Horacio Salomon

Enhanced fidelity of 3TC-selected mutant HIV-1 reverse transcriptase.

Monotherapy with (−)2′,3′-dideoxy-3′-thiacytidine (3TC) leads to the appearance of a drug-resistant variant of human immunodeficiency virus-type 1 (HIV-1) with the methionine-184 → valine (M184V) substitution in the reverse transcriptase (RT). Despite resulting drug resistance, treatment for more than 48 weeks is associated with a lower plasma viral burden than that at baseline. Studies to investigate this apparent contradiction revealed the following. (i) Titers of HIV-neutralizing antibodies remained stable in 3TC-treated individuals in contrast to rapid declines in those treated with azidothymidine (AZT). (ii) Unlike wild-type HIV, growth of M184V HIV in cell culture in the presence of d4T, AZT, Nevirapine, Delavirdine, or Saquinavir did not select for variants displaying drug resistance. (iii) There was an increase in fidelity of nucleotide insertion by the M184V mutant compared with wild-type enzyme.

Diverse BF recombinants have spread widely since the introduction of HIV-1 into South America.

ObjectiveTo describe the genetic diversity of HIV-1 in South America by full genome sequencing and analysis. MethodsPurified peripheral blood mononuclear cell DNA from HIV-infected individuals in Argentina, Uruguay and Bolivia was used to amplify full HIV-1 genomes. These were sequenced using the ABI 3100 automated sequencer and phylogenetically analysed. ResultsTwenty-one HIV-1 strains from three South American countries, 17 of which were pre-screened by envelope heteroduplex mobility assay (HMA), were studied. Ten out of 10 HMA subtype F and four out of seven HMA subtype B strains were actually BF recombinants upon full genome analysis. Two BF recombinants from Argentina and two from Uruguay had the same structure, representing a new circulating recombinant form termed CRF12_BFARMA159. Twelve other BF recombinants had structures related to CRF12 but with additional segments of subtype B; each was unique. BF recombinants were temporally and geographically widespread, found as early as 1986–1987 in vertically infected Argentinian children and in Argentina, Uruguay, and Bolivia.

Development of HIV-1 resistance to (-)2'-deoxy-3'-thiacytidine in patients with AIDS or advanced AIDS-related complex

Objective: To determine the rate of development of in vitro HIV resistance to (‐)2′‐deoxy‐3′‐thiacytidine (3TC) and relate the effect of dose to emergence of resistance. Methods: HIV‐infected men and non‐pregnant women, aged ≥ 18 years, with a CD4 count ≤ 300 × 106/l cells were fòllowed in a Phase I/II study, in which they were evaluated for tolerance to 3TC and effect of this agent with regard to viral susceptibility. Peripheral blood and plasma samples were collected at regular intervals for analysis. HIV was isolated using umbilical cord blood mononuclear cells as targets. These cells were also used in determinations of median inhibitory drug concentration. Specific amplification of the 184 mutation site, associated with HIV resistance to 3TC, was performed by polymerase chain reaction, using specific primer pairs, on DNA harvested from infected peripheral blood mononuclear cells (PBMC) of donors or, alternatively, on DNA that had been reverse transcribed from plasma‐associated HIV RNA. Results: Phenotypic resistance was detected in approximately one‐third of individuals studied, who were followed between 8 and 56 weeks. Development of 3TC resistance occurred independently of dose, although time of first appearance of resistant HIV‐1 variants appeared reduced at high 3TC doses. Amino‐acid changes at codon 184 in HIV‐1 reverse transcriptase were associated with, and preceded, the development of phenotypic 3TC resistance. Most commonly, a Met to Ile substitution appeared transiently before being superceded by a Val substitution at codon 184. Conclusions: In vitro resistance to 3TC developed in a high proportion of subjects who received prolonged monotherapy with this drug. The development of resistance to 3TC was associated with appearance of mutated viral forms and the disappearance of wild‐type virus, with regard to codon 184, in both patient plasma and PBMC. AIDS 1995, 9:351‐357

Two HIV-1 epidemics in Argentina: different genetic subtypes associated with different risk groups.

