Structures of Human Antibodies Bound to SARS-CoV-2 Spike Reveal Common Epitopes and Recurrent Features of Antibodies.

Neutralizing antibody responses to coronaviruses mainly target the receptor-binding domain (RBD) of the trimeric spike. Here, we characterized polyclonal immunoglobulin Gs (IgGs) and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their focus on RBD epitopes, recognition of alpha- and beta-coronaviruses, and contributions of avidity to increased binding/neutralization of IgGs over Fabs. Using electron microscopy, we examined specificities of polyclonal plasma Fabs, revealing recognition of both S1A and RBD epitopes on SARS-CoV-2 spike. Moreover, a 3.4 Å cryo-electron microscopy (cryo-EM) structure of a neutralizing monoclonal Fab-spike complex revealed an epitope that blocks ACE2 receptor binding. Modeling based on these structures suggested different potentials for inter-spike crosslinking by IgGs on viruses, and characterized IgGs would not be affected by identified SARS-CoV-2 spike mutations. Overall, our studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.

Recurrent Potent Human Neutralizing Antibodies to Zika Virus in Brazil and Mexico.

Antibodies to Zika virus (ZIKV) can be protective. To examine the antibody response in individuals who develop high titers of anti-ZIKV antibodies, we screened cohorts in Brazil and Mexico for ZIKV envelope domain III (ZEDIII) binding and neutralization. We find that serologic reactivity to dengue 1 virus (DENV1) EDIII before ZIKV exposure is associated with increased ZIKV neutralizing titers after exposure. Antibody cloning shows that donors with high ZIKV neutralizing antibody titers have expanded clones of memory B cells that express the same immunoglobulin VH3-23/VK1-5 genes. These recurring antibodies cross-react with DENV1, but not other flaviviruses, neutralize both DENV1 and ZIKV, and protect mice against ZIKV challenge. Structural analyses reveal the mechanism of recognition of the ZEDIII lateral ridge by VH3-23/VK1-5 antibodies. Serologic testing shows that antibodies to this region correlate with serum neutralizing activity to ZIKV. Thus, high neutralizing responses to ZIKV are associated with pre-existing reactivity to DENV1 in humans.

mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants.

Here we report on the antibody and memory B cell responses of a cohort of 20 volunteers who received the Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccine against SARS-CoV-21-4. Eight weeks after the second injection of vaccine, volunteers showed high levels of IgM and IgG anti-SARS-CoV-2 spike protein (S) and receptor-binding-domain (RBD) binding titre. Moreover, the plasma neutralizing activity and relative numbers of RBD-specific memory B cells of vaccinated volunteers were equivalent to those of individuals who had recovered from natural infection5,6. However, activity against SARS-CoV-2 variants that encode E484K-, N501Y- or K417N/E484K/N501-mutant S was reduced by a small-but significant-margin. The monoclonal antibodies elicited by the vaccines potently neutralize SARS-CoV-2, and target a number of different RBD epitopes in common with monoclonal antibodies isolated from infected donors5-8. However, neutralization by 14 of the 17 most-potent monoclonal antibodies that we tested was reduced or abolished by the K417N, E484K or N501Y mutation. Notably, these mutations were selected when we cultured recombinant vesicular stomatitis virus expressing SARS-CoV-2 S in the presence of the monoclonal antibodies elicited by the vaccines. Together, these results suggest that the monoclonal antibodies in clinical use should be tested against newly arising variants, and that mRNA vaccines may need to be updated periodically to avoid a potential loss of clinical efficacy.

