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ABSTRACT In the discovery of secondary metabolites, analysis of sequence data is a promising exploration path that remains largely underutilized due to the lack of computational platforms that enable such a systematic approach on a large scale. In this work, we present IMG-ABC (https://img.jgi.doe.gov/abc), an atlas of biosynthetic gene clusters within the Integrated Microbial Genomes (IMG) system, which is aimed at harnessing the power of “big” genomic data for discovering small molecules. IMG-ABC relies on IMG's comprehensive integrated structural and functional genomic data for the analysis of biosynthetic gene clusters (BCs) and associated secondary metabolites (SMs). SMs and BCs serve as the two main classes of objects in IMG-ABC, each with a rich collection of attributes. A unique feature of IMG-ABC is the incorporation of both experimentally validated and computationally predicted BCs in genomes as well as metagenomes, thus identifying BCs in uncultured populations and rare taxa. We demonstrate the strength of IMG-ABC's focused integrated analysis tools in enabling the exploration of microbial secondary metabolism on a global scale, through the discovery of phenazine-producing clusters for the first time in Alphaproteobacteria. IMG-ABC strives to fill the long-existent void of resources for computational exploration of the secondary metabolism universe; its underlying scalable framework enables traversal of uncovered phylogenetic and chemical structure space, serving as a doorway to a new era in the discovery of novel molecules. IMPORTANCE IMG-ABC is the largest publicly available database of predicted and experimental biosynthetic gene clusters and the secondary metabolites they produce. The system also includes powerful search and analysis tools that are integrated with IMG's extensive genomic/metagenomic data and analysis tool kits. As new research on biosynthetic gene clusters and secondary metabolites is published and more genomes are sequenced, IMG-ABC will continue to expand, with the goal of becoming an essential component of any bioinformatic exploration of the secondary metabolism world.

HIV-1 evolution in the cerebrospinal fluid (CSF) and plasma may result in discordant drug resistance mutations (DRMs) in the compartments. Single-genome amplification (SGA) was used to generate partial HIV-1 polymerase genomes in paired CSF and plasma samples from 12 HIV-1-positive participants in the CNS HIV Antiretroviral Therapy Effects Research (CHARTER) study who were classified as neurocognitively unimpaired or with various degrees of HIV-associated neurocognitive disorders (HAND). Subjects were viremic on combination antiretroviral therapy (cART). HIV-1 DRMs and phylogenetic characteristics were determined using the Stanford HIVdb program and phylogenetic analyses. Individual DRMs were identified more frequently in plasma than in paired CSF ( P =  0.0078). Significant differences in the ratios of DRMs in CSF and plasma were found in 3 individuals with HAND (3/7 = 43%). Two HAND subjects (2/7 = 29%) demonstrated one DRM in CSF not identified in paired plasma. Longitudinal analyses ( n  = 4) revealed significant temporal differences in the ratios of DRMs in the compartments. Statistically significant differences in the frequency of DRMs in the CSF and plasma are readily found in those on nonsuppressive cART. While compartment-based DRM discordance was largely consistent with increased drug-selective pressures in the plasma, overrepresentation of DRMs in the central nervous system (CNS) can occur. Underlying mechanisms of HAND are complex and multifactorial. The clinical impact of DRM discordance on viral persistence and HAND pathogenesis remains unclear and warrants further investigation in larger, longitudinal cohorts. IMPORTANCE Several antiretroviral agents do not efficiently enter the CNS, and independent evolution of HIV-1 viral variants in the CNS and plasma can occur. We used single-genome amplification (SGA) in cross-sectional and longitudinal analyses to uniquely define both the identity and relative proportions of drug resistance mutations (DRMs) on individual HIV-1 polymerase genomes in the cerebrospinal fluid (CSF) and plasma in individuals with incomplete viral suppression and known neurocognitive status. Statistically significant differences in the ratio of DRMs in the CSF and plasma were readily found in those on nonsuppressive cART, and overrepresentation of DRMs in the CNS can occur. Although questions about the clinical significance of DRM discordance remain, in the quest for viral eradication, it is important to recognize that a significant, dynamic, compartment-based DRM ratio imbalance can exist, as it has the potential to go unnoticed in the setting of standard clinical drug resistance testing., (Copyright © 2020 American Society for Microbiology.)