Summary: This study determined the risk behaviors and viral subtypes of HIV‐1 found in 134 heterosexual HIV‐seroprevalent maternity patients, 41 of their sexual partners (men who have sex with women [MSW]), and 95 homosexual men (men who have sex with men [MSM]) from Buenos Aires, Argentina. Peripheral blood mononuclear cells (PBMCs) were purified from blood and used for DNA extraction, amplification, and genotyping by the envelope heteroduplex mobility assay (env HMA). Most of the women had been infected by having sex with an already infected partner (84%), whereas most of the male partners had been infected via drug use (76%). Both the patients and their sexual partners were poorly educated, only 30% having completed secondary school. The MSM study subjects, however, were significantly better educated and had a lower prevalence of injecting drug use. Env HMA subtype F was found in 77% (103 of 134) of the maternity patients, with similar rates in their partners (73%). Most of the remaining samples were env subtype B. All but one of the couples was concordant in subtype. In the MSM risk group, however, only 10% were env HMA subtype F. Ninety percent of the MSM samples were subtype B. There are at least two independent epidemics of HIV‐1 infection in Buenos Aires, Argentina. One, in heterosexual men and women, is dominated by env subtype F whereas the other, in homosexual men, is dominated by env subtype B, as determined by env HMA.

Clinical correlates of in vitro HIV-1 resistance to zidovudine. Results of the Multicentre Canadian AZT Trial

ObjectiveTo describe the rate of development of in vitro HIV resistance to zidovudine (ZDV) and its prognostic implications within the Multicentre Canadian AZT Trial (MCAT). MethodsHIV-infected subjects in Centers for Disease Control (CDC) stages IIB, III and IVC-2 with CD4 cell counts > 270 x 106/I were treated with ZDV as part of a dose-range study. Participating volunteers underwent prospective clinical and laboratory evaluations at regular intervals. Viral cultures and sensitivity testing were performed every 12 weeks in a predefined subset of 50 volunteers. An isolate was designated ZDV-resistant if it had a median inhibitory concentration (IC50) for ZDV at least 50-fold higher than that of virus isolated from the same subject before initiation of antiviral chemotherapy. The relationship between resistance and subsequent disease progression was studied using the Mantel and Byar method, for which, at each instance of disease progression, 2 x 2 tables classifying progression versus resistance status were constructed. The observed number of progressions was compared with that expected under the null hypothesis using Mantel-Haenszel methods adjusted for baseline CD4: CD8 ratio. ResultsThe Kaplan-Meier estimate for the cumulative development of in vitro resistance was 64% [95% confidence interval (CI), 41–78] at 180 weeks. Baseline CD4: CD8 ratio was negatively associated (P = 0.10) with the subsequent development of resistance (proportional hazard, 0.44; 95% CI, 0.17–1.10). After adjusting for baseline CD4:CD8 ratio, the numbers of observed and expected progressions following the development of resistance were 15 and 7.6, respectively (P = 0.008). A similar relative risk of progression between resistant and non-resistant states was found in the two CD4:CD8 strata; observed and expected progressions were 4 and 2.3 and 11 and 5.2 in the high and low CD4: CD8 strata, respectively. ConclusionsIn vitro resistance to ZDV developed in 64% of subjects after 180 weeks of ZDV therapy. Lower CD4: CD8 ratio at baseline was associated with faster development of resistance. In addition, the development of resistance was found to be a marker of subsequent disease progression. This association persisted after adjustment for baseline CD4: CD8 ratio. Whether in vitro resistance to ZDV is merely a surrogate marker or a determinant of disease progression remains to be established.

Resistance to antiretroviral drugs in patients with primary HIV-1 infection

The widespread use of antiretroviral agents (ARVs) and the growing occurrence of HIV strains resistant to these drugs have given rise to serious concerns regarding the transmission of resistant viruses to newly infected persons. Plasma viral RNA from 80 individuals newly infected between 1997 and 1999 was genotyped by automated sequencing to analyze the profile of viruses resistant to nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs) and to protease inhibitors (PIs). The prevalence of mutations that conferred primary resistance to PIs (L10I, D30Y, V82A, L90M) was 15% of the cohort. RT genotypic variants, associated with high-level resistance to ARVs, were observed in 21% of individuals, including NRTI, NNRTI and multidrug (MDR) resistance in 6, 5, and 10% of cases, respectively. The phenotypic susceptibility of viral isolates to ARVs was also assayed and showed transmission of high-level resistance to ZDV, 3TC, and PIs in those individuals with MDR. The transmission of drug-resistant HIV genotypic variants is a serious problem that merits further attention by public health officials, virologists, and clinicians.

