Laura Romano

Antiretroviral Resistance Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Protease from Paired Cerebrospinal Fluid and Plasma Samples

Twenty-four adults infected with human immunodeficiency virus type 1 (HIV-1) with central nervous system symptoms were studied for antiretroviral resistance mutations in HIV-1 RNA obtained from paired cerebrospinal fluid (CSF) and plasma samples. Paired sequences were obtained from 21 and 13 patients for reverse transcriptase (RT) and for protease, respectively. Mutations conferring resistance to the RT inhibitors zidovudine, lamivudine, or nevirapine were detected in 14 patients, including 11 pretreated and 3 drug-naive subjects. The mutation patterns in the 2 compartments were different in most patients. Genotypic resistance to protease inhibitors was detected in both plasma and CSF from 1 patient treated with multiple protease inhibitors. However, accessory protease inhibitor resistance mutations at polymorphic sites were different in plasma and CSF in several patients. Partially independent evolution of viral quasispecies occurs in plasma and CSF, raising the possibility that compartmentalization of drug resistance may affect response to antiretroviral treatment.

Broad Nucleoside-Analogue Resistance Implications for Human Immunodeficiency Virus Type 1 Reverse-Transcriptase Mutations at Codons 44 and 118

Two large, independent human immunodeficiency virus type 1 resistance databases containing >7700 reverse-transcriptase (RT) sequences were used to analyze the epidemiology of amino acid substitutions at codons 44 and 118, which confer moderate lamivudine resistance in the presence of zidovudine resistance. As expected, E44A/D and V118I mutations were strongly associated with M41L, D67N, L210W, and T215Y but also with other mutations, including K43E/N/Q, T69D, V75M, H208Y, R211K, and K219R. Both E44D and V118I were more frequently associated with stavudine and didanosine than with zidovudine and lamivudine treatment. However, selection of E44A/D and V118I was also detected in association with a switch to other nucleoside RT inhibitors, including zalcitabine and abacavir. Site-directed mutagenesis confirmed that 44D and 118I can decrease phenotypic susceptibility not only to lamivudine but also to most other nucleoside analogues, particularly stavudine and abacavir. Thus, substitutions at RT codons 44 and 118 have broad implications in nucleoside RT inhibitor resistance in the setting of several nucleoside-associated mutations.

Antigenicity and immunogenicity of the V3 domain of HIV type 1 glycoprotein 120 expressed on the surface of Streptococcus gordonii.

Five different V3 domains of HIV-1 gp120 were expressed on the surface of the gram-positive bacterium Streptococcus gordonii, a model live vector for vaccine delivery. Sera of HIV-1-infected individuals and human monoclonal antibodies specifically recognized the gp120 sequences on the bacterial surface. Recombinant V3 from the reference HIV-1 strain MN was also shown to retain a conformation that allowed reaction with a conformation-specific monoclonal antibody. A V3-specific serum antibody response was detected in mice immunized both by subcutaneous injection and by vaginal colonization. V3-specific IgG2a antibodies, suggestive of a Th1 response, were found in the sera of mice colonized by recombinant bacteria.

Evaluation of the presence of 2-LTR HIV-1 unintegrated DNA as a simple molecular predictor of disease progression.

In a preliminary cross‐sectional analysis of 109 human immunodeficiency virus type 1 (HIV‐1)‐infected subjects the presence of 2‐long terminal repeat (LTR) unintegrated circular HIV‐1 DNA in peripheral blood mononuclear cells (PBMC) was found to be associated with both symptomatic infection (P = 0.0037) and low CD4 counts (P = 0.0004). To investigate the prognostic significance of the presence of 2‐LTR HIV‐1 DNA, a subset of 23 2‐LTR‐negative and 25 2‐LTR‐positive asymptomatic individuals were followed up for 12–24 months. The two groups did not differ in terms of baseline CD4 counts, zidovudine (ZDV) therapy, and duration of HIV‐1 infection. Longitudinal analysis of CD4 values did not indicate a significantly different CD4 outcome between the two groups. However, when only ZDV‐treated subjects were considered, a significant (P = 0.042) decrease in CD4 counts was found at month 24 with respect to baseline in 2‐LTR‐positive (n = 12) but not in 2‐LTR‐negative (n = 11) patients. Moreover, when >40% CD4 loss from baseline and/or development of CDC stage B or C symptoms were considered as indicators of disease progression, there was a significantly higher number of events in the whole 2‐LTR‐positive group than in the whole 2‐LTR‐negative group (P = 0.0197 at month 12, P = 0.0299 at month 18, P = 0.0373 at month 24). Thus, the presence of 2‐LTR‐HIV‐1 DNA in PBMC merits further investigation as a simple, qualitative, molecular predictor of disease progression and decreased response to antiretroviral therapy. J. Med. Virol. 52:20–25, 1997.