Unlabelled: To improve our understanding of the similarities and differences between neutralizing antibodies elicited by simian-human immunodeficiency virus (SHIV)-infected rhesus macaques and human immunodeficiency virus type 1 (HIV-1)-infected humans, we examined the plasma of 13 viremic macaques infected with SHIVSF162P3Nand 85 HIV-1-infected humans with known times of infection. We identified 5 macaques (38%) from 1 to 2 years postinfection (p.i.) with broadly neutralizing antibodies (bnAbs) against tier 2 HIV-1. In comparison, only 2 out of 42 (5%) human plasma samples collected in a similar time frame of 1 to 3 years p.i. exhibited comparable neutralizing breadths and potencies, with the number increasing to 7 out of 21 (30%) after 3 years p.i. Plasma mapping with monomeric gp120 identified only 2 out of 9 humans and 2 out of 4 macaques that contained gp120-reactive neutralizing antibodies, indicating distinct specificities in these plasma samples, with most of them recognizing the envelope trimer (including gp41) rather than the gp120 monomer. Indeed, a total of 20 gp120-directed monoclonal antibodies (MAbs) isolated from a human subject (AD358) and a Chinese rhesus macaque (GB40) displayed no or limited neutralizing activity against tier 2 strains. These isolated MAbs, mapped to the CD4-binding site, the V3 loop, the inner domain, and the C5 region of gp120, revealed genetic similarity between the human and macaque immunoglobulin genes used to encode some V3-directed MAbs. These results also support the use of envelope trimer probes for efficient isolation of HIV-1 bnAbs., Importance: HIV-1 vaccine research can benefit from understanding the development of broadly neutralizing antibodies (bnAbs) in rhesus macaques, commonly used to assess vaccine immunogenicity and efficacy. Here, we examined 85 HIV-1-infected humans and 13 SHIVSF162P3N-infected macaques for bnAbs and found that, similar to HIV-1-infected humans, bnAbs in SHIV-infected macaques are also rare, but their development might have been faster in some of the studied macaques. Plasma mapping with monomeric gp120 indicated that most bnAbs bind to the envelope trimer rather than the gp120 monomer. In support of this, none of the isolated gp120-reactive monoclonal antibodies (MAbs) displayed the neutralization breadth observed in the corresponding plasma. However, the MAb sequences revealed similarity between human and macaque genes used to encode some V3-directed MAbs. Our study sheds light on the timing and development of bnAbs in SHIV-infected macaques in comparison to HIV-1-infected humans and highlights the use of envelope trimer probes for efficient recovery of bnAbs., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Human leukocyte antigen (HLA) class I-associated polymorphisms in HIV-1 that persist upon transmission to HLA-mismatched hosts may spread in the population as the epidemic progresses. Transmission of HIV-1 sequences containing such adaptations may undermine cellular immune responses to the incoming virus in future hosts. Building upon previous work, we investigated the extent of HLA-associated polymorphism accumulation in HIV-1 polymerase (Pol) through comparative analysis of linked HIV-1/HLA class I genotypes sampled during historic (1979 to 1989; n = 338) and modern (2001 to 2011; n = 278) eras from across North America (Vancouver, BC, Canada; Boston, MA; New York, NY; and San Francisco, CA). Phylogenies inferred from historic and modern HIV-1 Pol sequences were star-like in shape, with an inferred most recent common ancestor (epidemic founder virus) sequence nearly identical to the modern North American subtype B consensus sequence. Nevertheless, modern HIV-1 Pol sequences exhibited roughly 2-fold-higher patristic (tip-to-tip) genetic distances than historic sequences, with HLA pressures likely driving ongoing diversification. Moreover, the frequencies of published HLA-associated polymorphisms in individuals lacking the selecting HLA class I allele was on average ∼2.5-fold higher in the modern than in the historic era, supporting their spread in circulation, though some remained stable in frequency during this time. Notably, polymorphisms restricted by protective HLA alleles appear to be spreading to a greater relative extent than others, though these increases are generally of modest absolute magnitude. However, despite evidence of polymorphism spread, North American hosts generally remain at relatively low risk of acquiring an HIV-1 polymerase sequence substantially preadapted to their HLA profiles, even in the present era., Importance: HLA class I-restricted cytotoxic T-lymphocyte (CTL) escape mutations in HIV-1 that persist upon transmission may accumulate in circulation over time, potentially undermining host antiviral immunity to the transmitted viral strain. We studied >600 experimentally collected HIV-1 polymerase sequences linked to host HLA information dating back to 1979, along with phylogenetically reconstructed HIV-1 sequences dating back to the virus' introduction into North America. Overall, our results support the gradual spread of many-though not all-HIV-1 polymerase immune escape mutations in circulation over time. This is consistent with recent observations from other global regions, though the extent of polymorphism accumulation in North America appears to be lower than in populations with high seroprevalence, older epidemics, and/or limited HLA diversity. Importantly, the risk of acquiring an HIV-1 polymerase sequence at transmission that is substantially preadapted to one's HLA profile remains relatively low in North America, even in the present era., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Host and viral factors influence the HIV-1 infection course. Reduced Nef function has been observed in HIV-1 controllers during the chronic phase, but the kinetics and mechanisms of Nef attenuation in such individuals remain unclear. We examined plasma RNA-derived Nef clones from 10 recently infected individuals who subsequently suppressed viremia to less than 2,000 RNA copies/ml within 1 year postinfection (acute controllers) and 50 recently infected individuals who did not control viremia (acute progressors). Nef clones from acute controllers displayed a lesser ability to downregulate CD4 and HLA class I from the cell surface and a reduced ability to enhance virion infectivity compared to those from acute progressors (all P<0.01). HLA class I downregulation activity correlated inversely with days postinfection (Spearman's R=-0.85, P=0.004) and positively with baseline plasma viral load (Spearman's R=0.81, P=0.007) in acute controllers but not in acute progressors. Nef polymorphisms associated with functional changes over time were identified in follow-up samples from six controllers. For one such individual, mutational analyses indicated that four polymorphisms selected by HLA-A*31 and B*37 acted in combination to reduce Nef steady-state protein levels and HLA class I downregulation activity. Our results demonstrate that relative control of initial HIV-1 viremia is associated with Nef clones that display reduced function, which in turn may influence the course of HIV-1 infection. Transmission of impaired Nef sequences likely contributed in part to this observation; however, accumulation of HLA-associated polymorphisms in Nef that impair function also suggests that CD8+ T-cell pressures play a role in this phenomenon., Importance: Rare individuals can spontaneously control HIV-1 viremia in the absence of antiretroviral treatment. Understanding the host and viral factors that contribute to the controller phenotype may identify new strategies to design effective vaccines or therapeutics. The HIV-1 Nef protein enhances viral pathogenesis through multiple mechanisms. We examined the function of plasma HIV-1 RNA-derived Nef clones isolated from 10 recently infected individuals who subsequently controlled HIV viremia compared to the function of those from 50 individuals who failed to control viremia. Our results demonstrate that early Nef clones from HIV controllers displayed lower HLA class I and CD4 downregulation activity, as well as a reduced ability to enhance virion infectivity. The accumulation of HLA-associated polymorphisms in Nef during the first year postinfection was associated with impaired protein function in some controllers. This report highlights the potential for host immune responses to modulate HIV pathogenicity and disease outcome by targeting cytotoxic T lymphocyte (CTL) epitopes in Nef., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

