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J Martinez-Picado

Publications and source records attributed to J Martinez-Picado.

12 recordsLinked to original sources
PubMed2004

HIV evolution: CTL escape mutation and reversion after transmission.

Within-patient HIV evolution reflects the strong selection pressure driving viral escape from cytotoxic T-lymphocyte (CTL) recognition. Whether this intrapatient accumulation of escape mutations translates into HIV evolution at the population level has not been evaluated. We studied over 300 patients drawn from the B- and C-clade epidemics, focusing on human leukocyte antigen (HLA) alleles HLA-B57 and HLA-B5801, which are associated with long-term HIV control and are therefore likely to exert strong selection pressure on the virus. The CTL response dominating acute infection in HLA-B57/5801-positive subjects drove positive selection of an escape mutation that reverted to wild-type after transmission to HLA-B57/5801-negative individuals. A second escape mutation within the epitope, by contrast, was maintained after transmission. These data show that the process of accumulation of escape mutations within HIV is not inevitable. Complex epitope- and residue-specific selection forces, including CTL-mediated positive selection pressure and virus-mediated purifying selection, operate in tandem to shape HIV evolution at the population level.

Adult↗
PubMed2000

Virological and immunological effects of treatment interruptions in HIV-1 infected patients with treatment failure.

OBJECTIVE: To analyse the immunological and virological effects of treatment interruptions in HIV-1-infected patients with treatment failure and multidrug-resistant virus. METHODS: Drug susceptibility was assessed using Antivirogram and genotypic analysis was based on population and clonal sequencing for 48 patients who had interrupted treatment (> or = 2 months). RESULTS: Treatment interruption resulted in viral load increases (mean 0.7 log 10 copies/ ml; P = 0.0001) and CD4 cell count decreases (mean 89 x 10(6) cells/l; P = 0.0001). A complete shift to wild-type virus at the phenotypic, genotypic and clonal level was observed in 28/45 patients. These patients differed from those that did not show a shift to wild type in baseline CD4 cell counts (192 versus 59 x 10(6) cells/l; P= 0.007) and in the relationship between baseline viral load and CD4 cell count (no correlation versus a significant negative correlation; P= 0.008). Response to re-initiation of treatment fell with increasing viral load [relative hazard (RH) 0.33; P= 0.001] and with increasing total number of drugs with reduced susceptibility (RH 0.51; P = 0.0003); it improved with the number of new drugs received (RH 2.12; P = 0.0002) and a shift to wild type (RH 5.22, P = 0.006). CONCLUSIONS: Changes in surrogate markers suggest that treatment provided benefit in spite of virological failure and resistant virus. Although patients with a shift to wildtype virus responded better in the short term to treatment re-initiation, the long-term effects are not known and the risk of immune deterioration needs to be carefully considered.

Adult↗
PubMed2000

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.

Drug Resistance, Microbial↗
PubMed2000

Antiretroviral resistance during successful therapy of HIV type 1 infection.

HIV type 1 (HIV-1) drug resistance mutations were selected during antiretroviral therapy successfully suppressing plasma HIV-1 RNA to <50 copies/ml. New resistant mutant subpopulations were identified by clonal sequencing analyses of viruses cultured from blood cells. Drug susceptibility tests showed that biological clones of virus with the mutations acquired during successful therapy had increased resistance. Each of the five subjects with new resistant mutants had evidence of some residual virus replication during highly active antiretroviral therapy (HAART), based on transient episodes of plasma HIV-1 RNA > 50 copies/ml and virus env gene sequence changes. Each had received a suboptimal regimen before starting HAART. Antiretroviral-resistant HIV-1 can be selected from residual virus replication during HAART in the absence of sustained rebound of plasma HIV-1 RNA.

Anti-HIV Agents↗
PubMed2000

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.

Anti-HIV Agents↗
PubMed2000

Structured treatment interruption in chronically HIV-1 infected patients after long-term viral suppression.

