Characteristics of study subjects with primary HIV-1 infection evaluated for drug resistance mutations.

<p>OLA: oligonucleotide ligation assay; CS: consensus sequencing; IQR: interquartile range; NS: not significant at p = .05; ARV: antiretroviral; PI: protease inhibitor; NRTI: nucleoside reverse transcriptase inhibitor; NNRTI: non-nucleoside reverse transcriptase inhibitor therapy.</p><p>*differences between groups were not significant in analyses that were both unadjusted and adjusted for time from infection to the date of sampling.</p

HIV-1 drug resistance in ARV-naïve subjects with primary HIV-1 infection.

<p>ARV: antiretroviral; OLA: oligonucleotide ligation assay; PBMC: peripheral blood mononuclear cells.</p><p>+ = subjects with ≥1 mutation or mixture.</p><p>− = subjects without mutations or with indeterminate results.</p><p>Numbers represent subjects in whom HIV-1 drug resistance was/was not detected in plasma and PBMC specimens that had been obtained a median of 29 (IQR 19–66) and 31 (IQR 19–66) days after HIV-1 infection, respectively; all specimens were collected within six months of infection. McNemar's exact tests compare only subjects with discordant results (indicated in bold).</p

Time to suppression of plasma HIV-1 RNA levels among previously ARV-naïve subjects with and without minority variant drug resistance mutations.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028952#pone-0028952-g001" target="_blank">Figure 1:</a> The median time to virologic suppression (HIV-1 RNA<50 copies/mL) was 110 (IQR 62–147) days for 63 treated subjects without detectable mutations (solid line), 84 (IQR 56–109) days for 10 subjects with minority variant mutations treated with ≥3 active ARVs (dashed line), and 104 (60–162) days for nine subjects with minority variant mutations treated with <3 active ARVs (dotted line) (p = .9).</p

HIV-1 drug resistance detected by consensus sequencing and OLA in ARV-naïve subjects and virologic response to ARV therapy.

<p>In this table, the subset of subjects who received antiretroviral (ARV) therapy are grouped based on whether they had drug resistance detected by consensus sequencing (Group I), drug resistance detected by OLA but who received at least three active ARV agents (Group II), or drug resistance detected by OLA who received fewer than three active agents (Group III). ARVs are highlighted in grey if subjects had mutations conferring at least intermediate level resistance to that ARV. K70R, L74V, T215F, and V82S/T were not detected in any treated subjects.</p><p>CS: consensus sequencing; OLA: oligonucleotide ligation assay; VL: viral load (HIV-1 RNA level); ARV: antiretroviral; VF: virologic failure; IDV: indinavir, HU: hydroxyurea, ABC: abacavir, EFV: efavirenz, NVP: nevirapine, r-: ritonavir-boosted, LPV: lopinavir, ATZ: atazanavir; DNS: did not suppress.</p>1<p>Log₁₀ copies/mL.</p>2<p>Antiretroviral medications were switched on day 5 due to side effects.</p>3<p>Subject #69234 subsequently discontinued medications two months later due to adherence difficulties.</p>4<p>OLA probes did not test for M41L and T215D.</p>5<p>OLA on PBMC for subject 56710 yielded indeterminate results for T215Y.</p>6<p>DNS: did not suppress prior to discontinuing ARVs or study censorship. Subjects #26973, 44378, and 78882 were followed for 104, 44, and 63 days, respectively, while receiving ARVs.</p

Superior Control of HIV-1 Replication by CD8+ T Cells Targeting Conserved Epitopes: Implications for HIV Vaccine Design

<div><p>A successful HIV vaccine will likely induce both humoral and cell-mediated immunity, however, the enormous diversity of HIV has hampered the development of a vaccine that effectively elicits both arms of the adaptive immune response. To tackle the problem of viral diversity, T cell-based vaccine approaches have focused on two main strategies (i) increasing the breadth of vaccine-induced responses or (ii) increasing vaccine-induced responses targeting only conserved regions of the virus. The relative extent to which set-point viremia is impacted by epitope-conservation of CD8⁺ T cell responses elicited during early HIV-infection is unknown but has important implications for vaccine design. To address this question, we comprehensively mapped HIV-1 CD8⁺ T cell epitope-specificities in 23 ART-naïve individuals during early infection and computed their conservation score (CS) by three different methods (prevalence, entropy and conseq) on clade-B and group-M sequence alignments. The majority of CD8⁺ T cell responses were directed against variable epitopes (p<0.01). Interestingly, increasing breadth of CD8⁺ T cell responses specifically recognizing conserved epitopes was associated with lower set-point viremia (r = - 0.65, p = 0.009). Moreover, subjects possessing CD8⁺ T cells recognizing at least one conserved epitope had 1.4 log₁₀ lower set-point viremia compared to those recognizing only variable epitopes (p = 0.021). The association between viral control and the breadth of conserved CD8⁺ T cell responses may be influenced by the method of CS definition and sequences used to determine conservation levels. Strikingly, targeting variable versus conserved epitopes was independent of HLA type (p = 0.215). The associations with viral control were independent of functional avidity of CD8⁺ T cell responses elicited during early infection. Taken together, these data suggest that the next-generation of T-cell based HIV-1 vaccines should focus on strategies that can elicit CD8⁺ T cell responses to multiple conserved epitopes of HIV-1.</p></div

Breadth of HIV-1-specific CD8⁺ T cell responses to conserved Gag epitopes correlate with lower viremia.