An attenuation of the HIV-1 replication capacity (RC) has been observed for immune-mediated escape mutations in Gag restricted by protective HLA alleles. However, the extent to which escape mutations affect other viral proteins during natural infection is not well understood. We generated recombinant viruses encoding plasma HIV-1 RNA integrase sequences from antiretroviral-naïve individuals with early (n = 88) and chronic (n = 304) infections and measured the in vitro RC of each. In contrast to data from previous studies of Gag, we observed little evidence that host HLA allele expression was associated with integrase RC. A modest negative correlation was observed between the number of HLA-B-associated integrase polymorphisms and RC in chronic infection (R = -0.2; P = 0.003); however, this effect was not driven by mutations restricted by protective HLA alleles. Notably, the integrase variants S119R, G163E, and I220L, which represent uncommon polymorphisms associated with HLA-C*05, -A*33, and -B*52, respectively, correlated with lower RC (all q < 0.2). We identified a novel C*05-restricted epitope (HTDNGSNF(114-121)) that likely contributes to the selection of the S119R variant, the polymorphism most significantly associated with lower RC in patient sequences. An NL4-3 mutant encoding the S119R polymorphism displayed a ~35%-reduced function that was rescued by a single compensatory mutation of A91E. Together, these data indicate that substantial HLA-driven attenuation of integrase is not a general phenomenon during HIV-1 adaptation to host immunity. However, uncommon polymorphisms selected by HLA alleles that are not conventionally regarded to be protective may be associated with impaired protein function. Vulnerable epitopes in integrase might therefore be considered for future vaccine strategies.

Mutations that allow escape from CD8 T-cell responses are common in HIV-1 and may attenuate pathogenesis by reducing viral fitness. While this has been demonstrated for individual cases, a systematic investigation of the consequence of HLA class I-mediated selection on HIV-1 in vitro replication capacity (RC) has not been undertaken. We examined this question by generating recombinant viruses expressing plasma HIV-1 RNA-derived Gag-Protease sequences from 66 acute/early and 803 chronic untreated subtype B-infected individuals in an NL4-3 background and measuring their RCs using a green fluorescent protein (GFP) reporter CD4 T-cell assay. In acute/early infection, viruses derived from individuals expressing the protective alleles HLA-B*57, -B*5801, and/or -B*13 displayed significantly lower RCs than did viruses from individuals lacking these alleles (P < 0.05). Furthermore, acute/early RC inversely correlated with the presence of HLA-B-associated Gag polymorphisms (R = -0.27; P = 0.03), suggesting a cumulative effect of primary escape mutations on fitness during the first months of infection. At the chronic stage of infection, no strong correlations were observed between RC and protective HLA-B alleles or with the presence of HLA-B-associated polymorphisms restricted by protective alleles despite increased statistical power to detect these associations. However, RC correlated positively with the presence of known compensatory mutations in chronic viruses from B*57-expressing individuals harboring the Gag T242N mutation (n = 50; R = 0.36; P = 0.01), suggesting that the rescue of fitness defects occurred through mutations at secondary sites. Additional mutations in Gag that may modulate the impact of the T242N mutation on RC were identified. A modest inverse correlation was observed between RC and CD4 cell count in chronic infection (R = -0.17; P < 0.0001), suggesting that Gag-Protease RC could increase over the disease course. Notably, this association was stronger for individuals who expressed B*57, B*58, or B*13 (R = -0.27; P = 0.004). Taken together, these data indicate that certain protective HLA alleles contribute to early defects in HIV-1 fitness through the selection of detrimental mutations in Gag; however, these effects wane as compensatory mutations accumulate in chronic infection. The long-term control of HIV-1 in some persons who express protective alleles suggests that early fitness hits may provide lasting benefits.