Evolution of antibody immunity to SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected 78 million individuals and is responsible for over 1.7 million deaths to date. Infection is associated with the development of variable levels of antibodies with neutralizing activity, which can protect against infection in animal models1,2. Antibody levels decrease with time, but, to our knowledge, the nature and quality of the memory B cells that would be required to produce antibodies upon reinfection has not been examined. Here we report on the humoral memory response in a cohort of 87 individuals assessed at 1.3 and 6.2 months after infection with SARS-CoV-2. We find that titres of IgM and IgG antibodies against the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 decrease significantly over this time period, with IgA being less affected. Concurrently, neutralizing activity in plasma decreases by fivefold in pseudotype virus assays. By contrast, the number of RBD-specific memory B cells remains unchanged at 6.2 months after infection. Memory B cells display clonal turnover after 6.2 months, and the antibodies that they express have greater somatic hypermutation, resistance to RBD mutations and increased potency, indicative of continued evolution of the humoral response. Immunofluorescence and PCR analyses of intestinal biopsies obtained from asymptomatic individuals at 4 months after the onset of coronavirus disease 2019 (COVID-19) revealed the persistence of SARS-CoV-2 nucleic acids and immunoreactivity in the small bowel of 7 out of 14 individuals. We conclude that the memory B cell response to SARS-CoV-2 evolves between 1.3 and 6.2 months after infection in a manner that is consistent with antigen persistence.

Convergent antibody responses to SARS-CoV-2 in convalescent individuals.

During the coronavirus disease-2019 (COVID-19) pandemic, severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) has led to the infection of millions of people and has claimed hundreds of thousands of lives. The entry of the virus into cells depends on the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2. Although there is currently no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-21-5. Here we report on 149 COVID-19-convalescent individuals. Plasma samples collected an average of 39 days after the onset of symptoms had variable half-maximal pseudovirus neutralizing titres; titres were less than 50 in 33% of samples, below 1,000 in 79% of samples and only 1% of samples had titres above 5,000. Antibody sequencing revealed the expansion of clones of RBD-specific memory B cells that expressed closely related antibodies in different individuals. Despite low plasma titres, antibodies to three distinct epitopes on the RBD neutralized the virus with half-maximal inhibitory concentrations (IC50 values) as low as 2 ng ml-1. In conclusion, most convalescent plasma samples obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.

Temporal relations between peripheral and central arousals in good and poor sleepers.

Good sleepers and patients with insomnia symptoms (poor sleepers) were tracked with two measures of arousal; conventional polysomnography (PSG) for electroencephalogram (EEG) assessed cortical arousals, and a peripheral arterial tonometry device was used for the detection of peripheral nervous system (PNS) arousals associated with vasoconstrictions. The relationship between central (cortical) and peripheral (autonomic) arousals was examined by evaluating their close temporal dynamics. Cortical arousals almost invariably were preceded and followed by peripheral activations, while large peripheral autonomic arousals were followed by cortical arousals only half of the time. The temporal contiguity of these two types of arousals was altered in poor sleepers, and poor sleepers displayed a higher number of cortical and peripheral arousals compared with good sleepers. Given the difference in the number of peripheral autonomic arousals between good and poor sleepers, an evaluation of such arousals could become a means of physiologically distinguishing poor sleepers.

Plasma microRNA Interindividual Variability in Healthy Individuals, Pregnant Women, and an Individual with a Stably Altered Neuroendocrine Phenotype.

BACKGROUND: Extracellular RNAs (exRNAs) in biofluids are amenable to quantitative analysis and proposed as noninvasive biomarkers for monitoring organ function. Cell-lineage-specific microRNAs (miRNAs) are present in plasma as soluble ribonucleoproteins or enclosed in exRNA carriers and transported through the vasculature. However, more extensive studies of healthy individuals are needed to gain insights into the variability of plasma miRNA abundance and composition. METHODS: The exRNA composition of platelet-depleted plasma collected twice from 236 healthy individuals was characterized by small RNA sequencing. Plasma of pregnant women featuring dramatically increased placental miRNAs and samples from subject P12 with noticeably increased epithelial- and neuroendocrine-origin miRNAs were included for comparison. The miRNA content of 10 000g and 100 000g pellet fractions of plasma generated by ultracentrifugation was also determined. Data analysis methods included Pearson correlation, differential gene expression, and unsupervised clustering. RESULTS: The abundance changes for more variable miRNAs in plasma of normal individuals correlated between coexpressed cell-lineage-specific miRNAs of the liver, neuroendocrine organs, epithelial cells, and muscle. ExRNA of pellet fractions contained <2% of total plasma miRNA with modest enrichment of lineage-specific and variable miRNAs compared to supernatant. The abundance fold changes of miRNAs observed in pregnancy and P12 compared to normal exceeded interquartile variability of healthy individuals. The neuroendocrine miRNA signature of P12 persisted for more than 4 years and was absent in other individuals. CONCLUSIONS: This study defines the framework and effect size for screening of extensive plasma collections for miRNA phenotypes and biomarker discovery.

