Dried blood spots (DBS) may be a promising alternative specimen type to plasma for measuring the viral load (VL) in HIV-infected individuals in resource-limited settings. However, characterization of assay performance using DBS is incomplete. In this prospective study, the VL was measured in parallel using plasma and DBS specimens collected at the same time from 157 HIV-1-infected individuals. DBS were prepared by dispensing 50 μl of blood onto filter paper cards and were stored desiccated at −20°C. Nucleic acid extraction from plasma and DBS was performed automatically using the Abbott m2000sp instrument, and the VL was measured using the RealTime HIV-1 VL assay, which has a lower limit of detection of 40 HIV RNA copies/ml. The correlation between plasma and DBS results was good (R = 0.91; P < 0.001). The mean difference in the VL (DBS minus plasma) was 0.35 log copies (standard deviation [SD], 0.47 log copies). A total of 40 (26%) paired specimens had a difference of >0.5 log copy, and in 12 (7.8%) it was >1 log copy. the VL from DBS was measurable in 95.7% of specimens with a plasma VL of >2.74 log copies (550 HIV RNA copies/ml). In summary, the VL can reliably be measured using DBS with the Abbott RealTime HIV-1 assay. The estimated lower limit of detection of this automated methodology on DBS is 550 copies/ml, a threshold that may be acceptable for periodic VL monitoring in patients on antiretroviral therapy in resource-limited settings, where early detection of virologic treatment failure is often problematic.

Resistance-associated mutations in the HIV-1 protease modify viral fitness through changes in the catalytic activity and altered binding affinity for substrates and inhibitors. In this report, we examine the effects of 31 mutations at 26 amino acid positions in protease to determine their impact on infectivity and protease inhibitor sensitivity. We found that primary resistance mutations individually decrease fitness and generally increase sensitivity to protease inhibitors, indicating that reduced virion-associated protease activity reduces virion infectivity and the reduced level of per virion protease activity is then more easily titrated by a protease inhibitor. Conversely, mutations at more variable positions (compensatory mutations) confer low-level decreases in sensitivity to all protease inhibitors with little effect on infectivity. We found significant differences in the observed effect on infectivity with a pseudotype virus assay that requires the protease to cleave the cytoplasmic tail of the amphotropic murine leukemia virus (MuLV) Env protein. Additionally, we were able to mimic the fitness loss associated with resistance mutations by directly reducing the level of virion-associated protease activity. Virions containing 50% of a D25A mutant protease were 3- to 5-fold more sensitive to protease inhibitors. This level of reduction in protease activity also resulted in a 2-fold increase in sensitivity to nonnucleoside inhibitors of reverse transcriptase and a similar increase in sensitivity to zidovudine (AZT), indicating a pleiotropic effect associated with reduced protease activity. These results highlight the interplay between enzyme activity, viral fitness, and inhibitor mechanism and sensitivity in the closed system of the viral replication complex.

GS-8374 is a novel bis-tetrahydrofuran HIV-1 protease (PR) inhibitor (PI) with a unique diethylphosphonate moiety. It was selected from a series of analogs containing various di(alkyl)phosphonate substitutions connected via a linker to the para position of a P-1 phenyl ring. GS-8374 inhibits HIV-1 PR with high potency (Ki = 8.1 pM) and with no known effect on host proteases. Kinetic and thermodynamic analysis of GS-8374 binding to PR demonstrated an extremely slow off rate for the inhibitor and favorable contributions of both the enthalpic and entropic components to the total free binding energy. GS-8374 showed potent antiretroviral activity in T-cell lines, primary CD4+ T cells (50% effective concentration [EC50] = 3.4 to 11.5 nM), and macrophages (EC50 = 25.5 nM) and exhibited low cytotoxicity in multiple human cell types. The antiviral potency of GS-8374 was only moderately affected by human serum protein binding, and its combination with multiple approved antiretrovirals showed synergistic effects. When it was tested in a PhenoSense assay against a panel of 24 patient-derived viruses with high-level PI resistance, GS-8374 showed lower mean EC50s and lower fold resistance than any of the clinically approved PIs. Similar to other PIs, in vitro hepatic microsomal metabolism of GS-8374 was efficiently blocked by ritonavir, suggesting a potential for effective pharmacokinetic boosting in vivo. In summary, results from this broad in vitro pharmacological profiling indicate that GS-8374 is a promising candidate to be further assessed as a new antiretroviral agent with potential for clinical efficacy in both treatment-naïve and -experienced patients.