Human immunodeficiency virus type 1 (HIV-1) controllers maintain viremia at <2,000 RNA copies/ml without antiretroviral therapy. Viruses from controllers with chronic infection were shown to exhibit impaired replication capacities, in part associated with escape mutations from cytotoxic-T-lymphocyte (CTL) responses. In contrast, little is known about viruses during acute/early infection in individuals who subsequently become HIV controllers. Here, we examine the viral replication capacities, HLA types, and virus sequences from 18 HIV-1 controllers identified during primary infection. gag-protease chimeric viruses constructed using the earliest postinfection samples displayed significantly lower replication capacities than isolates from persons who failed to control viremia (P = 0.0003). Protective HLA class I alleles were not enriched in these early HIV controllers, but viral sequencing revealed a significantly higher prevalence of drug resistance mutations associated with impaired viral fitness in controllers than in noncontrollers (6/15 [40.0%] versus 10/80 [12.5%], P = 0.018). Moreover, of two HLA-B57-positive (B57(+)) controllers identified, both harbored, at the earliest time point tested, signature escape mutations within Gag that likewise impair viral replication capacity. Only five controllers did not express "protective" alleles or harbor viruses with drug resistance mutations; intriguingly, two of them displayed typical B57 signature mutations (T242N), suggesting the acquisition of attenuated viruses from B57(+) donors. These data indicate that acute/early stage viruses from persons who become controllers have evidence of reduced replication capacity during the initial stages of infection which is likely associated with transmitted or acquired CTL escape mutations or transmitted drug resistance mutations. These data suggest that viral dynamics during acute infection have a major impact on HIV disease outcome.

We evaluated the human immunodeficiency virus type 1 (HIV-1) integrase coding region of the pol gene for the presence of natural polymorphisms in patients during early infection (AHI) and with triple-class drug-resistant HIV-1 (MDR). We analyzed selected recombinant viruses containing patient-derived HIV-1 integrase for susceptibility to a panel of strand transfer integrase inhibitors (InSTI). A pretreatment sequence analysis of the integrase coding region was performed for 112 patients identified during acute or early infection and 15 patients with triple-class resistance. A phenotypic analysis was done on 10 recombinant viruses derived from nine patients against a panel of six diverse InSTI. Few of the polymorphisms associated with in vitro InSTI resistance were identified in the samples from newly infected individuals or those patients with MDR HIV-1. We identified polymorphisms V72I, L74I, T97A, V151I, M154I/L, E157Q, V165I, V201I, I203M, T206S, and S230N. V72I was the most common, seen in 63 (56.3%) of the AHI samples. E157Q was the only naturally occurring mutation thought to contribute to resistance to elvitegravir, raltegravir, and L-870,810. None of the patient-derived viruses demonstrated any significant decrease in susceptibility to the drugs tested. In summary, the integrase coding region contains as much natural variation as that seen in protease, but mutations associated with high-level resistance to existing InSTI are rarely, if ever, present in integrase naïve patients, especially those being used clinically. Most of the highly prevalent polymorphisms have little effect on InSTI susceptibility in the absence of specific primary mutations. Baseline testing for integrase susceptibility in InSTI-naïve patients is not currently warranted.

Human immunodeficiency virus effectively evades CD8(+) T-cell responses through the development of CD8 escape mutations. Recent reports documenting reversion of transmitted mutations and the impact of specific escape mutations upon viral replication suggest that complex forces limit the accumulation of CD8 escape mutations at the population level. However, the presence of compensatory mutations capable of alleviating the impact of CD8 escape mutations on replication capacity may enable their persistence in an HLA-mismatched host. Herein, we illustrate the long-term stability of stereotypic escape mutations in the immunodominant HLA-B27-restricted epitope KK10 in p24/Gag following transmission when accompanied by a specific compensatory mutation.

During acute human immunodeficiency virus type 1 (HIV-1) infection, early host cellular immune responses drive viral evolution. The rates and extent of these mutations, however, remain incompletely characterized. In a cohort of 98 individuals newly infected with HIV-1 subtype B, we longitudinally characterized the rates and extent of HLA-mediated escape and reversion in Gag, Pol, and Nef using a rational definition of HLA-attributable mutation based on the analysis of a large independent subtype B data set. We demonstrate rapid and dramatic HIV evolution in response to immune pressures that in general reflect established cytotoxic T-lymphocyte (CTL) response hierarchies in early infection. On a population level, HLA-driven evolution was observed in approximately 80% of published CTL epitopes. Five of the 10 most rapidly evolving epitopes were restricted by protective HLA alleles (HLA-B*13/B*51/B*57/B*5801; P = 0.01), supporting the importance of a strong early CTL response in HIV control. Consistent with known fitness costs of escape, B*57-associated mutations in Gag were among the most rapidly reverting positions upon transmission to non-B*57-expressing individuals, whereas many other HLA-associated polymorphisms displayed slow or negligible reversion. Overall, an estimated minimum of 30% of observed substitutions in Gag/Pol and 60% in Nef were attributable to HLA-associated escape and reversion events. Results underscore the dominant role of immune pressures in driving early within-host HIV evolution. Dramatic differences in escape and reversion rates across codons, genes, and HLA restrictions are observed, highlighting the complexity of viral adaptation to the host immune response.

