, 51 on nelfinavir, 11 on ritonavir, and nine on saquinavir. HIV-1 RNA, averaged at 4 and 8 weeks, decreased by 1.19 log10 for the 78 GART patients and -0.61 log10 for the 75 no-GART patients (treatment difference: -0.53 log, 95% confidence interval, -0.77 to -0.29; P = 0.00001). Overall, the best virologic responses occurred in patients who received three or more drugs to which their HIV-1 appeared to be susceptible. Conclusion In patients failing triple drug therapy, GART with expert advice was superior to no-GART as measured by short-term viral load responses.

Peripheral blood samples from HIV-seropositive individuals enrolled in a pilot clinical trial investigating the use of allogeneic dendritic cell therapy were evaluated for mixed chimerism. In this study, dendritic cells from HLA-identical, HIV-seronegative siblings were used. Patients received an infusion of dendritic cells pulsed with HIV MN gp160 protein or with peptides from HLA-A2 restricted epitopes of env, gag, and pol proteins every month for 6–9 months. Of the five allogeneic dendritic cell recipients, two showed increases in HIV antigen-specific immune responses. Allele-specific polymorphisms were identified in three sib-pairs that allowed infused donor cells to be detected using sensitive PCR-based molecular methods. Analysis of blood samples from patients showed similar patterns of donor cell persistence after the first infusion, in that cells were detectable for at least 1 week. Also, differences were observed in the kinetics of cell survival between the first and subsequent infusion cycles in all three patients. This suggests variation in HIV-specific immune responses detected among these three patients was not due to differences in persistence of infused donor cells.

The frequency of protease and reverse transcriptase (RT) gene mutations was determined in HIV-1 strains from 153 patients entering the CPCRA 046 (GART) study who were failing triple-drug regimens consisting of one protease inhibitor (PI) and two RT inhibitors. Population-based sequence analyses showed that nearly all patients had similar RT gene mutations regardless of prior drug exposure, although the M184V mutation was significantly less prevalent in patients not recently treated with lamivudine. Whilst typical inhibitor-specific (‘signature’) protease gene mutations were found in patients failing their first PI, these mutations were significantly less likely to be found in patients exposed to two or more PIs. Protease gene mutations associated with multi-PI resistance were more likely to be observed in patients treated with more than one PI. These results suggest sequential treatment with PIs select for a relatively limited number of protease gene mutations that likely originated during early PI therapy. These protease gene mutations and a similarly limited set of RT gene mutations appear to be responsible for treatment failure in antiretroviral therapy.

Background —Coronary artery disease occurs in an accelerated fashion in the donor heart after heart transplantation (TxCAD), but the cause is poorly understood. The risk of developing TxCAD is increased by cytomegalovirus (CMV) infection and decreased by use of calcium blockers. Our group observed that prophylactic administration of ganciclovir early after heart transplantation inhibited CMV illness, and we now propose to determine whether this therapy also prevents TxCAD. Methods and Results —One hundred forty-nine consecutive patients (131 men and 18 women aged 48±13 years) were randomized to receive either ganciclovir or placebo during the initial 28 days after heart transplantation. Immunosuppression consisted of muromonab-CD3 (OKT-3) prophylaxis and maintenance with cyclosporine, prednisone, and azathioprine. Mean follow-up time was 4.7±1.3 years. In a post hoc analysis of this trial designed to assess efficacy of ganciclovir for prevention of CMV disease, we compared the actuarial incidence of TxCAD, defined by annual angiography as the presence of any stenosis. Because calcium blockers have been shown to prevent TxCAD, we analyzed the results by stratifying patients according to use of calcium blockers. TxCAD could not be evaluated in 28 patients because of early death or limited follow-up. Among the evaluable patients, actuarial incidence of TxCAD at follow-up (mean, 4.7 years) in ganciclovir-treated patients (n=62) compared with placebo (n=59) was 43±8% versus 60±10% ( P 40 years (relative risk, 2.7; CI, 1.3 to 5.5; P P =0.04). Stratification on the basis of calcium blocker use revealed differences in TxCAD incidence when ganciclovir and placebo were compared: no calcium blockers (n=53), 32±11% (n=28) for ganciclovir versus 62±16% (n=25) for placebo ( P P =NS). Conclusions —TxCAD incidence appears to be lower in patients treated with ganciclovir who are not treated with calcium blockers. Given the limitations imposed by post hoc analysis, a randomized clinical trial is required to address this issue.

