Low-Abundance HIV Drug-Resistant Viral Variants in Treatment-Experienced Persons Correlate with Historical Antiretroviral Use

BACKGROUND: It is largely unknown how frequently low-abundance HIV drug-resistant variants at levels under limit of detection of conventional genotyping (<20% of quasi-species) are present in antiretroviral-experienced persons experiencing virologic failure. Further, the clinical implications of low-abundance drug-resistant variants at time of virologic failure are unknown. METHODOLOGY/PRINCIPAL FINDINGS: Plasma samples from 22 antiretroviral-experienced subjects collected at time of virologic failure (viral load 1380 to 304,000 copies/mL) were obtained from a specimen bank (from 2004-2007). The prevalence and profile of drug-resistant mutations were determined using Sanger sequencing and ultra-deep pyrosequencing. Genotypes were interpreted using Stanford HIV database algorithm. Antiretroviral treatment histories were obtained by chart review and correlated with drug-resistant mutations. Low-abundance drug-resistant mutations were detected in all 22 subjects by deep sequencing and only in 3 subjects by Sanger sequencing. In total they accounted for 90 of 247 mutations (36%) detected by deep sequencing; the majority of these (95%) were not detected by standard genotyping. A mean of 4 additional mutations per subject were detected by deep sequencing (p<0.0001, 95%CI: 2.85-5.53). The additional low-abundance drug-resistant mutations increased a subject's genotypic resistance to one or more antiretrovirals in 17 of 22 subjects (77%). When correlated with subjects' antiretroviral treatment histories, the additional low-abundance drug-resistant mutations correlated with the failing antiretroviral drugs in 21% subjects and correlated with historical antiretroviral use in 79% subjects (OR, 13.73; 95% CI, 2.5-74.3, p = 0.0016). CONCLUSIONS/SIGNIFICANCE: Low-abundance HIV drug-resistant mutations in antiretroviral-experienced subjects at time of virologic failure can increase a subject's overall burden of resistance, yet commonly go unrecognized by conventional genotyping. The majority of unrecognized resistant mutations correlate with historical antiretroviral use. Ultra-deep sequencing can provide important historical resistance information for clinicians when planning subsequent antiretroviral regimens for highly treatment-experienced patients, particularly when their prior treatment histories and longitudinal genotypes are not available

Initial antibodies binding to HIV-1 gp41 in acutely infected subjects are polyreactive and highly mutated

Many HIV-1 envelope-reactive antibodies shortly after HIV-1 transmission may arise from crow-reactive memory B cells previously stimulated by non-HIV-1 host or microbial antigen

HIV drug resistance mutation detection by standard sequencing (ss) and ultra-deep sequencing (uds).

<p><b>NOTE:</b> (), % abundance by uds.</p><p>AZT, zidovudine; ABC, abacavir; 3TC, lamivudine; FTC, emtricitabine; DDI, didanosine.</p><p>Mutations in bold, IAS-USA major mutations in low abundance.</p><p>Mutation in <i>italic</i>, mutations detected by uds only.</p><p>TDF, tenofovir; D4T, stavudine; EFV, efavirenz; LPV, lopinavir; SQV, saquinavir; fAMP, fosamprenavir; NLF, nelfinavir; ATV, atazanavir; r, ritonavir boosting.</p

High-resolution antibody dynamics of vaccine-induced immune responses

The adaptive immune system confers protection by generating a diverse repertoire of antibody receptors that are rapidly expanded and contracted in response to specific targets. Next-generation DNA sequencing now provides the opportunity to survey this complex and vast repertoire. In the present work, we describe a set of tools for the analysis of antibody repertoires and their application to elucidating the dynamics of the response to viral vaccination in human volunteers. By analyzing data from 38 separate blood samples across 2 y, we found that the use of the germ-line library of V and J segments is conserved between individuals over time. Surprisingly, there appeared to be no correlation between the use level of a particular VJ combination and degree of expansion. We found the antibody RNA repertoire in each volunteer to be highly dynamic, with each individual displaying qualitatively different response dynamics. By using combinatorial phage display, we screened selected VH genes paired with their corresponding VL library for affinity against the vaccine antigens. Altogether, this work presents an additional set of tools for profiling the human antibody repertoire and demonstrates characterization of the fast repertoire dynamics through time in multiple individuals responding to an immune challenge