Three neutralizing monoclonal antibodies (MAbs), 2G12, 2F5, and 4E10, with activity in vitro and in vivo were administered in an open-label, nonrandomized, proof-of-concept study to attempt to prevent viral rebound after interruption of antiretroviral therapy (ART). Ten human immunodeficiency virus type 1-infected individuals identified and treated with ART during acute and early infection were enrolled. The first six patients were administered 1.0 g of each of the three MAbs per infusion. The remaining four patients received 2G12 at 1.0 g/infusion and 2.0 g/infusion of 2F5 and 4E10. The MAbs were well tolerated. Grade I post-partial thromboplastin time prolongations were noted. Viral rebound was observed in 8/10 subjects (28 to 73 days post-ART interruption), and 2/10 subjects remained aviremic over the course of the study. In seven of eight subjects with viral rebound, clear resistance to 2G12 emerged, whereas reductions in the susceptibilities of plasma-derived recombinant viruses to 2F5 and 4E10 were neither sustained nor consistently measured. Viral rebound was associated with a preferential depletion of CD4(+) T cells within the gastrointestinal tract. Though safe, the use of MAbs generally delayed, but did not prevent, virologic rebound. Consideration should be given to further pilot studies with alternative combinations of MAbs and perhaps additional novel treatment modalities.

During acute and early human immunodeficiency virus type 1 (HIV-1) infection (AEI) more than 50% of CD4+ T cells are preferentially depleted from the gastrointestinal (GI) lamina propria. To better understand the underlying mechanisms, we studied virological and immunological events within the peripheral blood (PB) and GI tract during AEI. A total of 32 AEI subjects and 18 uninfected controls underwent colonic biopsy. HIV-1 viral DNA and RNA levels were quantified in CD4+ T cells derived from the GI tract and PB by using real-time PCR. The phenotype of infected cells was characterized by using combinations of immunohistochemistry and in situ hybridization. Markers of immunological memory, activation, and proliferation were examined by flow cytometry and immunohistochemistry, and the host-derived cytotoxic cellular response was examined by using immunohistochemistry. GI CD4+ T cells harbored, on average, 13-fold higher HIV-1 viral DNA levels and 10-fold higher HIV-1 RNA levels than PB CD4+ T cells during AEI. HIV-1 RNA was detected in both "activated" and "nonactivated" mucosal CD4+ T cells. A significantly higher number of activated and proliferating T cells were detected in the GI tract compared to the PB, and a robust cytotoxic response (HIV-1 specificity not determined) was detected in the GI tract as early as 18 days postinfection. Mucosal CD4+ T-cell depletion is multifactorial. Direct viral infection likely accounts for the earliest loss of CD4+ T cells. Subsequently, ongoing infection of susceptible CD4+ T cells, along with activation-induced cellular death and host cytotoxic cellular response, are responsible for the persistence of the lesion.

As the AIDS epidemic continues unabated, the development of a human immunodeficiency virus (HIV) vaccine is critical. Ideally, an effective vaccine should elicit cell-mediated and neutralizing humoral immune responses. We have determined the in vitro susceptibility profile of sexually transmitted viruses from 91 patients with acute and early HIV-1 infection to three monoclonal antibodies, 2G12, 2F5, and 4E10. Using a recombinant virus assay to measure neutralization, we found all transmitted viruses were neutralized by 4E10, 80% were neutralized by 2F5, and only 37% were neutralized by 2G12. We propose that the induction of 4E10-like antibodies should be a priority in designing immunogens to prevent HIV-1 infection.

The meaning of viral blips in human immunodeficiency virus type 1 (HIV-1)-infected patients treated with seemingly effective highly active antiretroviral therapy (HAART) is still controversial and under investigation. Blips might represent low-level ongoing viral replication in the presence of drug or simply release of virions from the latent reservoir. Patients treated early during HIV-1 infection are more likely to have a lower total body viral burden, a homogenous viral population, and preserved HIV-1-specific immune responses. Consequently, viral blips may be less frequent in them than in patients treated during chronic infection. To test this hypothesis, we compared the occurrence of viral blips in 76 acutely infected patients (primary HIV infection [PHI] group) who started therapy within 6 months of the onset of symptoms with that in 47 patients who started HAART therapy during chronic infection (chronic HIV infection [CHI] group). Viral blip frequency was approximately twofold higher in CHI patients (0.122 +/- 0.12/viral load [VL] sample, mean +/- standard deviation) than in PHI patients (0.066 +/- 0.09/VL sample). However, in both groups, viral blip frequency did not increase with longer periods of observation. Also, no difference in viral blip frequency was observed between treatment subgroups, and the occurrence of a blip was not associated with a recent change in CD4(+) T-cell count. Finally, in PHI patients the VL set point was a significant predictor of blip frequency during treatment.

The initial virus strains from as many as 12% of individuals with primary human immunodeficiency virus (HIV) infection have a 50% inhibitory concentration

Although intermittent episodes of low-level viremia are often observed in well-suppressed highly active antiretroviral therapy (HAART)-treated patients, the timing and amplitude of viral blips have never been examined in detail. We analyze here the dynamics of viral blips, i.e., plasma VL measurements of >50 copies/ml, in 123 HAART-treated patients monitored for a mean of 2.6 years (range, 5 months to 5.3 years). The mean (+/- the standard deviation) blip frequency was 0.09 +/- 0.11/sample, with about one-third of patients showing no viral blips. The mean viral blip amplitude was 158 +/- 132 human immunodeficiency virus type 1 (HIV-1) RNA copies/ml. Analysis of the blip frequency and amplitude distributions suggest that two blips less than 22 days apart have a significant chance of being part of the same episode of viremia. The data are consistent with a hypothetical model in which each episode of viremia consists of a phase of VL rise, followed by two-phase exponential decay. Thus, the term "viral blip" may be a misnomer, since viral replication appears to be occurring over an extended period. Neither the frequency nor the amplitude of viral blips increases with longer periods of observation, but the frequency is inversely correlated with the CD4(+)-T-cell count at the start of therapy, suggesting that host-specific factors but not treatment fatigue are determinants of blip frequency.