The safety and antiviral activity of the second-generation nonnucleoside inhibitor HBY 097 was investigated in asymptomatic or mildly symptomatic human immunodeficiency virus (HIV)-1-infected patients in a randomized, double-blinded, dose-escalation study. Mean maximum virus load decreases ranged from -1.31 log10 copies/mL of plasma at week 1 in the group receiving HBY 097 monotherapy (250 mg three times daily) to -2.19 log10 copies/mL at week 4 in the group receiving zidovudine plus HBY 097 (750 mg three times daily). After 12 weeks, these patients had viral RNA copy numbers 1.05 log10 below baseline. Genotypic analysis of resistance development revealed reverse transcriptase K103N variants in most patients, which was associated with less durable efficacy of HBY 097 treatment. Fewer patients receiving combination therapy with high-dose HBY 097 developed the K103N variant (P

Peptide/MHC tetrameric complexes were used to enumerate the frequency of HLA class I-restricted epitope-specific CD8+ T cells in 18 HLA-A*0201 HIV type 1-infected asymptomatic patients. HLA-A*0201 molecules were complexed to HIV Gag p17 (amino acids 77–85) and reverse transcriptase (amino acids 464–472) peptides, biotinylated, and bound to streptavidin-phycoerythrin to form tetramers. We show in this study that 17 of 18 HIV-1-infected asymptomatic patients have circulating frequencies of 1/50–1/1000 CD8+ T cells that recognize both Gag and Pol CTL epitopes or either epitope alone. The functional nature of these cells is open to interpretation, as we show that despite relatively high frequencies of fresh epitope-specific CD8+ T cells, variant epitope sequences in viral plasma progeny were rare. In addition, the majority of tetramer-positive cells did not display discernible fresh CTL activity; only after restimulation with specific peptide in culture was there an expansion of epitope-specific CD8+ cells, correlating with high CTL activity. These data suggest that fresh tetramer-stained cells probably represent memory precursors; we demonstrate, with the application of highly active antiretroviral therapy, that the interruption of chronic antigenic stimulation causes significant reductions in the frequency of these cells in five of six patients. In conclusion, this study provides evidence that persistently replicating viral populations are probably required to maintain high frequencies of HIV-1 epitope-specific CD8+ T cells in asymptomatic chronically infected individuals

The relationships among treatment regimens, plasma human immunodeficiency virus (HIV) RNA levels, and resistance mutations to saquinavir (codons 48 and 90) and zidovudine (codon 215) were examined in a cohort of 144 patients from the AIDS Clinical Trials Group 229 study. After 24‐40 weeks of therapy, no patients who had received the two-drug combination (zidovudine plus saquinavir) had only codon 48 mutations, 45.8% had only codon 90 mutations, and 8.3% had both codon 48 and 90 mutations. Mutations developed by patients who had received the three-drug combination (zidovudine and zalcitabine plus saquinavir) were codon 48 alone in 1.4%, codon 90 alone in 33.3%, and both codons 48 and 90 in 4.2%. The difference between the groups showed a trend toward reduced mutations with three versus two drugs but did not reach significance ( , two-sided x 2 ). Higher baseline HIV RNA P 5 .11 levels correlated with the development of protease mutations. Mutations at codon 215 were present in 82% of all patients at baseline and in 87% after therapy.

The effect of patient preimmunization virus sequences on CTL responses during gp160 immunization were studied. Ten HLA-A2+, HIV+ asymptomatic patients with CD4+ T cells500/mm3 were given two courses of HIV-1 MN rgp160 vaccine over a 2-year period. Envelope epitope-specific CTL responses, using PBMCs, were measured against peptide-coated autologous B lymphoblastoid cell lines. Optimum CTL epitopes were determined by HLA-A2-binding affinity of 9- to 10-mer peptides containing the HLA-A2.1-binding motif. Ten of the high- or intermediate-binding peptides were conserved among50% of reported clade B HIV strains. These peptide-specific CTL activities and the patient virus sequences in peptide-coding regions were monitored. Six patients showed envelope peptide-specific CTL responses, which correlated with the presence of whole envelope antigen-specific CTL responses. Five of these patients, who showed responses to epitopes in the gp41 region (aa 814-824), had preimmunization virus similar to the vaccine sequence in this region. Three patients who did not show these epitope-specific responses had initially different sequences in the HIV gene encoding that region. The epitope-specific CTL responses appear to reflect recall responses, as only patients infected with virus containing the vaccine sequence developed them and they could be recalled with a second set of vaccine injections. This appears to be reminiscent of the concept of T cell "original antigenic sin." This vaccine was also immunogenic as measured by gp160-specific lymphocyte-proliferative responses. However, increased immune responses did not impact the HIV load or CTL epitope sequences during therapy.