The Abbott RealTime HIV-1 viral load assay uses primers and probes targeted to integrase, which is also the target of integrase inhibitors such as raltegravir. Viral loads of 42 raltegravir-susceptible and 40 raltegravir-resistant specimens were determined using RealTime HIV-1 and Roche Monitor (v1.5). The differences in viral load measurements between assays were comparable in the two groups, demonstrating that the RealTime HIV-1 assay can tolerate raltegravir-selected mutations.

Patterns of HIV-1 protease inhibitor (PI) resistance-associated mutations (RAMs) and effects on PI susceptibility associated with the L76V mutation were studied in a large database. Of 20,501 sequences with ≥1 PI RAM, 3.2% contained L76V; L76V was alone in 0.04%. Common partner mutations included M46I, I54V, V82A, I84V, and L90M. L76V was associated with a 2- to 6-fold decrease in susceptibility to lopinavir, darunavir, amprenavir, and indinavir and a 7- to 8-fold increase in susceptibility to atazanavir and saquinavir.

Abstract

We screened 150 individuals from two recent seroconverter cohorts and found that six (4%) had CXCR4-using viruses. Clonal analysis of these six individuals, along with a seventh individual identified during clinical care as a recent seroconverter, revealed the presence of both X4- and dual-tropic variants in these recently infected adults. The ability of individual CXCR4-using variants to infect cells expressing CD4/CXCR4 or CD4/CCR5 varied dramatically. These data demonstrate that virus populations in some newly infected individuals can consist of either heterogeneous populations containing both CXCR4-using and CCR5-tropic viruses, or homogeneous populations containing only CXCR4-using viruses. The presence of CXCR4-using viruses at early stages of infection suggests that testing for viral tropism before using CCR5 antagonists may be important even in persons with known recent infection. The presence of CXCR4-using viruses in a subset of newly infected individuals could impact the efficacies of vaccine and microbicide strategies that target CCR5-tropic viruses.

Background

Over 150 000 Malawians have started antiretroviral therapy (ART), in which first-line therapy is stavudine/lamivudine/nevirapine. We evaluated drug resistance patterns among patients failing first-line ART on the basis of clinical or immunological criteria in Lilongwe and Blantyre, Malawi.

Methods

Patients meeting the definition of ART failure (new or progressive stage 4 condition, CD4 cell count decline more than 30%, CD4 cell count less than that before treatment) from January 2006 to July 2007 were evaluated. Among those with HIV RNA of more than 1000 copies/ml, genotyping was performed. For complex genotype patterns, phenotyping was performed.

Results

Ninety-six confirmed ART failure patients were identified. Median (interquartile range) CD4 cell count, log10 HIV-1 RNA, and duration on ART were 68 cells/μl (23–174), 4.72 copies/ml (4.26–5.16), and 36.5 months (26.6–49.8), respectively. Ninety-three percent of samples had nonnucleoside reverse transcriptase inhibitor mutations, and 81% had the M184V mutation. The most frequent pattern included M184V and nonnucleoside reverse transcriptase inhibitor mutations along with at least one thymidine analog mutation (56%). Twenty-three percent of patients acquired the K70E or K65R mutations associated with tenofovir resistance; 17% of the patients had pan-nucleoside resistance that corresponded to K65R or K70E and additional resistance mutations, most commonly the 151 complex. Emergence of the K65R and K70E mutations was associated with CD4 cell count of less than 100 cells/μl (odds ratio 6.1) and inversely with the use of zidovudine (odds ratio 0.18). Phenotypic susceptibility data indicated that the nucleoside reverse transcriptase inhibitor backbone with the highest activity for subsequent therapy was zidovudine/lamivudine/tenofovir, followed by lamivudine/tenofovir, and then abacavir/didanosine.

Conclusion

When clinical and CD4 cell count criteria are used to monitor first-line ART failure, extensive nucleoside reverse transcriptase inhibitor and nonnucleoside reverse transcriptase inhibitor resistance emerges, with most patients having resistance profiles that markedly compromise the activity of second-line ART.

Abstract

Detailed comparisons of HIV drug resistance assays are needed to identify the most useful assays for research studies, and to facilitate comparison of results from studies that use different methods. We analyzed nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance in 40 HIV-infected Ugandan infants who had received nevirapine (NVP)-based prophylaxis using the following assays: an FDA-cleared HIV genotyping assay (the ViroSeq HIV-1 Genotyping System v2.0), a commercially available HIV genotyping assay (GeneSeq HIV), a commercially available HIV phenotyping assay (PhenoSense HIV), and a sensitive point mutation assay (LigAmp). ViroSeq and GeneSeq HIV results (NVP resistance yes/no) were similar for 38 (95%) of 40 samples. In 6 (15%) of 40 samples, GeneSeq HIV detected mutations in minor subpopulations that were not detected by ViroSeq, which identified two additional infants with NVP resistance. LigAmp detected low-level mutations in 12 samples that were not detected by ViroSeq; however, LigAmp testing identified only one additional infant with NVP resistance. GeneSeq HIV and PhenoSense HIV determinations of susceptibility differed for specific NNRTIs in 12 (31%) of the 39 samples containing mixtures at relevant mutation positions. PhenoSense HIV did not detect any infants with NVP resistance who were not identified with GeneSeq HIV testing. In this setting, population sequencing-based methods (ViroSeq and GeneSeq HIV) were the most informative and had concordant results for 95% of the samples. LigAmp was useful for the detection and quantification of minority variants. PhenoSense HIV provided a direct and quantitative measure of NNRTI susceptibility.