Gammadelta T cells are primarily found in the gastrointestinal mucosa and play an important role in the first line of defense against viral, bacterial, and fungal pathogens. We sought to examine the impact of human immunodeficiency virus type 1 (HIV-1) infection on mucosal as well as peripheral blood gammadelta T-cell populations. Our results demonstrate that HIV-1 infection is associated with significant expansion of Vdelta1 and contraction of Vdelta2 cell populations in both the mucosa and peripheral blood. Such changes were observed during acute HIV-1 infection and persisted throughout the chronic phase, without apparent reversion after treatment with highly active antiretroviral therapy (HAART). Despite an increase in the expression of CCR9 and CD103 mucosal homing receptors on peripheral blood gammadelta T cells in infected individuals, mucosal and peripheral blood gammadelta T cells appeared to be distinct populations, as reflected by distinct CDR3 length polymorphisms and sequences in the two compartments. Although the underlying mechanism responsible for triggering the expansion of Vdelta1 gammadelta T cells remains unknown, HIV-1 infection appears to have a dramatic impact on gammadelta T cells, which could have important implications for HIV-1 pathogenesis.

It is believed that replication capacity is an important determinant of human immunodeficiency virus type 1 (HIV-1) pathogenicity and transmissibility. To explore this, we conducted a comprehensive analysis of the replication properties of nine drug-resistant and nine drug-susceptible viral isolates derived from patients with primary HIV-1 infection. Viral isolates were tested for single-cycle infectivity in the GHOST cell line. The infectivity of isolates carrying resistance-associated mutations was significantly higher than that of drug-susceptible isolates. Additionally, the growth kinetics of these isolates were determined in CD4+ T lymphocytes. Drug-resistant isolates replicated as well as drug-susceptible viruses. Insertion of the resistance-conferring regions into an NL4-3-based molecular background resulted in chimeras that displayed a modest but significant reduction in replication capacity compared to the drug-susceptible chimeric viruses. Of note, two multidrug-resistant isolates and one protease inhibitor-resistant isolate displayed higher rates of infectivity and growth kinetics than the other drug-resistant or drug-susceptible isolates. These distinct replicative features, however, were not seen in the corresponding chimeras, indicating that changes within the C-terminal region of Gag as well as within the protease and reverse transcriptase genes contribute to but are not sufficient for the level of compensatory adaptation observed. These findings suggest that some drug-resistant viruses isolated during primary infection possess unique adaptive changes that allow for both high viral replication capacity and resistance to one or more classes of antiretroviral drugs. Further studies are needed to elucidate the precise regions that are essential for these characteristics.

Mathematical models provide an understanding of in vivo replication kinetics of human immunodeficiency virus type 1 (HIV-1). With a novel intervention designed for increased potency, we have more accurately deduced the half-lives of virus-producing CD4(+) T cells, 0.7 day, and the generation time of HIV-1 in vivo, approximately 2 days, confirming the dynamic nature of HIV-1 replication.

The emergence of antiretroviral (ARV) drug-resistant human immunodeficiency virus type 1 (HIV-1) quasispecies is a major cause of treatment failure. These variants are usually replaced by drug-sensitive ones when the selective pressure of the drugs is removed, as the former have reduced fitness in a drug-free environment. This was the rationale for the design of structured ARV treatment interruption (STI) studies for the management of HIV-1 patients with treatment failure. We have studied the origin of drug-sensitive HIV-1 quasispecies emerging after STI in patients with treatment failure due to ARV drug resistance. Plasma and peripheral blood mononuclear cell samples were obtained the day of treatment interruption (day 0) and 30 and 60 days afterwards. HIV-1 pol and env were partially amplified, cloned, and sequenced. At day 60 drug-resistant variants were replaced by completely or partially sensitive quasispecies. Phylogenetic analyses of pol revealed that drug-sensitive variants emerging after STI were not related to their immediate temporal ancestors but formed a separate cluster, demonstrating that STI leads to the recrudescence and reemergence of a sequestrated viral population rather than leading to the back mutation of drug-resistant forms. No evidence for concomitant changes in viral tropism was seen, as deduced from env sequences. This study demonstrates the important role that the reemergence of quasispecies plays in HIV-1 population dynamics and points out the difficulties that may be found when recycling ARV therapies with patients with treatment failure.

In order to boost immune responses in persons in whom highly active antiretroviral therapy (HAART) was initiated within 120 days of the onset of symptoms of newly acquired human immunodeficiency virus type 1 (HIV-1) infection, we administered vaccines containing a canarypox virus vector, vCP1452, with HIV-1 genes encoding multiple HIV-1 proteins, and recombinant gp160. Fifteen HIV-1-infected subjects who achieved sustained suppression of plasma viremia for at least 2 years were enrolled. While continuing antiretroviral therapy, each subject received at least four intramuscular injections of the vaccines on days 0, 30, 90, and 180. Adverse events were mild, with the most common being transient tenderness at the vCP1452 injection site. Of the 14 patients who completed vaccination, 13 had significant increases in anti-gp120 or anti-p24 antibody titers, and 9 had transient augmentation of their T-cell proliferation responses to gp160 and/or p24. HIV-1-specific CD8(+) T cells were quantified using an intracellular gamma interferon staining assay. Among 11 patients who had increased CD8(+) T-cell responses, seven had responses to more than one HIV-1 antigen. In summary, vaccination with vCP1452 and recombinant gp160 appears safe and immunogenic in newly HIV-1-infected patients on HAART.