OBJECTIVE: To investigate the virological and immunological impact of a structured treatment interruption (STI) in a cohort of HIV-1 chronically infected patients with a further long-lasting effective virus suppression. METHODS: Twelve HIV-1 chronically infected adults who had maintained viral suppression (< 20 copies/ml) for more than 2 years, as well as a CD4:CD8 ratio > 1 for a median time of 22 months, were included in the study. Participants interrupted their antiretroviral treatment during a maximum period of 30 days or until a viral load rebound > 3000 copies/ml was detected. The same prior antiretroviral regimen was resumed after STI. Kinetics of plasma viral rebound was evaluated every 2 days during the treatment interruption period. Flow cytometry and cell proliferation assays were performed before and after STI. Genotypic resistance was assessed at the time of treatment resumption. RESULTS: No adverse events occurred during the interruption period. In two patients no viral rebound was detected after 30 days of treatment interruption. In the remaining 10 patients, viral load became detectable (> 20 copies/ml) at a median time of 14 days after treatment interruption. Afterwards, viral load increased exponentially with a mean t1/2 of 1.6 days. Treatment was successfully resumed in all patients. No resistance-conferring mutations associated with the pre-interruption antiretroviral regimen were detected. The percentage of CD4 and CD8 lymphocytes did not vary during the STI period; however, the level of expression of T-cell activation antigen CD38 on CD8 T cells increased significantly in response to viral rebound. Four patients gained T-helper cell responses to recall antigens (tuberculin and tetanus toxoid), two of who developed an HIV-specific response to p24. CONCLUSIONS: STI in chronically HIV-1-infected patients is not associated with reductions in CD4 T lymphocytes or to clinical complications in this group of patients after 2 years of effective plasma viral suppression. Viral load rebounds in most but not all patients, without evidence of selection of resistance-conferring mutations. Thereafter, viraemia can be effectively controlled by antiretroviral agent reintroduction. HIV-specific T-helper cell responses may be achieved after one cycle of treatment interruption suggesting some degree of immune-stimulation. These data do not discard consecutive cycles of STI as a therapeutic strategy to boost HIV-specific immunity in order to maintain viral replication under effective control.

Adult↗
PubMed2000

Comparison of sequencing by hybridization and cycle sequencing for genotyping of human immunodeficiency virus type 1 reverse transcriptase.

The performances of two methods of nucleotide sequencing were compared for the detection of drug resistance mutations in human immunodeficiency virus type 1 reverse transcriptase (RT) in viruses isolated from highly RT inhibitor-experienced individuals. Of 11,677 amino acids deduced from population PCR products by both cycle sequencing and sequencing by hybridization to high-density arrays of oligonucleotide probes, 97.4% were concordant by both methods, 0.8% were discordant, and 1.7% had an ambiguous determination by at least one method. A higher rate of discordance (3.9%) was observed among RT inhibitor resistance-associated codons. In 45% of the isolates, RT codon 67 was deduced as the wild-type Asp by hybridization sequencing but as the zidovudine resistance-associated Asn by cycle sequencing. In other resistance-associated codon discordances, cycle sequencing also more commonly called a known resistance-associated amino acid than hybridization sequencing did. The nucleotide sequence in the vicinity of several codons with discordant calls influenced population-based hybridization sequencing. For isolates evaluated by additional sequencing of molecular clones of PCR products by both methods, the discordance between methods was less frequent (0.4% of all 5,994 amino acids and 0 of 494 drug resistance-associated codons). At positions which were discordant or ambiguous in the population sequences, the results of sequencing of clones by both methods were usually in agreement with the population cycle sequencing result. In summary, most RT codons were highly concordant by both methods of population-based sequencing, with discordances due in large part to genetic mixtures within or adjacent to discordant codons.

Anti-HIV Agents↗
PubMed1999

Improved CD4 lymphocyte outgrowth in response to effective antiretroviral therapy.

CD4 lymphocyte regenerative capacity was evaluated by use of an ex vivo outgrowth assay in human immunodeficiency virus (HIV)-1-infected subjects enrolled in a clinical trial (Merck 039). CD4 lymphocytes were selectively expanded in vitro by T cell receptor triggering, which also induces HIV production from latently infected cells. CD4 cell expansion and lack of virus production in cultures correlated well with clinical responses and were best in those receiving an aggressive antiretroviral three-drug regimen. Twelve clinical responders receiving triple-drug therapy monitored for 60 weeks had both excellent ex vivo CD4 cell expansion and lack of HIV replication, often in the absence of added drug in culture. Breakthrough viruses recovered from drug-containing arms of the cultures showed phenotypic resistance to the drugs used in vivo. This CD4 lymphocyte outgrowth assay correlates well with clinical outcome in subjects receiving potent antiretroviral regimens and may predict the emergence of early drug resistance.