Detailed comparisons of HIV drug resistance assays are needed to identify the most useful assays for research studies, and to facilitate comparison of results from studies that use different methods. We analyzed nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance in 40 HIV-infected Ugandan infants who had received nevirapine (NVP)-based prophylaxis using the following assays: an FDA-cleared HIV genotyping assay (the ViroSeq HIV-1 Genotyping System v2.0), a commercially available HIV genotyping assay (GeneSeq HIV), a commercially available HIV phenotyping assay (PhenoSense HIV), and a sensitive point mutation assay (LigAmp). ViroSeq and GeneSeq HIV results (NVP resistance yes/no) were similar for 38 (95%) of 40 samples. In 6 (15%) of 40 samples, GeneSeq HIV detected mutations in minor subpopulations that were not detected by ViroSeq, which identified two additional infants with NVP resistance. LigAmp detected low-level mutations in 12 samples that were not detected by ViroSeq; however, LigAmp testing identified only one additional infant with NVP resistance. GeneSeq HIV and PhenoSense HIV determinations of susceptibility differed for specific NNRTIs in 12 (31%) of the 39 samples containing mixtures at relevant mutation positions. PhenoSense HIV did not detect any infants with NVP resistance who were not identified with GeneSeq HIV testing. In this setting, population sequencing-based methods (ViroSeq and GeneSeq HIV) were the most informative and had concordant results for 95% of the samples. LigAmp was useful for the detection and quantification of minority variants. PhenoSense HIV provided a direct and quantitative measure of NNRTI susceptibility.

Background

We previously reported the existence of CXCR4-using HIV-1 in 6–14 week-old Ugandan infants. Whether these viruses were transmitted from the mother perinatally or evolved after transmission is not known. In the current study, we investigated the origin of the CXCR4-using viruses in these infants by comparing HIV-1 envelope clones from the infants to those from their mothers at or near the time of delivery.

Methods

Envelope clones were isolated from five Ugandan infant plasma samples that harbored CXCR4-using viruses, collected at the time of HIV diagnosis (four at birth, one at week 6), and from their mothers at delivery. Coreceptor usage and phylogenetic relatedness of HIV-1 populations in mother–infant pairs were analyzed in detail using the Trofile assay and sequence analysis of envelope clones, respectively.

Results

X4-tropic clones were identified in two mother–infant pairs and dual-tropic clones were found in three pairs, either alone or in combination with R5-tropic viruses. Dual-tropic clones varied in their ability to infect CXCR4-expressing cells. In each mother–infant pair, X4-tropic or dual-tropic clones shared similar phenotypic profiles and V3 sequence patterns; gp160 sequences of X4-tropic and dual-tropic clones from infants were phylogenetically indistinguishable from those of their mothers. The virus populations were phylogenetically homogenous in three infants and segregated according to coreceptor tropism in the remaining two infants.

Conclusions

This study demonstrates that X4-tropic and dual-tropic HIV-1 can be transmitted from mother to infant, before, during or shortly after delivery, and establishes vertical transmission as an important source of CXCR4-using viruses in infants.

Abstract

The role specific reverse transcriptase (RT) drug resistance mutations play in influencing phenotypic susceptibility to RT inhibitors in virus strains with complex resistance interaction patterns was assessed using recombinant viruses that consisted of RT-PCR-amplified pol fragments derived from plasma HIV-1 RNA from two treatment-experienced patients. Specific modifications of key RT amino acids were performed by site-directed mutagenesis. A panel of viruses with defined genotypic resistance mutations was assessed for phenotypic drug resistance. Introduction of M184V into several different clones expressing various RT resistance mutations uniformly decreased susceptibility to abacavir, lamivudine, and didanosine, and increased susceptibility to zidovudine, stavudine, and tenofovir; replication capacity was decreased. The L74V mutation had similar but slightly different effects, contributing to decreased susceptibility to abacavir, lamivudine, and didanosine and increased susceptibility to zidovudine and tenofovir, but in contrast to M184V, L74V contributed to decreased susceptibility to stavudine. In virus strains with the nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations K101E and G190S, the L74V mutation increased replication capacity, consistent with published observations, but replication capacity was decreased in strains without NNRTI resistance mutations. K101E and G190S together tend to decrease susceptibility to all nucleoside RT inhibitors, but the K103N mutation had little effect on nucleoside RT inhibitor susceptibility. Mutational interactions can have a substantial impact on drug resistance phenotype and replication capacity, and this has been exploited in clinical practice with the development of fixed-dose combination pills. However, we are the first to report these mutational interactions using molecularly cloned recombinant strains derived from viruses that occur naturally in HIV-infected individuals.