Magnitude, Breadth, and Functional Profile of T-Cell Responses during Human Immunodeficiency Virus Primary Infection with B and BF Viral Variants

ABSTRACT The molecular pattern of the human immunodeficiency virus (HIV) epidemic in Argentina provides an appropriate scenario to study cellular immune responses in patients with non-clade B infection. We aimed to map T-cell responses in patients infected with BF recombinant variants and compare them with those of clade B patients. Sixteen recently infected patients were enrolled and grouped by viral subtype. Nef-specific responses were evaluated with a peptide matrix-based gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay using B and BF overlapping peptides. Cross-clade and clade-specific responses were found. A correlation between B versus BF Nef-specific responses was identified. Detailed analysis at the single-peptide level revealed that BF patients show a narrower response but greater magnitude. Nef immunodominant responses agreed with previous publications, although the B loop was targeted at an unexpectedly high frequency. The putative HLA allele(s) restricting each positive response was determined. Single-peptide level screening with two different peptide sets uncovered discordant responses (mostly caused by peptide offsetting) and allowed detection of increased breadth. Positive responses identified by ELISPOT assay were further studied by intracellular cytokine staining. These were almost exclusively mediated by CD8 T cells. Characterization of concordant responses revealed that cells show distinct functional profiles, depending on the peptide presented. Last, quality (in terms of polyfunctionality) of T cells was associated with better viral replication containment. Overall, interclade differences in the frequency of epitopes recognized, structural domains targeted, and magnitude of responses were identified. Screening T-cell responses with multiple sets increased sensitivity. Further support for the notion of polyfunctional CD8+ T-cell requirement to better control viral replication is also provided.

Drug resistance among HIV-infected pregnant women receiving antiretrovirals for prophylaxis.

Objective:To quantify primary resistance mutations (PRMs) among HIV-1-infected women receiving antiretroviral therapy (ART) for prevention of mother-to-child transmission (MTCT). Methods:Peripheral blood mononuclear cell samples from HIV-1-infected women enrolled in a prospective cohort study in Argentina, the Bahamas, Brazil, and Mexico (NISDI Perinatal Study) were assayed for PRMs. Eligible women were those enrolled by March 2005 and diagnosed with HIV-1 infection during the current pregnancy, and who received ART for MTCT prophylaxis and were followed for 6–12 weeks postpartum. Results:Of 819 women, 198 met the eligibility criteria. At enrollment, 98% were asymptomatic, 62% had plasma viral load < 1000 copies/ml, 53% had CD4+ cell count ≥ 500 cells/μl, and 78% were ART-exposed (mean duration, 8.0 weeks; 95% confidence interval, 7.1–8.9). The most complex ART regimen during pregnancy was usually (81%) a three-drug regimen [two nucleoside reverse transcriptase inhibitors (NRTIs) + one protease inhibitor or two NRTIs + one non-nucleoside reverse transcriptase inhibitor). PRMs were observed in samples from 19 (16%) of 118 women that were amplifiable at one or both time points [11/76 (14%) at enrollment; 14/97 (14%) at 6–12 weeks]. The occurrence of PRMs was not associated with clinical, immunological, or virological disease stage at either time point, whether ART-naive versus exposed at enrollment, or the most complex or number of antiretroviral drug regimens received during pregnancy (P > 0.1). Of 55 women with amplifiable samples at both time points, PRMs were detected in 11 samples (20%). Conclusions:PRMs occurred among 16.1% of relatively healthy HIV-1-infected mothers from Latin American and Caribbean countries receiving MTCT prophylaxis.

Intersubtype BF recombinants of HIV-1 in a population of injecting drug users in Argentina.

Summary:The presence of recombinant intersubtypes of HIV-1 in Argentina has been reported since the mid-1990s. In this study, sequences of a region of the gag, pol, and vpu genes of HIV-1 were analyzed in samples of 21 injection drug users (IDUs) residing in the suburbs of the city of Buenos Aires. Genomic characterization and identification of recombination sites were made comparing the 3 regions with reference isolation sequences of subtypes B, F, C, A, and B/F recombinants: CRF12_BF and non-CRF12_BF sequences. Subtype assignment of the analyzed segments was phylogenetically confirmed. All the samples turned out to be BF recombinants in at least 1 of the 3 studied genes. Twelve samples (57%) had the same pattern as the Argentinean CRF12_BF, whereas in the rest, the pattern differed in at least 1 of the 3 genes. The relation of these fragments to the CRF12_BF was phylogenetically verified. These results indicate the predominance of BF recombinants and the presence of a high percentage of sequences closely related to the CRF12_BF in the IDU population in Argentina and suggest a possible association between viral variants and the transmission route.