Our group has been studying HIV in genital fluids for several years. Our first findings demonstrated that both RNA PCR and proviral DNA PCR could be utilized to quantify virus within seminal interstitial fluid and non-spermatozoa mononuclear cells, respectively. It became clear that these techniques were more sensitive than viral culture and we also reported that both disease progression and treatment influenced sperm viral concentrations generally parallel to virus concentrations in blood. A more recent study from our group has demonstrated that mutations seen in the virus in the blood are seen in the seminal plasma viral as well as in the proviral forms within non-spermatozoa cells. We have also studied female patients for 2-month periods looking at RNA in plasma and cervical secretions, as well as proviral DNA in cervical and vaginal samples by polymerase chain reaction (PCR) amplification techniques. The HIV RNA levels again appears to be the most sensitive and well-related to systemic viral load. Thus, genital secretion of cell-free virus and cells containing proviral DNA in both sexes parallels systemic virus levels, is a site for measurement of transmission of drug-resistant virus and should be monitored in therapy as well as in pathogenesis studies.

An understanding of the mechanisms of virologic cross-resistance between human immunodeficiency virus type 1 protease inhibitors is important for the establishment of effective treatment strategies for patients who no longer respond to their initial protease inhibitor. Protease gene sequencing results from patients treated with saquinavir showed significant increases in the frequency of the G48V protease mutation in patients receiving higher doses of the drug. In addition, all six patients who developed the G48V mutation during saquinavir therapy developed the V82A mutation either on continued saquinavir or after a switch to nelfinavir or indinavir. In vitro susceptibility assays showed that all 13 isolates with reduced susceptibilities to two or more protease inhibitors had either the G48V or L90M mutation, along with an average of six other protease mutations. Reduced susceptibility to nelfinavir was found in 14 isolates, but only 1 possessed the D30N mutation. These results suggest that mutations selected in vivo by initial saquinavir therapy may provide more cross-resistance to the other protease inhibitors than has been previously reported.

This study explores whether previous failures on antiretroviral drug regimens preclude the possibility of immune restoration. This was assessed by evaluating T cell subset changes in individuals who received a salvage regimen of highly active antiretroviral therapy (HAART) after initially failing protease inhibitor monotherapy. Ten HIV-1-infected asymptomatic patients received a regimen of indinavir, zidovudine, and 3TC after failing saquinavir monotherapy. Changes in absolute numbers of naive, memory, and activated CD4+ and CD8+ T cells expressing a selection of CD45RA, CD62L, CD45RO, HLA-DR, and CD38 markers were monitored prospectively over 6 months. These measurements were correlated with plasma viral load along with alterations in a selected CD8+ V alpha/Vbeta T cell receptor (TCR) repertoire. Over 6 months there was a progressive increase in numbers of CD4+ memory (CD45RA-CD62L+) and naive (CD45RA+CD62L+) T cells, which displayed a modest inverse correlation with viral load. Two phases of CD8+ memory cell changes were identified, consisting of a transient increase in CD45RA+CD62L- numbers after 2 months and thereafter a progressive rise in CD45RA-CD62L+ cells until 6 months. A strong correlation existed between reduced viral load and loss of activated CD8+CD38+HLA-DR+ cell numbers. There was also a temporary broadening of the CD8+ V alpha/Vbeta TCR repertoire at 8 weeks, which became skewed after 6 months in parallel with reduced viral suppression. Closer analysis of naive and memory cell subset proportions in individual patients revealed that enlarged pools of naive subsets were evident in those patients with rebounds in viral load. Overall, drug-experienced patients responding to HAART displayed increased numbers of naive and memory CD4+ subsets, and reduced CD8+ cell activation with a loss of TCR skewing.

To test the hypothesis that human cytomegalovirus (CMV) gB genotype may differ with geographic origin or patient demographics, CMV DNA was amplified for gB typing from immunocompromised patients in Italy and Africa and compared with previously reported frequencies in California. Increased gB2 frequency occurred in Italian homosexual AIDS patients, as compared with both Italian heterosexual injection drug users with AIDS and heterosexual Zimbabwe AIDS patients. Occurrence of gB3 in Italy was higher in injection drug users than in homosexual AIDS patients. The incidence of gB4 was higher overall in the Italian as compared with the California patients. Therefore geographic and demographic differences in patients affect gB distribution and should be considered before associations of gB genotypes and virulence are made.