Lamivudine therapy selects for the M184V mutation. Although this mutation reduces the replicative capacity of human immunodeficiency virus in vitro, its impact on viral fitness in vivo has not been well defined. We used quantitative allele-specific PCR to precisely calculate the fitness differences between the mutated M184V virus and one that had reverted to the wild type in a cohort of patients by selectively interrupting reverse transcriptase inhibitor therapy, and we found that the M184V variants were consistently 4 to 8% less fit than the wild type in the absence of drug. After a lag phase of variable duration, wild-type variants emerged due to continued evolution of pol and back mutation rather than through emergence of an archived wild-type variant.

Objective

To analyze antiretroviral drug susceptibility in HIV from recently infected adults in Rakai, Uganda, prior to the availability of antiretroviral drug treatment.

Methods

Samples obtained at the time of HIV seroconversion (1998–2003) were analyzed using the GeneSeq HIV and PhenoSense HIV assays (Monogram Biosciences, Inc., South San Francisco, California, USA).

Results

Test results were obtained for 104 samples (subtypes: 26A, 1C, 66D, 9A/D, 1C/D, 1 intersubtype recombinant). Mutations used for genotypic surveillance of transmitted antiretroviral drug resistance were identified in six samples: three had nucleoside reverse transcriptase inhibitor (NRTI) surveillance mutations (two had M41L, one had K219R), and three had protease inhibitor surveillance mutations (I47V, F53L, N88D); none had nonnucleoside reverse transcriptase inhibitor (NNRTI) surveillance mutations. Other resistance-associated mutations were identified in some samples. However, none of the samples had a sufficient number of mutations to predict reduced antiretroviral drug susceptibility. Ten (9.6%) of the samples had reduced phenotypic susceptibility to at least one drug (one had partial susceptibility to didanosine, one had nevirapine resistance, and eight had resistance or partial susceptibility to at least one protease inhibitor). Fifty-three (51%) of the samples had hypersusceptibility to at least one drug (seven had zidovudine hypersusceptibility, 28 had NNRTI hypersusceptibility, 34 had protease inhibitor hypersusceptibility). Delavirdine hyper-susceptibility was more frequent in subtype A than D. In subtype D, efavirenz hypersusceptibility was associated with substitutions at codon 11 in HIV-reverse transcriptase.

Conclusion

Phenotyping detected reduced antiretroviral drug susceptibility and hypersusceptibility in HIV from some antiretroviral-naive Ugandan adults that was not predicted by genotyping. Phenotyping may complement genotyping for analysis of antiretroviral drug susceptibility in populations with nonsubtype B HIV infection.

In a phase I/II evaluation of the CXCR4 antagonist AMD3100, human immunodeficiency virus RNA levels were significantly reduced in a single study subject who harbored CXCR4 (X4)-tropic virus, but not in subjects who harbored either dual/mixed (DM)-tropic or CCR5 (R5)-tropic virus (C. W. Hendrix et al., J. Acquir. Immune Defic. Syndr. 37:1253-1262, 2004). In this study, we analyzed the envelope clones of DM-tropic virus in baseline and treated virus populations from 14 subjects. Ten subjects exhibited significant reductions in CXCR4-mediated infectivity after 10 days of AMD3100 therapy relative to baseline (X4 suppressor group), while four subjects had no reduction of CXCR4-mediated infectivity (X4 nonsuppressor group). The baseline viruses of the X4 suppressor group infected CXCR4-expressing cells less efficiently than those of the X4 nonsuppressor group. Clonal analysis indicated that the baseline viruses from the X4 suppressor group contained a higher proportion of R5-tropic variants mixed with CXCR4-using variants, while the X4 nonsuppressor group was enriched for CXCR4-using variants. AMD3100 suppressed X4-tropic variants in all subjects studied, but not all dualtropic variants. Furthermore, dualtropic variants that used CXCR4 efficiently were suppressed by AMD3100, while dualtropic variants that used CXCR4 poorly were not. This study demonstrated that AMD3100 has the ability to suppress both X4-tropic and certain dualtropic variants in vivo. The suppression of CXCR4-using variants by AMD3100 is dependent on both the tropism composition of the virus population and the efficiency of CXCR4 usage of individual variants.