Didanosine Compared with Continued Zidovudine Therapy for HIV-Infected Patients with 200 to 500 CD4 Cells/mm3: A Double-Blind, Randomized, Controlled Trial

Zidovudine (3-azido-3-deoxythymidine) has been shown in placebo-controlled studies [1, 2] to prolong survival in patients with the acquired immunodeficiency syndrome (AIDS), to delay the development of AIDS in those with AIDS-related complex, and to delay the development of AIDS and AIDS-related complex in patients with asymptomatic human immunodeficiency virus (HIV) infection. The duration of the clinical benefit afforded by zidovudine monotherapy, however, appears to be limited [3-6]. The underlying mechanism or mechanisms responsible for disease progression during zidovudine therapy must still be definitively established. However, current evidence suggests that the development of viral resistance to zidovudine is at least partly responsible for the short duration of benefit [7, 8]. Didanosine (2,3-dideoxyinosine) is a newer nucleoside analog that has been shown to be effective in vitro against HIV [9]. Didanosine has in vitro activity against viral isolates that have high-level resistance to zidovudine [10]. Early clinical trials showed that didanosine can have a persistent beneficial effect on surrogate markers of HIV infection, such as CD4 counts, p24 antigen levels, and constitutional symptoms [11-14]. The investigators who did these studies found that peripheral neuropathy and pancreatitis were the dose-limiting toxicities of didanosine. More recently, controlled studies have shown that a switch to didanosine can improve clinical outcome in persons with advanced HIV disease who have received zidovudine [15, 16]. More specifically, Kahn and colleagues [15] showed such a benefit in patients with AIDS or AIDS-related complex who were clinically stable while receiving zidovudine and who had CD4 counts of 300 cells/mm3 or less and in asymptomatic HIV-infected patients with CD4 counts of 200 cells/mm3 or less. Spruance and coworkers [16] showed a similar benefit in patients with CD4 counts of 300 cells/mm3 or less and signs of clinical deterioration while receiving zidovudine therapy. No clinical data are available on the role of didanosine in stable patients in earlier stages of HIV disease who have received zidovudine. We therefore specifically compared the safety and efficacy of didanosine with that of continued zidovudine therapy in clinically stable HIV-infected persons who had CD4 counts between 200 and 500 cells/mm3 and had received zidovudine for at least 6 months. We hypothesized that a change to a second effective antiretroviral agent before the anticipated development of high-level resistance to zidovudine would prevent resistance and consequently delay the progression of HIV disease. Methods Study Design Randomization was stratified by the study center and by the CD4 cell count at study enrollment (more than or less than 300 cells/mm3). Successfully screened patients were randomly assigned using computer-generated random numbers. Randomization was done at a central location to ensure that patients, research personnel, and pharmacists remained blinded to the treatment allocation. All study participants provided informed consent. The study protocol and informed consent were approved by the review boards of the participating institutions and by the Canadian HIV Trials Network (CTN), with which our study is registered as protocol CTN-002. Patients Eligible study participants were male and nonpregnant female patients 12 years of age or older. Other entry criteria were the following: 1) HIV infection documented by enzyme-linked immunosorbent assay; 2) two sequential prerandomization CD4 counts between 200 and 500 cells/mm3 obtained at least 72 hours apart within 30 days of randomization, with the most recent measurement done within 14 days of randomization; 3) zidovudine therapy received for at least 6 months before randomization at a dose of at least 500 mg/d for the month immediately preceding study entry; 4) zidovudine therapy at 500 mg/d or greater for at least 21 of the previous 26 weeks; 5) a Karnofsky performance status of greater than 60 at study entry; 6) a hemoglobin level greater than 85 g/L or a hematocrit greater than 0.25 (in the absence of blood transfusion in the preceding 2 weeks); 7) a neutrophil count greater than 0.75 109/L; 8) a platelet count greater than 50 109/L; 9) serum aminotransferase and alkaline phosphatase levels greater than five times the upper limit of normal; 10) a serum creatinine level greater than 1.5 times the upper limit of normal; 11) a serum uric acid level less than 530 mol/L; and 12) a serum amylase level less than 2.1 times the upper limit of normal. The following are the normal values for chemical variables: aspartate aminotransferase, as high as 0.67 kat/L; alkaline phosphatase, 0.58 to 1.75 kat/L; creatinine, 40 to 120 mol/L; and amylase, 0.50 to 1.83 kat/L. Study participants were required to take adequate birth control measures during the study. The following were the exclusion