Antiretroviral therapy with protease inhibitors in human immunodeficiency virus type 1– and human herpesvirus 8–coinfected patients

Human herpesvirus 8 (HHV‐8) is believed to play a role in the pathogenesis of Kaposis sarcoma (KS) and possibly in other proliferative disorders often associated with human immunodeficiency virus type 1 (HIV‐1) infection. Recent case reports have indicated resolution of KS and clearance of HHV‐8 DNA from peripheral blood mononuclear cells (PBMC) in HIV‐1–infected subjects following highly effective antiretroviral therapy, including HIV‐1 protease inhibitors (PI), suggesting a possible activity for these compounds on HHV‐8 replication. In the present study, the time course of PBMC HHV‐8 DNA levels, plasma HIV‐1 RNA load, and CD4+ T‐cell counts were followed up in six coinfected subjects (four with and two without KS) under antiretroviral therapy with PI. A specific anti–HHV‐8 role for PI was not consistently found, since fluctuation of HHV‐8 viral load over time appeared to be independent of treatment. Nevertheless, our data support the hypothesis that KS patients may significantly benefit from PI therapy as an indirect consequence of partial restoration of immune functions following effective anti–HIV‐1 combination therapy. J. Med. Virol. 57:140–144, 1999.

Development and significance of the HIV-1 reverse transcriptase M184V mutation during combination therapy with lamivudine, zidovudine, and protease inhibitors.

To analyze the emergence and role of the lamivudine (3TC)-selected HIV-1 reverse transcriptase (RT) M184V mutation under triple therapy, we performed a retrospective study of 40 nucleoside RT inhibitor-pretreated and 16 drug-naive patients who were switched to combined treatment with zidovudine (ZDV) plus 3TC plus a protease inhibitor (PI). Plasma viral load and pol genotype were analyzed at baseline and after 24 and 48 weeks of combination therapy. Emergence of the M184V RT mutation at week 48 was detected in 3 of 16 (18.7%) initially drug-naive subjects as opposed to 21 of 40 (52.5%) ZDV-pretreated patients. Multivariate logistic analysis detected HIV-1 RNA load at week 24 as the best predictor of subsequent selection of the M184V mutant (p = .0121). Among ZDV-resistant study subjects at week 24 (n = 17), those with mutant RT M184V codon had a more favorable HIV-1 RNA slope than those with wild-type RT 184M codon (p = .0551). This trend was observed, although in a less evident manner, even in pretreated ZDV-sensitive patients. These findings suggest that development of the 3TC-resistance M184V mutation under triple therapy with 3TC, ZDV, and a PI may have unexpected beneficial effects in vivo in addition to those associated with resensitization of ZDV-resistant virus to ZDV.

Detection of genotypically drug-resistant HIV-1 variants and non-B subtypes in recently infected antiretroviral-naive adults in Italy

Sezione di Microbiologia, Dipartimento di Biologia Molecolare, Universita di Siena, Siena, Italy; U.O. di Malattie Infettive, Azienda Ospedaliera di Careggi, Firenze, Firenze, Italy; U.O. di Malattie Infettive, Azienda Ospedaliera di Prato, Prato, Italy; U.O. di Malattie Infettive, Azienda Ospedaliera di Grosseto, Grosseto, Italy; and Servizio di Microbiologia e Virologia, Azienda Ospedaliera Senese, Siena, Italy.