Entecavir (ETV) was developed for the treatment of chronic hepatitis B virus (HBV) infection and is globally approved for that indication. Initial preclinical studies indicated that ETV had no significant activity against human immunodeficiency virus type 1 (HIV-1) in cultured cell lines at physiologically relevant ETV concentrations, using traditional anti-HIV assays. In response to recent clinical observations of anti-HIV activity of ETV in HIV/HBV-coinfected patients not receiving highly active antiretroviral therapy (HAART), additional investigative studies were conducted to expand upon earlier results. An extended panel of HIV-1 laboratory and clinical strains and cell types was tested against ETV, along with a comparison of assay methodologies and resistance profiling. These latest studies confirmed that ETV has only weak activity against HIV, using established assay systems. However, a >100-fold enhancement of antiviral activity (equivalent to the antiviral activity of lamivudine) could be obtained when assay conditions were modified to reduce the initial viral challenge. Also, the selection of a M184I virus variant during the passage of HIV-1 at high concentrations of ETV confirmed that ETV can exert inhibitory pressure on the virus. These findings may have a significant impact on how future assays are performed with compounds to be used in patients infected with HIV. These results support the recommendation that ETV therapy should be administered in concert with HAART for HIV/HBV-coinfected patients.

Following interruption of antiretroviral therapy among individuals with acquired drug resistance, preexisting drug-sensitive virus emerges relatively rapidly. In contrast, wild-type virus is not archived in individuals infected with drug-resistant human immunodeficiency virus (HIV) and thus cannot emerge rapidly in the absence of selective drug pressure. Fourteen recently HIV-infected patients with transmitted drug-resistant virus were followed for a median of 2.1 years after the estimated date of infection (EDI) without receiving antiretroviral therapy. HIV drug resistance and pol replication capacity (RC) in longitudinal plasma samples were assayed. Resistance mutations were characterized as pure populations or mixtures. The mean time to first detection of a mixture of wild-type and drug-resistant viruses was 96 weeks (1.8 years) (95% confidence interval, 48 to 192 weeks) after the EDI. The median time to loss of detectable drug resistance using population-based assays ranged from 4.1 years (conservative estimate) to longer than the lifetime of the individual (less conservative estimate). The transmission of drug-resistant virus was not associated with virus with reduced RC. Sexual transmission of HIV selects for highly fit drug-resistant variants that persist for years. The prolonged persistence of transmitted drug resistance strongly supports the routine use of HIV resistance genotyping for all newly diagnosed individuals.

Many studies have demonstrated that the third variable region (V3) of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) is a major determinant of coreceptor tropism. Other regions in the surface gp120 subunit of Env can modulate coreceptor tropism in a manner that is not fully understood. In this study, we evaluated the effect of env determinants outside of V3 on coreceptor usage through the analysis of (i) patient-derived env clones that differ in coreceptor tropism, (ii) chimeric env sequences, and (iii) site-directed mutants. The introduction of distinct V3 sequences from CXCR4-using clones into an R5-tropic env backbone conferred the inefficient use of CXCR4 in some but not all cases. Conversely, in many cases, X4- and dual-tropic env backbones containing the V3 sequences of R5-tropic clones retained the ability to use CXCR4, suggesting that sequences outside of the V3 regions of these CXCR4-using clones were responsible for CXCR4 use. The determinants of CXCR4 use in a set of dual-tropic env sequences with V3 sequences identical to those of R5-tropic clones mapped to the gp41 transmembrane (TM) subunit. In one case, a single-amino-acid substitution in the fusion peptide of TM was able to confer CXCR4 use; however, TM substitutions associated with CXCR4 use varied among different env sequences. These results demonstrate that sequences in TM can modulate coreceptor specificity and that env sequences other than that of V3 may facilitate efficient CXCR4-mediated entry. We hypothesize that the latter plays an important role in the transition from CCR5 to CXCR4 coreceptor use.