Treatment of HIV infection with zidovudine (ZDV) may select for changes in the genetic sequence of the viral reverse transcriptase (RT) that imparts drug resistance. The presence of a 2-bp mutation at codon 215 of RT (from threonine to phenylalanine or tyrosine) was assessed in plasma viral RNA in 85 subjects treated with ZDV in the AIDS Clinical Trials Group (ACTG) 175 virology substudy. Median CD4 cell numbers, HIV plasma RNA levels, and infectious titers of virus were significantly different over 56 weeks of treatment among 58 subjects with the wild-type threonine at codon 215 virus at study entry compared with the 27 subjects with mutations to phenylalanine or tyrosine (MUT) virus. Thirty percent (13 of 44 subjects) with wild-type virus at study entry developed a new codon 215 mutation. Genotypic resistance at codon 215 in plasma HIV RNA is associated with the subsequent immunologic and virologic failure of ZDV monotherapy in subjects with 200 to 500 CD4 cells/mm3.

Cytomegalovirus (CMV) retinitis in patients infected with human immunodeficiency virus (HIV) is a significant clinical problem. Seventy-five patients with CD4 T cell counts100/mm3 were monitored prospectively every 2 months for CMV DNA burden. The target for DNA amplification was a 162-bp fragment from the CMV immediate early gene. CMV DNA burden, at levels ofor =320 in white blood cells oror =32 in plasma (P = .001), particularly when sustained (P = .005 and .008, respectively), distinguished patients who developed retinitis from those who remained free of disease. Progression to retinitis was not consistently accompanied by increases in CMV burden, indicating that quantitation of CMV burden beyond threshold levels is not necessary to predict risk for development of retinitis. Virus isolation from WBC, but not urine, was also significantly associated with risk for retinitis (P = .001).

Therapeutic vaccination has been proposed as a strategy to augment immune mechanisms to control viral replication and slow clinical progression of HIV infection to disease. Following recombinant gp160 (r-gp160) immunization in three clinical trials, plasma HIV-1 RNA and cellular proviral DNA were assessed by quantitative polymerase chain reaction (PCR) in 76 HIV-seropositive subjects with CD4+ T cell counts > or = 300/mm3. Immunization increased HIV-specific cellular immune responses (e.g., cytotoxic T lymphocyte [CTL] activities, lymphocyte proliferative responses); however, there were no significant effects of immunization or cellular immune responses on measures of plasma RNA or cellular DNA viral load.

Human immunodeficiency virus type 1 (HIV-1) drug susceptibility testing is often curtailed because such testing is expensive and time consuming. A colorimetric tetrazolium dye method previously used for high-throughput antiviral drug screening was adapted to assess the susceptibility of 16 HIV-1 isolates to zidovudine, didanosine, lamivudine, and nevirapine in MT-2 cells. Cell viability was assessed colorimetrically, and all measurements and calculations were automated. Each HIV-1 isolate was tested in > or = 5 assays to determine the reproducibility of the assay in HIV-1 isolates with known reverse-transcriptase mutations. The drug susceptibility of several mutant HIV-1 strains whose drug susceptibilities had not previously been well defined was also determined. Data on HIV-1 replication from the susceptibility assays indicated that some mutant HIV-1 isolates may have been less cytopathic in MT-2 cells than wild type HIV-1 isolates.

Cytomegalovirus isolates can be grouped into 4 gB and 2 gH genotypes. gB genotypes were studied in patients infected with human immunodeficiency virus (HIV) and in allograft transplantation recipients. In allograft recipients, the distribution of gB 1, -2, -3, and -4 in leukocytes and urine, respectively, was 36%, 21%, 43%, and 0% and 39%, 30%, 17%, and 13%. However, in leukocytes of HIV-infected patients with

The potential benefit of T cell-based vaccination for HIV-1 infection remains to be determined. Cytotoxic T lymphocytes (CTLs) appear to clear substantial populations of HIV-1 virus in vivo, although CTL activity may contribute to the decline in CD4+ T cell count observed in the course of the disease. To investigate further the role of specific CTL responses in the control of HIV-1 replication, we raised primary CTL lines against a panel of conserved HIV-1 epitopes using blood-derived dendritic cells as antigen-presenting cells (APCs). Specific primary human CTL responses were induced against HLA-A*0201-restricted peptides with dendritic cells from HIV-1-seronegative donors. This method of immunization elicited cytotoxic activities capable of recognizing endogenously processed antigen. The CTL induction protocol was extended in order to explore the capacity of HLA-matched allogeneic dendritic cells to evoke novel CTL responses in T cells from an HIV-seropositive asymptomatic individual. Allogeneic peptide-pulsed dendritic cells from a healthy sibling were capable of eliciting a CTL response directed against an HIV epitope (env814: SLLNATDIAV) that was initially not detected in the CTL effector population of the HIV-1-infected patient. The possibility of manipulating CTL specificity directed against multiple conserved HIV-1 epitopes represents a significant step in the evaluation of T cell-based vaccination for treatment of disease.