Broad diversity of neutralizing antibodies isolated from memory B cells in HIV-infected individuals.

Antibodies to conserved epitopes on the human immunodeficiency virus (HIV) surface protein gp140 can protect against infection in non-human primates, and some infected individuals show high titres of broadly neutralizing immunoglobulin (Ig)G antibodies in their serum. However, little is known about the specificity and activity of these antibodies. To characterize the memory antibody responses to HIV, we cloned 502 antibodies from HIV envelope-binding memory B cells from six HIV-infected patients with broadly neutralizing antibodies and low to intermediate viral loads. We show that in these patients, the B-cell memory response to gp140 is composed of up to 50 independent clones expressing high affinity neutralizing antibodies to the gp120 variable loops, the CD4-binding site, the co-receptor-binding site, and to a new neutralizing epitope that is in the same region of gp120 as the CD4-binding site. Thus, the IgG memory B-cell compartment in the selected group of patients with broad serum neutralizing activity to HIV is comprised of multiple clonal responses with neutralizing activity directed against several epitopes on gp120.

Persistent cellular immunity to SARS-CoV-2 infection.

SARS-CoV-2 is responsible for an ongoing pandemic that has affected millions of individuals around the globe. To gain further understanding of the immune response in recovered individuals, we measured T cell responses in paired samples obtained an average of 1.3 and 6.1 mo after infection from 41 individuals. The data indicate that recovered individuals show persistent polyfunctional SARS-CoV-2 antigen-specific memory that could contribute to rapid recall responses. Recovered individuals also show enduring alterations in relative overall numbers of CD4+ and CD8+ memory T cells, including expression of activation/exhaustion markers, and cell division.

mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants.

To date severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected over 100 million individuals resulting in over two million deaths. Many vaccines are being deployed to prevent coronavirus disease 2019 (COVID-19) including two novel mRNA-based vaccines 1,2 . These vaccines elicit neutralizing antibodies and appear to be safe and effective, but the precise nature of the elicited antibodies is not known 3-6 . Here we report on the antibody and memory B cell responses in a cohort of 20 volunteers who received either the Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccines. Consistent with prior reports, 8 weeks after the second vaccine injection volunteers showed high levels of IgM, and IgG anti-SARS-CoV-2 spike protein (S) and receptor binding domain (RBD) binding titers 3,5,6 . Moreover, the plasma neutralizing activity, and the relative numbers of RBD-specific memory B cells were equivalent to individuals who recovered from natural infection 7,8 . However, activity against SARS-CoV-2 variants encoding E484K or N501Y or the K417N:E484K:N501Y combination was reduced by a small but significant margin. Consistent with these findings, vaccine-elicited monoclonal antibodies (mAbs) potently neutralize SARS-CoV-2, targeting a number of different RBD epitopes in common with mAbs isolated from infected donors. Structural analyses of mAbs complexed with S trimer suggest that vaccine- and virus-encoded S adopts similar conformations to induce equivalent anti-RBD antibodies. However, neutralization by 14 of the 17 most potent mAbs tested was reduced or abolished by either K417N, or E484K, or N501Y mutations. Notably, the same mutations were selected when recombinant vesicular stomatitis virus (rVSV)/SARS-CoV-2 S was cultured in the presence of the vaccine elicited mAbs. Taken together the results suggest that the monoclonal antibodies in clinical use should be tested against newly arising variants, and that mRNA vaccines may need to be updated periodically to avoid potential loss of clinical efficacy.

Identification of Novel Therapeutic Targets for Fibrolamellar Carcinoma Using Patient-Derived Xenografts and Direct-from-Patient Screening.