GS-9148 [(5-(6-amino-purin-9-yl)-4-fluoro-2,5-dihydro-furan-2-yloxymethyl)phosphonic acid] is a novel ribose-modified human immunodeficiency virus type 1 (HIV-1) nucleotide reverse transcriptase (RT) inhibitor (NRTI) selected from a series of nucleoside phosphonate analogs for its favorable in vitro biological properties including (i) a low potential for mitochondrial toxicity, (ii) a minimal cytotoxicity in renal proximal tubule cells and other cell types, (iii) synergy in combination with other antiretrovirals, and (iv) a unique resistance profile against multiple NRTI-resistant HIV-1 strains. Notably, antiviral resistance analysis indicated that neither the K65R, L74V, or M184V RT mutation nor their combinations had any effect on the antiretroviral activity of GS-9148. Viruses carrying four or more thymidine analog mutations showed a substantially smaller change in GS-9148 activity relative to that observed with most marketed NRTIs. GS-9131, an ethylalaninyl phosphonoamidate prodrug designed to maximize the intracellular delivery of GS-9148, is a potent inhibitor of multiple subtypes of HIV-1 clinical isolates, with a mean 50% effective concentration of 37 nM. Inside cells, GS-9131 is readily hydrolyzed to GS-9148, which is further phosphorylated to its active diphosphate metabolite (A. S. Ray, J. E. Vela, C. G. Boojamra, L. Zhang, H. Hui, C. Callebaut, K. Stray, K.-Y. Lin, Y. Gao, R. L. Mackman, and T. Cihlar, Antimicrob. Agents Chemother. 52:648-654, 2008). GS-9148 diphosphate acts as a competitive inhibitor of RT with respect to dATP (Ki = 0.8 μM) and exhibits low inhibitory potency against host polymerases including DNA polymerase γ. Oral administration of GS-9131 to beagle dogs at a dose of 3 mg/kg of body weight resulted in high and persistent levels of GS-9148 diphosphate in peripheral blood mononuclear cells (with a maximum intracellular concentration of >9 μM and a half-life of >24 h). This favorable preclinical profile makes GS-9131 an attractive clinical development candidate for the treatment of patients infected with NRTI-resistant HIV.

Despite the success of highly active antiretroviral therapy, the current emergence and spread of drug-resistant variants of human immunodeficiency virus (HIV) stress the need for new inhibitors with distinct properties. We designed, produced, and screened a library of compounds based on an original l-lysine scaffold for their potentials as HIV type 1 (HIV-1) protease inhibitors (PI). One candidate compound, PL-100, emerged as a specific and noncytotoxic PI that exhibited potent inhibition of HIV-1 protease and viral replication in vitro (Ki, ∼36 pM, and 50% effective concentration [EC50], ∼16 nM, respectively). To confirm that PL-100 possessed a favorable resistance profile, we performed a cross-resistance study using a panel of 63 viral strains from PI-experienced patients selected for the presence of primary PI mutations known to confer resistance to multiple PIs now in clinical use. The results showed that PL-100 retained excellent antiviral activity against almost all of these PI-resistant viruses and that its performance in this regard was superior to those of atazanavir, amprenavir, indinavir, lopinavir, nelfinavir, and saquinavir. In almost every case, the increase in the EC50 for PL-100 observed with viruses containing multiple mutations in protease was far less than that obtained with the other drugs tested. These data underscore the potential for PL-100 to be used in the treatment of drug-resistant HIV disease and argue for its further development.

In human immunodeficiency virus type 1 (HIV-1) subtype B, CXCR4 coreceptor use ranges from ∼20% in early infection to ∼50% in advanced disease. Coreceptor use by non-subtype B HIV is less well characterized. We studied coreceptor tropism of subtype A and D HIV-1 collected from 68 pregnant, antiretroviral drug-naive Ugandan women (HIVNET 012 trial). None of 33 subtype A or 10 A/D-recombinant viruses used the CXCR4 coreceptor. In contrast, nine (36%) of 25 subtype D viruses used both CXCR4 and CCR5 coreceptors. Clonal analyses of the nine subtype D samples with dual or mixed tropism revealed heterogeneous viral populations comprised of X4-, R5-, and dual-tropic HIV-1 variants. In five of the six samples with dual-tropic strains, V3 loop sequences of dual-tropic clones were identical to those of cocirculating R5-tropic clones, indicating the presence of CXCR4 tropism determinants outside of the V3 loop. These dual-tropic variants with R5-tropic-like V3 loops, which we designated “dual-R,” use CCR5 much more efficiently than CXCR4, in contrast to dual-tropic clones with X4-tropic-like V3 loops (“dual-X”). These observations have implications for pathogenesis and treatment of subtype D-infected individuals, for the association between V3 sequence and coreceptor tropism phenotype, and for understanding potential mechanisms of evolution from exclusive CCR5 use to efficient CXCR4 use by subtype D HIV-1.