Anti-HIV Agents↗
PubMed1999

Human immunodeficiency virus type 1 cloning vectors for antiretroviral resistance testing.

Better detection of minority human immunodeficiency virus type 1 (HIV-1) populations containing gene mutations may improve the usefulness of antiretroviral resistance testing for clinical management. Molecular cloning of HIV-1 PCR products which might improve minority detection can be slow and difficult, and commercially available recombinant virus assays test drug susceptibility of virus pools. We describe novel plasmids and simple methods for rapid cloning of HIV-1 PCR products from patient specimens and their application to generate infectious recombinant virus clones for virus phenotyping and genotyping. Eight plasmids with differing deletions of sequences encoding HIV-1 protease, reverse transcriptase, or Gag p7/p1 and Gag p1/p6 cleavage sites were constructed for cloning HIV-1 PCR products. A simple HIV-1 sequence-specific uracil deglycosylase-mediated cloning method with the vectors and primers designed here was more rapid than standard ligase-mediated cloning. Pooled and molecularly cloned infectious recombinant viruses were generated with these vectors. Replicative viral fitness and drug susceptibility phenotypes of cloned infectious viruses containing patient specimen-derived sequences were measured. Clonal resistance genotyping analyses were also performed from virus isolates, plasma HIV-1 RNA, and infected cell DNA. Sequencing of a limited number of molecular clones detected minorities of resistant virus not identified in the pooled population PCR product sequence and linkage of minority mutations.

Anti-HIV Agents↗
PubMed1999

Replicative fitness of protease inhibitor-resistant mutants of human immunodeficiency virus type 1.

The relative replicative fitness of human immunodeficiency virus type 1 (HIV-1) mutants selected by different protease inhibitors (PIs) in vivo was determined. Each mutant was compared to wild type (WT), NL4-3, in the absence of drugs by several methods, including clonal genotyping of cultures infected with two competing viral variants, kinetics of viral antigen production, and viral infectivity/virion particle ratios. A nelfinavir-selected protease D30N substitution substantially decreased replicative capacity relative to WT, while a saquinavir-selected L90M substitution moderately decreased fitness. The D30N mutant virus was also outcompeted by the L90M mutant in the absence of drugs. A major natural polymorphism of the HIV-1 protease, L63P, compensated well for the impairment of fitness caused by L90M but only slightly improved the fitness of D30N. Multiply substituted indinavir-selected mutants M46I/L63P/V82T/I84V and L10R/M46I/L63P/V82T/I84V were just as fit as WT. These results indicate that the mutations which are usually initially selected by nelfinavir and saquinavir, D30N and L90M, respectively, impair fitness. However, additional mutations may improve the replicative capacity of these and other drug-resistant mutants. Hypotheses based on the greater fitness impairment of the nelfinavir-selected D30N mutant are suggested to explain observations that prolonged responses to delayed salvage regimens, including alternate PIs, may be relatively common after nelfinavir failure.

Adaptation, Biological↗
PubMed1996

Species-Specific Detection of Vibrio anguillarum in Marine Aquaculture Environments by Selective Culture and DNA Hybridization.

Methods for specific detection of Vibrio anguillarum in complex microbial communities within diverse marine aquaculture environments were evaluated. A system for the detection of culturable cells based on the combined use of a selective medium and a nonradioactively labeled oligodeoxynucleotide complementary to 16S rRNA was developed. Four hundred fourteen bacterial cultures were evaluated in order to assess the specificity of the method. When both the selective medium and the specific probe gave positive results, the cultures were always identified as V. anguillarum. The selectivity for colony hybridization was 1 V. anguillarum cell in 10,000 total bacterial cells in environmental samples. The utility of the method was also compared with detection by dot blot hybridization of either raw DNA purified from environmental samples or PCR-amplified DNA of 16S rRNA genes, using universal eubacterial primers. The post-PCR hybridization was more sensitive (8 x 10(sup2) cells) than direct hybridization of the whole purified DNA (10(sup6) cells). However, the selective medium-probe combined method was as sensitive as post-PCR hybridization, albeit more specific.

Journal Article↗