Both Human Immunodeficiency Virus Cellular DNA Sequencing and Plasma RNA Sequencing Are Useful for Detection of Drug Resistance Mutations in Blood Samples from Antiretroviral-Drug-Naive Patients

ABSTRACT Genotypic antiretroviral testing is recommended for newly infected drug-naive subjects, and the material of choice is plasma RNA. Since drug resistance mutations (DRMs) may persist longer in cellular DNA than in plasma RNA, we investigated whether the use of peripheral blood mononuclear cell (PBMC) human immunodeficiency virus (HIV) DNA increases the sensitivity of genotypic testing in antiretroviral-drug-naive subjects. We compared the rate of primary drug resistance in plasma RNA and PBMC DNA in 288 HIV type 1-infected drug-naive persons tested at a single clinical virology center from June 2004 to October 2006. Resistance in the plasma compartment to at least one drug was detected for 64 out of 288 (22.2%) naive patients and in the PBMC compartment for 56 (19.4%) patients. Overall, DRMs were found in 80 out of 288 (27.8%) patients. PBMC DRMs were present in plasma RNA from 16 subjects with wild-type virus infections. Another nine patients had additional DRMs in PBMCs with respect to those detected in plasma RNA. On the other hand, extra plasma DRMs were detected in PBMCs for 24 and 8 subjects with wild-type and drug-resistant virus, respectively. Resistance to more than one class of antiretroviral drug was detected by plasma and PBMC analysis for 25.0% and 36.2% of the subjects, respectively. Our data support the potential utility of genotypic resistance testing of PBMC DNA in conjunction with the currently recommended plasma RNA analysis.

Long-read direct infrared sequencing of crude PCR products for prediction of resistance to HIV-1 reverse transcriptase and protease inhibitors

Patients infected with human irnmunodeficiency virus type 1 (H1V-1) are being treated with a number of different combinations of antiretroviral compounds that target the essential viral enzymes reverse transcriptase and protease. Different sets of HIV-1 mutations that confer drug resistance have been well defined; they allow reasonabe prediction of the drug sensitivity pattern from analysis of the HIV-1 genotype in vivo. Since periodical monitoring of genotypic resistance is expected to improve clinical management in a large number of infected patients, practical and cost-effective methods are highly desirable to set at least medium-scale sequencing in clinical diagnostic settings. We present a complete protocol for direct sequencing of HIV-1 reverse transcriptase and protease-coding regions. Features making the system amenable to routine clinical use include:1.Highly robust presequencing steps (plasma RNA extraction, reverse transcription, and nested PCR);2.Direct use of the crude unpurified PCR product as the sequencing template; and3.Use of infrared-labeled sequencing primers consistently allowing long reads, thus obviating the need for sequencing of both DNA strands.

Frequency and Treatment-Related Predictors of Thymidine-Analogue Mutation Patterns in HIV-1 Isolates after Unsuccessful Antiretroviral Therapy

We investigated, in patients tested between 1991 and 2004, the patterns of mutually exclusive human immunodeficiency virus-1 thymidine-analogue mutations (TAMs) in 4039 reverse-transcriptase sequences with > or = 1 TAM. TAM pattern 1, which included M41L and L210W and excluded K70R and is coupled with more-extensive cross-resistance to drugs, became the most frequent pattern after 1996. In 1465 genotypes from 684 patients in whom highly active antiretroviral therapy (HAART) was unsuccessful, predictors of this pattern were the number of previous HAART regimens undergone (adjusted odds ratio [OR], 1.09 [95% confidence interval {CI}, 1.02-1.16]), use of stavudine/lamivudine (adjusted OR, 1.42 [95% CI, 1.05-1.99]), use of nevirapine (adjusted OR, 1.60 [95% CI, 1.14-2.24]), use of efavirenz (adjusted OR, 1.56 [95% CI, 1.08-2.27]), and use of ritonavir (adjusted OR, 1.35 [95% CI, 1.04-1.75]).