criteria: 1) the presence of an uncontrolled AIDS-defining illness; 2) known or suspected pulmonary Kaposi sarcoma or Kaposi sarcoma requiring systemic cytotoxic chemotherapy; 3) grade II or greater dementia; 4) active substance abuse; 5) antiretroviral therapy other than zidovudine; 6) any use of biological-response modifiers or corticosteroids within 30 days of entry or therapy with ribavirin within 90 days of entry; 7) previous participation in studies involving didanosine or zalcitabine; 8) grade II or greater neurologic, allergic, or renal toxicities; 9) any history of pancreatitis, intractable diarrhea, or malabsorption; 10) unexplained seizures within the previous 6 months or need for anticonvulsant agents; 11] treatment with neurotoxic drugs within 30 days of entry; and 12) past or current heart disease or requirement for cardiac medication. All study participants were encouraged to use prophylaxis for Pneumocystis carinii infection according to contemporary guidelines [17]. The use of megestrol acetate, foscarnet, aspirin, acetaminophen, nonsteroidal anti-inflammatory agents, oral acidifying agents, and oral acyclovir was discouraged. Treatment of opportunistic infections was permitted. In patients developing serious symptoms or laboratory abnormalities, study medications were withheld until the symptoms or laboratory abnormalities resolved; at this point, patients were encouraged to resume the study medication, according to a prespecified dose-reduction scheme. Treatment Regimens Zidovudine (Retrovir, Burroughs-Wellcome, Research Triangle Park, North Carolina) was provided in 100-mg capsules to be taken at a dosage of 600 mg/d divided into at least three daily doses. Didanosine (Videx, Bristol-Myers Squibb, Princeton, New Jersey) was provided in sachets containing 5.2 g of citrate-phosphate buffer and sucrose adjusted to yield a final net weight of 20 g. The contents of one sachet were to be dissolved in water and swallowed. Didanosine dosage was adjusted for weight: Patients weighing at least 60 kg received 500 mg/d in two divided doses; patients weighing less than 60 kg received 334 mg/d in two divided doses. The didanosine formulation was changed in October 1991 from 500- and 334-mg/d sachet formulations to 400- and 200-mg/d tablet formulations. Study participants were instructed to chew the didanosine tablets thoroughly either together or in rapid succession and then to rinse with approximately 120 mL of room-temperature drinking water, which was also to be swallowed. Alternatively, the two tablets were to be crushed and thoroughly dispersed in at least 120 mL of drinking water; this solution was to be drunk immediately, followed by approximately 120 mL of drinking water. Study participants were instructed to always take didanosine on an empty stomach, at least 2 hours after and 1 hour before meals. To maintain the double-blind nature of the protocol, patients assigned to receive didanosine were given identical zidovudine placebo, and those assigned to receive zidovudine were given identical didanosine placebo. Follow-up After completion of the baseline evaluation, patients were seen at biweekly intervals for the first 2 months and monthly thereafter. A safety profile, including a symptom-targeted questionnaire, hematologic assessment, and chemistry panel were done at each visit. The CD4 count and viral resistance studies were done at baseline; at weeks 2, 8, and 12; and every 3 months thereafter. Formal follow-up of this cohort, as per the study protocol, was completed on 12 October 1992. Long-term, off-protocol, follow-up information on survival, AIDS-defining illnesses, and CD4 lymphocyte counts was compiled on one occasion using standardized data collection forms in December 1993. This allowed us to collect additional follow-up information on all patients after study completion and information on the complete study period for patients who dropped out of the study. Study End Points The primary clinical end point was the occurrence of a new, previously undiagnosed AIDS-defining event (according to the revised 1987 criteria of the Centers for Disease Control and Prevention) or death [18]. Clinical end points were reviewed by study monitors at each clinical site and were confirmed in a blinded manner by the clinical end points committee. Didanosine was licensed by the Food and Drug Administration in the United States and by the Health Protection Branch in North America in the fall of 1991. At that time, didanosine became the standard therapy in Canada for persons with zidovudine intolerance or disease progression despite zidovudine therapy. Thus, in late 1991, while the study remained blinded and before any data were analyzed, a 33% decline in CD4 counts from baseline was added as a primary study end point to maintain consistency with prevailing clinical practice. Sensitivity Testing Samples for testing sensitivity to the study drugs were obtained from 102 of 120 patients (85%) enrolled at five clinical sites who were preselected on the basis of logistic issues. For