Deletions, insertions, and amino acid substitutions in the β3-β4 hairpin loop-coding region of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have been associated with resistance to nucleoside RT inhibitors when appearing in combination with other mutations in the RT-coding region. In this work, we have measured the in vivo fitness of HIV-1 variants containing a deletion of 3 nucleotides affecting codon 69 (Δ69) of the viral RT as well as the replication capacity (RC) ex vivo of a series of recombinant HIV-1 variants carrying an RT bearing the Δ69 deletion or the T69A mutation in a multidrug-resistant (MDR) sequence background, including the Q151M complex and substitutions M184V, K103N, Y181C, and G190A. Patient-derived viral clones having RTs with Δ69 together with S163I showed increased RCs under drug pressure. These data were consistent with the viral population dynamics observed in a long-term-treated HIV-1-infected patient. In the absence of drugs, viral clones containing T69A replicated more efficiently than those having Δ69, but only when patient-derived sequences corresponding to RT residues 248 to 527 were present. These effects could be attributed to a functional interaction between the C-terminal domain of the p66 subunit (RNase H domain) and the DNA polymerase domain of the RT. Finally, recombinant HIV-1 clones bearing RTs with MDR-associated mutations, including deletions at codon 69, showed increased susceptibilities to protease inhibitors in phenotypic assays. These effects correlated with impaired Gag cleavage and could be attributed to delayed maturation and decreased production of active protease in those variants.

Two novel human immunodeficiency virus protease mutations, I84C and I84A, were identified in patient isolates. The mutants with I84C displayed high-level resistance (median, at least 56-fold) to nelfinavir and saquinavir, but the majority remained susceptible to lopinavir. In contrast, isolates with the I84A mutation exhibited ≥33-fold median increased levels of resistance to nelfinavir, indinavir, amprenavir, ritonavir, lopinavir, saquinavir, and atazanavir. Isolates with the I84A or I84C mutation tended to be more resistant than the isolates with the I84V mutation. Modeling of the structure of the mutant proteases indicated that the I84V, I84C, and I84A mutations all create unoccupied volume in the active site, with I84A introducing the greatest change in the accessible surface area from that of the wild-type structure.

Background

HIV protease inhibitor (PI) therapy results in the rapid selection of drug resistant viral variants harbouring one or two substitutions in the viral protease. To combat PI resistance development, two approaches have been developed. The first is to increase the level of PI in the plasma of the patient, and the second is to develop novel PI with high potency against the known PI-resistant HIV protease variants. Both approaches share the requirement for a considerable increase in the number of protease mutations to lead to clinical resistance, thereby increasing the genetic barrier. We investigated whether HIV could yet again find a way to become less susceptible to these novel inhibitors.

Methods and Findings

We have performed in vitro selection experiments using a novel PI with an increased genetic barrier (RO033-4649) and demonstrated selection of three viruses 4- to 8-fold resistant to all PI compared to wild type. These PI-resistant viruses did not have a single substitution in the viral protease. Full genomic sequencing revealed the presence of NC/p1 cleavage site substitutions in the viral Gag polyprotein (K436E and/or I437T/V) in all three resistant viruses. These changes, when introduced in a reference strain, conferred PI resistance. The mechanism leading to PI resistance is enhancement of the processing efficiency of the altered substrate by wild-type protease. Analysis of genotypic and phenotypic resistance profiles of 28,000 clinical isolates demonstrated the presence of these NC/p1 cleavage site mutations in some clinical samples (codon 431 substitutions in 13%, codon 436 substitutions in 8%, and codon 437 substitutions in 10%). Moreover, these cleavage site substitutions were highly significantly associated with reduced susceptibility to PI in clinical isolates lacking primary protease mutations. Furthermore, we used data from a clinical trial (NARVAL, ANRS 088) to demonstrate that these NC/p1 cleavage site changes are associated with virological failure during PI therapy.

Conclusions

HIV can use an alternative mechanism to become resistant to PI by changing the substrate instead of the protease. Further studies are required to determine to what extent cleavage site mutations may explain virological failure during PI therapy.

Changes in the cleavage site of the Gag substrate for the HIV protease can convey resistance to protease inhibitors and might contribute to virologic failure during therapy that includes these drugs.

Editors' Summary

Background.

Twenty-five years ago, infection with the human immunodeficiency virus (HIV)—the causative agent of AIDS—was a death sentence. However, drugs that attack various stages of the HIV life cycle were soon developed that, although not curing the infection, kept it in check when used in combination and greatly increased the life expectancy of people infected with HIV. Unfortunately, viruses resistant to these drugs have rapidly emerged and antiviral therapy now fails in many patients. The use of HIV protease inhibitors (PIs) in combination therapies, for example, has led to the stepwise selection of viral variants resistant to these drugs. Resistance is first acquired when the viral protease changes so that PIs no longer bind to it and inhibit it efficiently. These changes often reduce the efficiency with which the protease binds its substrates—polyproteins called Gag and GagPol that it chops up into smaller proteins to make new viral particles. So the next step is the accumulation of changes elsewhere in the protease that make it work better, and sometimes changes in its substrate that make it easier to cut; these compensatory changes do not directly affect viral resistance to PIs.