To repurpose therapeutics for fibrolamellar carcinoma (FLC), we developed and validated patient-derived xenografts (PDX) from surgical resections. Most agents used clinically and inhibitors of oncogenes overexpressed in FLC showed little efficacy on PDX. A high-throughput functional drug screen found primary and metastatic FLC were vulnerable to clinically available inhibitors of TOPO1 and HDAC and to napabucasin. Napabucasin's efficacy was mediated through reactive oxygen species and inhibition of translation initiation, and specific inhibition of eIF4A was effective. The sensitivity of each PDX line inversely correlated with expression of the antiapoptotic protein Bcl-xL, and inhibition of Bcl-xL synergized with other drugs. Screening directly on cells dissociated from patient resections validated these results. This demonstrates that a direct functional screen on patient tumors provides therapeutically informative data within a clinically useful time frame. Identifying these novel therapeutic targets and combination therapies is an urgent need, as effective therapeutics for FLC are currently unavailable. SIGNIFICANCE: Therapeutics informed by genomics have not yielded effective therapies for FLC. A functional screen identified TOPO1, HDAC inhibitors, and napabucasin as efficacious and synergistic with inhibition of Bcl-xL. Validation on cells dissociated directly from patient tumors demonstrates the ability for functional precision medicine in a solid tumor.This article is highlighted in the In This Issue feature, p. 2355.

DRUL for school: Opening Pre-K with safe, simple, sensitive saliva testing for SARS-CoV-2.

To address the need for simple, safe, sensitive, and scalable SARS-CoV-2 tests, we validated and implemented a PCR test that uses a saliva collection kit use at home. Individuals self-collected 300 µl saliva in vials containing Darnell Rockefeller University Laboratory (DRUL) buffer and extracted RNA was assayed by RT-PCR (the DRUL saliva assay). The limit of detection was confirmed to be 1 viral copy/µl in 20 of 20 replicate extractions. Viral RNA was stable in DRUL buffer at room temperature up to seven days after sample collection, and safety studies demonstrated that DRUL buffer immediately inactivated virus at concentrations up to 2.75x106 PFU/ml. Results from SARS-CoV-2 positive nasopharyngeal (NP) swab samples collected in viral transport media and assayed with a standard FDA Emergency Use Authorization (EUA) test were highly correlated with samples placed in DRUL buffer. Direct comparison of results from 162 individuals tested by FDA EUA oropharyngeal (OP) or NP swabs with co-collected saliva samples identified four otherwise unidentified positive cases in DRUL buffer. Over six months, we collected 3,724 samples from individuals ranging from 3 months to 92 years of age. This included collecting weekly samples over 10 weeks from teachers, children, and parents from a pre-school program, which allowed its safe reopening while at-risk pods were quarantined. In sum, we validated a simple, sensitive, stable, and safe PCR-based test using a self-collected saliva sample as a valuable tool for clinical diagnosis and screening at workplaces and schools.

Evolution of Antibody Immunity to SARS-CoV-2.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected 78 million individuals and is responsible for over 1.7 million deaths to date. Infection is associated with development of variable levels of antibodies with neutralizing activity that can protect against infection in animal models. Antibody levels decrease with time, but the nature and quality of the memory B cells that would be called upon to produce antibodies upon re-infection has not been examined. Here we report on the humoral memory response in a cohort of 87 individuals assessed at 1.3 and 6.2 months after infection. We find that IgM, and IgG anti-SARS-CoV-2 spike protein receptor binding domain (RBD) antibody titers decrease significantly with IgA being less affected. Concurrently, neutralizing activity in plasma decreases by five-fold in pseudotype virus assays. In contrast, the number of RBD-specific memory B cells is unchanged. Memory B cells display clonal turnover after 6.2 months, and the antibodies they express have greater somatic hypermutation, increased potency and resistance to RBD mutations, indicative of continued evolution of the humoral response. Analysis of intestinal biopsies obtained from asymptomatic individuals 4 months after coronavirus disease-2019 (COVID-19) onset, using immunofluorescence, or polymerase chain reaction, revealed persistence of SARS-CoV-2 nucleic acids and immunoreactivity in the small bowel of 7 out of 14 volunteers. We conclude that the memory B cell response to SARS-CoV-2 evolves between 1.3 and 6.2 months after infection in a manner that is consistent with antigen persistence.

Primary HIV-1 infection is associated with preferential depletion of CD4+ T lymphocytes from effector sites in the gastrointestinal tract.