We calculated the relative efficacy of treatment, defined as the rate of decline of virus levels in plasma during treatment relative to the rate of decline during highly potent combination therapy, in human immunodeficiency virus type 1 (HIV-1) patients treated for 56 days with different doses of the protease inhibitor nelfinavir. Relative efficacies based on the rate of decline of HIV-1 RNA levels in plasma over the first 14 to 21 days correlated with drug dose and viral load reduction by day 56. Calculation of relative treatment efficacies over the first 2 to 3 weeks of treatment can allow rapid assessment of new antiretroviral agents and dosing regimens, reducing the need to keep subjects in clinical trials on monotherapy for prolonged periods of time. Relative efficacy may also serve as a measure of treatment efficacy in patients in initiating established therapies.

We have designed a novel, precise, and sensitive assay to measure unspliced (US) human immunodeficiency virus type 1 (HIV-1) mRNA in peripheral blood mononuclear cells of HIV-1-infected individuals by using real-time PCR and molecular beacons. Individuals were classified as either well suppressed (WS) or partially suppressed, based on longitudinal measurements of plasma HIV-1 RNA. The proportion of individuals with US mRNA undetectable over time was significantly higher among WS individuals; however, 30% of WS subjects still had detectable US mRNA after 24 months of effective antiviral therapy.

Little is known of the changes in human immunodeficiency virus type 1 (HIV-1)-specific effector cytotoxic T lymphocytes (CTL) after potent antiretroviral therapy. Using HLA/peptide tetrameric complexes, we show that after starting treatment, there are early rapid fluctuations in the HIV-1-specific CTL response which last 1 to 2 weeks. These fluctuations are followed by an exponential decay (median half-life, 45 days) of HIV-1-specific CTL which continues while viremia remains undetectable. These data have implications for the immunological control of drug-resistant virus.

Nelfinavir mesylate (formerly AG1343) is a potent and selective inhibitor of human immunodeficiency virus (HIV) protease approved for the treatment of individuals infected with HIV. Nucleotide sequence analysis of protease genes from plasma HIV type 1 (HIV-1) RNA revealed a unique aspartic acid (D)-to-asparagine (N) substitution at residue 30 (D30N) in 25 of 55 patients treated with nelfinavir for a median of 13 weeks. Although the appearance of D30N was occasionally associated with concurrent or sequential emergence of other changes (e.g., at residues 35, 36, 46, 71, 77, and 88), genotypic changes associated with phenotypic resistance to other protease inhibitors were not observed (e.g., at residues 48, 50, 82, and 84) or were only rarely observed (e.g., at residue 90). In phenotypic assays, viral isolates with high-level resistance to nelfinavir remained susceptible to indinavir, saquinavir, ritonavir, and amprenavir (formerly VX-478/141W94). Similar results were observed in phenotypic assays utilizing HIV-1 NL4-3, which contained the D30N substitution alone or in combination with substitutions at other residues (e.g., residues 46, 71, and 88). These data indicate that the initial pathway of resistance to nelfinavir is unique and suggest that individuals failing short courses of nelfinavir-containing regimens may respond to regimens containing other protease inhibitors.

Ten subjects received 600 to 1,200 mg of the human immunodeficiency virus type 1 (HIV-1) protease inhibitor ritonavir per day. Following 2 weeks of therapy, plasma HIV RNA levels decreased by a mean of 1. 57 (range, 0.89 to 1.96) log units. With continued therapy, HIV RNA levels began to rise in eight subjects. The initial rise in plasma RNA levels was temporally associated with the development and quantitative increase in the V82 resistance mutation. Doubling times of the V82A mutant virus were estimated to be 2.4 to 4.8 days. An L63P/A mutation was commonly present at baseline even in subjects with a durable virologic response. The concomitant acquisition of an L63P/A mutation with the V82A/F mutation at the time when plasma RNA levels rebounded suggests a role for the L63P/A mutation in improving the fitness of the V82A/F mutation. Subsequent additional genotypic changes at codons 54 and 84 were often associated with further increases in plasma RNA levels. Ongoing viral replication in the presence of drugs resulted in the appearance of additional genotypic changes, including the L90M saquinavir resistance mutation, and decreased phenotypic susceptibility. The relative fitness of the protease V82A ritonavir resistance mutation and reverse transcriptase T215Y/F zidovudine resistance mutation following drug withdrawal were estimated to be 96 to 98% that of the wild type. Durability of the virologic response was associated with plasma RNA levels at the nadir. A virologic response beyond 60 days was not observed unless plasma HIV RNA levels were suppressed below 2,000 copies/ml, consistent with estimates from V82A doubling times for selection of a single resistance mutation to dominate the replicating population.