Why Was This Study Done?

To prevent viruses with resistance to PIs emerging, drug doses are kept high in patients and new PIs are being developed with high potency against known PI-resistant HIV variants. Both approaches set a “high genetic barrier” to the development of PI resistance by ensuring that HIV has to incorporate many changes in its protease to become resistant. But, the HIV genome naturally changes—mutates—very rapidly, so novel HIV variants could emerge that are less susceptible to the new potent PIs without the virus having to leap this high genetic barrier. In this study, the researchers have investigated whether HIV can find an alternative route to PI resistance that does not involve the introduction of multiple changes into its protease.

What Did the Researchers Do and Find?

The researchers took wild-type HIV and treated it in the laboratory with a new PI regimen that has a high genetic barrier. By gradually increasing its concentration, the researchers selected three viral populations that were able to grow in 4- to 8-fold higher concentrations of the PI than wild-type virus. None of these populations had mutations in the viral protease. Instead, they all had mutations near one of the sites—the NC/p1 site—where the protease normally cuts the Gag polyprotein. These mutations, the researchers report, enhanced the overall efficiency with which the wild-type protease cleaved the polyprotein, and a selection experiment with another PI showed that the development of PI resistance through alterations near the NC/p1 cleavage site was not unique to one PI. The researchers also investigated the potential clinical significance of this new drug resistance mechanism by looking for the same mutations in nearly 30,000 patient samples. Many of the samples did indeed have these mutations. Finally, they showed that mutations at the NC/p1 cleavage site were associated with virological failure (increased viral replication) during PI therapy in an ongoing clinical trial.

What Do These Findings Mean?

These results suggest that increased polyprotein processing because of mutations in the natural substrate of the HIV protease might be a new mechanism by which HIV can become resistant to PIs. This strategy, which occurs in the laboratory and in patients, allows HIV to develop PI resistance without the need for multiple changes in its protease and so avoids the high genetic barrier to resistance that new PIs provide. Clinical studies are now needed to test which of the mutations seen in this study contribute to virological failure, whether the degree of this failure is clinically relevant, and whether these substrate mutations enhance the effect of protease mutations. If the clinical importance of the new mechanism is confirmed, genetic examination of both the polyprotein and the protease will be needed when trying to figure out why a PI-containing therapy is failing in individual patients. Furthermore, it will be necessary to test whether this mechanism can contribute to the development of resistance when evaluating new drugs.

Additional Information.

Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040036.

US National Institute of Allergy and Infectious Diseases factsheet on HIV infection and AIDS

US Department of Health and Human Services information on AIDS

US Centers for Disease Control and Prevention information on HIV/AIDS

Aidsmap information on HIV and AIDS provided by the charity NAM

BioAfrica, Bioinformatics for HIV Research, information on HIV-1 protease cleavage sites

Genotypic patterns associated with nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance in the absence of well-characterized resistance mutations were identified using a database (n > 47,000) of phenotype-genotype data. Among samples with no known NNRTI mutations, the most resistant samples contained K101P (n = 35) or a combination of K103R and V179D (n = 41). Site-directed mutagenesis confirmed the importance of these mutations.

We examined the population dynamics of human immunodeficiency virus type 1 pro variants during the evolution of resistance to the protease inhibitor ritonavir (RTV) in vivo. pro variants were followed in subjects who had added RTV to their previously failed reverse transcriptase inhibitor therapy using a heteroduplex tracking assay designed to detect common resistance-associated mutations. In most cases the initial variant appeared rapidly within 2 to 3 months followed by one or more subsequent population turnovers. Some of the subsequent transitions between variants were rapid, and some were prolonged with the coexistence of multiple variants. In several cases variants without resistance mutations persisted despite the emergence of new variants with an increasing number of resistance-associated mutations. Based on the rate of turnover of pro variants in the RTV-treated subjects we estimated that the mean fitness of newly emerging variants was increased 1.2-fold (range, 1.02 to 1.8) relative to their predecessors. A subset of pro genes was introduced into infectious molecular clones. The corresponding viruses displayed impaired replication capacity and reduced susceptibility to RTV. A subset of these clones also showed increased susceptibility to two nonnucleoside reverse transcriptase inhibitors and the protease inhibitor saquinavir. Finally, a significant correlation between the reduced replication capacity and reduced processing at the gag NC-p1 processing site was noted. Our results reveal a complexity of patterns in the evolution of resistance to a protease inhibitor. In addition, these results suggest that selection for resistance to one protease inhibitor can have pleiotropic effects that can affect fitness and susceptibility to other drugs.