Given its population of CCR5-expressing, immunologically activated CD4(+) T cells, the gastrointestinal (GI) mucosa is uniquely susceptible to human immunodeficiency virus (HIV)-1 infection. We undertook this study to assess whether a preferential depletion of mucosal CD4(+) T cells would be observed in HIV-1-infected subjects during the primary infection period, to examine the anatomic subcompartment from which these cells are depleted, and to examine whether suppressive highly active antiretroviral therapy could result in complete immune reconstitution in the mucosal compartment. Our results demonstrate that a significant and preferential depletion of mucosal CD4(+) T cells compared with peripheral blood CD4(+) T cells is seen during primary HIV-1 infection. CD4(+) T cell loss predominated in the effector subcompartment of the GI mucosa, in distinction to the inductive compartment, where HIV-1 RNA was present. Cross-sectional analysis of a cohort of primary HIV-1 infection subjects showed that although chronic suppression of HIV-1 permits near-complete immune recovery of the peripheral blood CD4(+) T cell population, a significantly greater CD4(+) T cell loss remains in the GI mucosa, despite up to 5 yr of fully suppressive therapy. Given the importance of the mucosal compartment in HIV-1 pathogenesis, further study to elucidate the significance of the changes observed here is critical.

Persistent Cellular Immunity to SARS-CoV-2 Infection.

SARS-CoV-2 is responsible for an ongoing pandemic that affected millions of individuals around the globe. To gain further understanding of the immune response in recovered individuals we measured T cell responses in paired samples obtained an average of 1.3 and 6.1 months after infection from 41 individuals. The data indicate that recovered individuals show persistent polyfunctional SARS-CoV-2 antigen specific memory that could contribute to rapid recall responses. In addition, recovered individuals show enduring immune alterations in relative numbers of CD4 + and CD8 + T cells, expression of activation/exhaustion markers, and cell division. SUMMARY: We show that SARS-CoV-2 infection elicits broadly reactive and highly functional memory T cell responses that persist 6 months after infection. In addition, recovered individuals show enduring immune alterations in CD4 + and CD8 + T cells compartments.

Structures of human antibodies bound to SARS-CoV-2 spike reveal common epitopes and recurrent features of antibodies.

Neutralizing antibody responses to coronaviruses focus on the trimeric spike, with most against the receptor-binding domain (RBD). Here we characterized polyclonal IgGs and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their degree of focus on RBD epitopes, recognition of SARS-CoV, MERS-CoV, and mild coronaviruses, and how avidity effects contributed to increased binding/neutralization of IgGs over Fabs. Electron microscopy reconstructions of polyclonal plasma Fab-spike complexes showed recognition of both S1A and RBD epitopes. A 3.4Å cryo-EM structure of a neutralizing monoclonal Fab-S complex revealed an epitope that blocks ACE2 receptor-binding on "up" RBDs. Modeling suggested that IgGs targeting these sites have different potentials for inter-spike crosslinking on viruses and would not be greatly affected by identified SARS-CoV-2 spike mutations. These studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.

A Combination of Human Broadly Neutralizing Antibodies against Hepatitis B Virus HBsAg with Distinct Epitopes Suppresses Escape Mutations.

Although there is no effective cure for chronic hepatitis B virus (HBV) infection, antibodies are protective and correlate with recovery from infection. To examine the human antibody response to HBV, we screened 124 vaccinated and 20 infected, spontaneously recovered individuals. The selected individuals produced shared clones of broadly neutralizing antibodies (bNAbs) that targeted 3 non-overlapping epitopes on the HBV S antigen (HBsAg). Single bNAbs protected humanized mice against infection but selected for resistance mutations in mice with prior established infection. In contrast, infection was controlled by a combination of bNAbs targeting non-overlapping epitopes with complementary sensitivity to mutations that commonly emerge during human infection. The co-crystal structure of one of the bNAbs with an HBsAg peptide epitope revealed a stabilized hairpin loop. This structure, which contains residues frequently mutated in clinical immune escape variants, provides a molecular explanation for why immunotherapy for HBV infection may require combinations of complementary bNAbs.

Convergent Antibody Responses to SARS-CoV-2 Infection in Convalescent Individuals.