A two-amino acid insertion near codon 70 of the HIV-1 protease gene was found in an individual failing protease inhibitor therapy. Susceptibility of this strain to protease inhibitors was similar to that of non-insert-containing strains with comparable resistance mutations and one or more other major PI mutations.

The observation that human immunodeficiency virus type 1 (HIV-1) mutations conferring resistance to one reverse transcriptase (RT) inhibitor may suppress resistance to other RT inhibitors provides a rationale for treating HIV-1 with certain RT inhibitor combinations. We examined phenotypic and genotypic changes during culture of a multinucleoside (zidovudine, didanosine, zalcitibine, and stavudine)-resistant HIV-1 strain with and without additional RT inhibitors (nevirapine and lamivudine). The development of nevirapine or lamivudine resistance by the multinucleoside-resistant strain was not accompanied by a reduction in zidovudine or didanosine resistance.

Background This double-blind study evaluated treatment with either a single nucleoside or two nucleosides in adults infected with human immunodeficiency virus type 1 (HIV-1) whose CD4 cell counts were from 200 to 500 per cubic millimeter. Methods We randomly assigned 2467 HIV-1–infected patients (43 percent without prior antiretroviral treatment) to one of four daily regimens: 600 mg of zidovudine; 600 mg of zidovudine plus 400 mg of didanosine; 600 mg of zidovudine plus 2.25 mg of zalcitabine; or 400 mg of didanosine. The primary end point was a >50 percent decline in the CD4 cell count, development of the acquired immunodeficiency syndrome (AIDS), or death. Results Progression to the primary end point was more frequent with zidovudine alone (32 percent) than with zidovudine plus didanosine (18 percent; relative hazard ratio, 0.50; P

Background We studied measures of human immunodeficiency virus (HIV) replication, the viral phenotype, and immune function (CD4 cell counts) and the relation of changes in these indicators to clinical outcomes in a subgroup of patients in a controlled trial of early antiretroviral treatment for HIV, the AIDS Clinical Trials Group Study 175. Methods The 391 subjects, each of whom entered the study with a single screening CD4 cell count of 200 to 500 per cubic millimeter, were randomly assigned to receive zidovudine alone, didanosine alone, zidovudine plus didanosine, or zidovudine plus zalcitabine. Plasma concentrations of HIV RNA were assessed in 366 subjects, and viral isolates from 332 subjects were assayed for the presence of the syncytium-inducing phenotype. Results After eight weeks, the mean (±SE) decrease from base line in the concentration of HIV RNA, expressed as the change in the base 10 log of the number of copies per milliliter, was 0.26±0.06 for patients treated with zidovudine alone, 0.65±0....

Immune responses provoked by human immunodeficiency virus (HIV) infection ultimately are insufficient to control the disease and do not include strong lymphocyte-proliferative responses to HIV antigens or antibodies to many viral epitopes. A randomized double-blind, placebo-controlled trial evaluated the immunogenicity of recombinant HIV envelope vaccine (rgp160) in HIV-infected subjects with > or = 400/mm3 CD4 T cells. Controls received hepatitis B vaccine. Of subjects receiving rgp160, 98% developed lymphocyte-proliferative responses to the immunogen, 33% to a different envelope protein, and 56% and 60% to p24 and p66, respectively. All doses of vaccine (20, 80, 320, 1280 microgram) induced new responses. New antibodies to epitopes on rgp160 developed only in recipients of higher doses of rgp160. CD4 T cell percentages declined less rapidly in recipients of rgp160 than in controls. Vaccination of HIV-infected subjects with rgp160 results in cellular and humoral immune responses to HIV that infection itself had not stimulated.

Background In patients with human immunodeficiency virus (HIV) infection, combined treatment with several agents may increase the effectiveness of antiviral therapy. We studied the safety and efficacy of saquinavir, an HIV-protease inhibitor, given with one or two nucleoside antiretroviral agents, as compared with the safety and efficacy of a combination of two nucleosides alone. Methods In this double-blind trial, patients with HIV infection were randomly assigned to receive either saquinavir (1800 mg per day) plus both zidovudine (600 mg per day) and zalcitabine (2.25 mg per day) or zidovudine plus either saquinavir or zalcitabine. The 302 patients enrolled had CD4+ counts of 50 to 300 cells per cubic millimeter and had previously received zidovudine for a median of 27 months. The study lasted 24 weeks, with an optional double-blind extension period of an additional 12 to 32 weeks. Results Ninety-six percent of the patients completed the 24-week study. In all three treatment groups, CD4+ cell counts ros...