Plasma human immunodeficiency virus type 1 (HIV-1) populations were genetically analyzed at their most variable locus, the envelope gene, during the rapid emergence of resistance to protease inhibitor monotherapy. Plasma virus populations remained genetically constant prior to drug treatment and during the 1 to 2 weeks following initiation of therapy, while viremia fell 10- to 100-fold. Concomitant with rapid plasma viremia rebounds associated with the emergence of drug-resistant virus, marked alterations were then detected at the env locus. Plasma population changes lasted only a few weeks before the reappearance of the pretreatment envelope variants. The emergence of resistance to single protease inhibitors was therefore associated with major but transient changes at a nonselected locus. Selection for resistance to single protease inhibitors thus appears to be more complex than the continued replication of a large, random, and therefore genetically representative sampling of the pretreatment plasma population. The possibility that drug-privileged anatomical sites containing distinct envelope variants and/or a small effective HIV-1 population size account for these results is discussed.

PNU-140690 is a member of a new class of nonpeptidic human immunodeficiency virus (HIV) protease inhibitors (sulfonamide-containing 5,6-dihydro-4-hydroxy-2-pyrones) discovered by structure-based design. PNU-140690 has excellent potency against a variety of HIV type 1 (HIV-1) laboratory strains and clinical isolates, including those resistant to the reverse transcriptase inhibitors zidovudine or delavirdine. When combined with either zidovudine or delavirdine, PNU-140690 contributes to synergistic antiviral activity. PNU-140690 is also highly active against HIV-1 variants resistant to peptidomimetic protease inhibitors, underscoring the structural distinctions between PNU-140690 and substrate analog protease inhibitors. PNU-140690 retains good antiviral activity in vitro in the presence of human plasma proteins, and preclinical pharmacokinetic studies revealed good oral bioavailability. Accordingly, PNU-140690 is a candidate for clinical evaluation.

We have studied the antibody responses to Env and Gag antigens of human immunodeficiency virus type 1 (HIV-1) in several cohorts of HIV-1-infected individuals: long-term nonprogressors, progressors to disease, acute seroconvertors, and recipients of HIV-1 protease inhibitors. We conclude that the antibody responses to Env and Gag antigens are differentially regulated and that changes in the plasma viral load in the measurable range (500 to 10(8) RNA copies per ml) do not directly affect the antibody responses to these HIV-1 proteins. We provide quantitative estimates of HIV-1-specific immunoglobulin G concentrations in plasma, which can be in excess of 1 mg/ml for both anti-gp120 and anti-p24 once the immune response to HIV-1 has stabilized after seroconversion. We discuss the apparent paradox that the absence of anti-Gag antibodies (which have, at best, limited antiviral activity) is indicative of disease progression, while the retention of anti-Env antibodies (which do have antiviral activity) is of limited (or no) prognostic value. We show that the disappearance of anti-Gag antibodies during disease progression is highly unlikely to be due to immune complexing; instead, we believe that it reflects the loss of T-cell help that is more necessary for the anti-Gag than the anti-Env response.

AG1343 ([3S-(3R*,4aR*,8aR*,2'S*,3'S*)]-2-[2' hydroxy-3'-phenylthiomethyl-4'-aza-5'-oxo-5'-(2''-methyl-3''-hydro xy-phenyl) pentyl]-decahydroiso-quinoline-3-N-t-butylcarboxamide methanesulfonic acid) is a selective, nonpeptidic inhibitor of human immunodeficiency virus (HIV) protease (Ki = 2 nM) that was discovered by protein structure-based drug design methodologies. AG1343 was effective against the replication of several laboratory and clinical HIV type 1 (HIV-1) or HIV-2 isolates including pyridinone- and zidovudine-resistant strains, with 50% effective concentrations ranging from 9 to 60 nM. In reversibility studies, inhibition of gag (p55) proteolytic processing in HIV-1 particles from cells treated with AG1343 was maintained for up to 36 h after drug removal. The ability of virus to develop resistance to AG1343 was studied by serial passage of HIV-1 NL4.3 in the presence of increasing concentrations of drug. After 28 passages, a variant with a 30-fold reduction in susceptibility to AG1343 was isolated. Molecular analysis of the protease from this variant indicated a double change from a Met to Ile at residue 46 and an Ile to Val or Ala at residue 84 (M46I+I84V, A). Consistent with these findings, reductions in susceptibility were observed for recombinant viruses constructed to contain the single I84V change or the double M46I+I84V substitutions. Resistance, however, was not detected for recombinant viruses containing other key mutations in HIV-1 protease, including a Val to Ile change at residue 32 or a Val to Ala or Phe at residue 82. The potent anti-HIV activity of AG1343 against several isolates suggests that AG1343 should perform well during ongoing human phase II clinical trials.

Inhibitors of the human immunodeficiency virus protease represent a promising new class of antiretroviral drugs for the treatment of AIDS. We now report the in vitro selection of viral variants with decreased sensitivity to a symmetry-based protease inhibitor, ABT-538, currently being tested in clinical trials. Molecular characterization of the variants shows that an isoleucine-to-valine substitution at position 84 results in a substantial decrease in sensitivity to the drug. Moreover, an additional mutation at position 82, valine to phenylalanine, further decreases viral susceptibility to ABT-538. Three-dimensional analysis of the protease-drug complex provides a structural explanation for the relative drug resistance induced by these two mutations. These findings emphasize the importance of closely monitoring patients receiving ABT-538 for the emergence of viral resistance and provide information that may prove useful in designing the next generation of protease inhibitors.