During the COVID-19 pandemic, SARS-CoV-2 infected millions of people and claimed hundreds of thousands of lives. Virus entry into cells depends on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S). Although there is no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-21-5. Here we report on 149 COVID-19 convalescent individuals. Plasmas collected an average of 39 days after the onset of symptoms had variable half-maximal neutralizing titers ranging from undetectable in 33% to below 1:1000 in 79%, while only 1% showed titers >1:5000. Antibody cloning revealed expanded clones of RBD-specific memory B cells expressing closely related antibodies in different individuals. Despite low plasma titers, antibodies to three distinct epitopes on RBD neutralized at half-maximal inhibitory concentrations (IC50s) as low as single digit ng/mL. Thus, most convalescent plasmas obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.

Detection of circulating extracellular mRNAs by modified small-RNA-sequencing analysis.

Extracellular mRNAs (ex-mRNAs) potentially supersede extracellular miRNAs (ex-miRNAs) and other RNA classes as biomarkers. We performed conventional small-RNA-sequencing (sRNA-seq) and sRNA-seq with T4 polynucleotide kinase (PNK) end-treatment of total exRNA isolated from serum and platelet-poor EDTA, ACD, and heparin plasma to study the effect on ex-mRNA capture. Compared to conventional sRNA-seq PNK-treatment increased the detection of informative ex-mRNAs reads up to 50-fold. The exRNA pool was dominated by hematopoietic cells and platelets, with additional contribution from the liver. About 60% of the 15- to 42-nt reads originated from the coding sequences, in a pattern reminiscent of ribosome-profiling. Blood sample type had a considerable influence on the exRNA profile. On average approximately 350 to 1,100 distinct ex-mRNA transcripts were detected depending on plasma type. In serum, additional transcripts from neutrophils and hematopoietic cells increased this number to near 2,300. EDTA and ACD plasma showed a destabilizing effect on ex mRNA and non-coding RNA ribonucleoprotein complexes compared to other plasma types. In a proof-of-concept study, we investigated differences between the exRNA profiles of patients with acute coronary syndrome (ACS) and healthy controls. The improved tissue resolution of ex mRNAs after PNK-treatment enabled us to detect a neutrophil-signature in ACS that escaped detection by ex miRNA analysis.

Evolving patterns of HIV-1 resistance to antiretroviral agents in newly infected individuals.

OBJECTIVE: To assess temporal changes in prevalence of transmitted HIV-1 drug resistance in a homogeneous cohort of newly infected individuals. METHODS: Pretreatment genotypic and phenotypic drug resistance was tested in 154 subjects with primary HIV-1 infection identified between 1995 and 2001 (group A; n = 76) and 1999 and 2001 (group B; n = 78). Sequence analysis was assessed by population-based sequencing. Virus susceptibility to antiretroviral agents was determined by the PhenoSense assay (ViroLogic). RESULTS: The frequency of resistance-associated mutations in protease (PR) and reverse transcriptase (RT) genes increased from 13.2% (1995-1998) to 19.7% (1999-2001). Although the overall prevalence of viruses with phenotypic resistance did not vary (1995-1998, 10.0%; 1999-2001, 10.8%), the distribution of drug classes changed [nucleoside RT inhibitor (NRTI): 8.3% to 2.7%; non-NRTI: 5.0% to 8.1%; protease inhibitors (PI): 1.7% to 5.4%]. The decrease of phenotypic resistance to NRTI in 1999-2001 was caused by the absence of transmitted lamivudine-resistant variants. Phenotypically susceptible variants with aspartic acid or serine residues at position 215 of RT (5.3%; P = 0.04) instead emerged. Hypersusceptibility to PI decreased from 18.3% to 5.4% (P = 0.02) while the amino acid substitutions in PR increased over time: M36I (6.6% to 19.7%) and A71V/T (3.9% to 15.8%). CONCLUSIONS: There was an increase in the number of HIV-1 variants with both genotypic and phenotypic resistance to non-NRTI and PI over time. Furthermore, viruses with altered genotypes compatible with thymidine analogue or PI exposure but susceptible phenotypes were seen in 1999-2001. The latter findings suggest transmission of viruses from subjects who have either changed or discontinued therapy.