<p>VL set point was compared to breadth of CD8⁺ T cell responses. (A and B) Correlation between breadth of CD8⁺ T cell responses against conserved Gag or variable Gag epitopes (clade-B) with plasma VL set point (Spearman Rank Correlation, r = −0.65, p = 0.009 and r = −0.32, p = 0.250 respectively). (A–B) The solid line represents a regression line. (C) The median plasma VL set point in individuals who mounted CD8⁺ T cell responses against at least one conserved (bCSp) Gag epitope (Mann Whitney, p = 0.019). (A–C) Subject possessing B*35Px, B*27 and B*57 allele are represented by red circles, green triangles and inverted green triangles respectively.</p

Transmission of HIV-1 drug resistance mutations within partner-pairs: A cross-sectional study of a primary HIV infection cohort

<div><p>Background</p><p>Transmission of human immunodeficiency virus type 1 (HIV-1) drug resistance mutations, particularly that of minority drug-resistant variants, remains poorly understood. Population-based studies suggest that drug-resistant HIV-1 is less transmissible than drug-susceptible viruses. We compared HIV-1 drug-resistant genotypes among partner-pairs in order to assess the likelihood of transmission of drug resistance mutations and investigate the role of minority variants in HIV transmission.</p><p>Methods and findings</p><p>From 1992–2010, 340 persons with primary HIV-1 infection and their partners were enrolled into observational research studies at the University of Washington Primary Infection Clinic (UWPIC). Out of 50 partner-pairs enrolled, 36 (72%) transmission relationships were confirmed by phylogenetic distance analysis of HIV-1 envelope (<i>env</i>) sequences, and 31 partner-pairs enrolled after 1995 met criteria for this study. Drug resistance mutations in the region of the HIV-1 polymerase gene (<i>pol</i>) that encodes protease and reverse transcriptase were assessed by 454-pyrosequencing. In 25 partner-pairs where the transmission direction could be determined, 12 (48%) transmitters had 1–4 drug resistance mutations (23 total) detected in their HIV-1 populations at a median frequency of 6.0% (IQR 1.5%–98.7%, range 1.0%–99.6%). Of 10 major mutations detected in five transmitters at a frequency >95%, 100% (95% CI 69.2%–100%) were detected in recipients. All of these transmitters were antiretroviral (ARV)-naïve at the time of specimen collection. Fourteen mutations (eight major mutations and six accessory mutations) were detected in nine transmitters at low frequencies (1.0%–11.8%); four of these transmitters had previously received ARV therapy. Two (14% [95% CI 1.8%–42.8%]) G73S accessory mutations were detected in both transmitter and recipient. This number is not significantly different from the number expected based on the observed frequencies of drug-resistant viruses in transmitting partners. Limitations of this study include the small sample size and uncertainties in determining the timing of virus transmission and mutation history.</p><p>Conclusions</p><p>Drug-resistant majority variants appeared to be commonly transmitted by ARV-naïve participants in our analysis and may contribute significantly to transmitted drug resistance on a population level. When present at low frequency, no major mutation was observed to be shared between partner-pairs; identification of accessory mutations shared within a pair could be due to transmission, laboratory artifact, or apolipoprotein B mRNA-editing enzyme, catalytic polypeptides (APOBECs), and warrants further study.</p></div

Method of determining CS influences significance of T cell association with and viral control.

<p>(A) The plasma VL set point was compared to breadth of conserved epitopes based on mCSp (Spearman Rank Correlation, r = −0.40, p = 0.139). (B, C) The plasma VL set point was compared to breadth of conserved epitopes based on bCSe (Spearman Rank Correlation, r = −0.52, p = 0.048) and mCSe (Spearman Rank Correlation, r = −0.52, p = 0.043) respectively. Subject possessing B*35Px, B*27 and B*57 allele are represented by red circles, green triangles and inverted green triangles respectively.</p

CD8⁺ T cell responses against conserved epitopes (bCSp) are associated with viral control.

<p>(A) The plasma VL set point was compared to breadth of conserved epitopes (Spearman Rank Correlation, r = −0.65, p = 0.009). The solid line represents a regression line. (B) The median plasma viral set point in individuals who mounted CD8⁺ T cell responses against at least one conserved epitope (Mann Whitney, p = 0.018). (A–B) Subjects possessing B*35Px, B*27 and B*57 alleles are represented by red circles, green triangles and inverted green triangles respectively.</p

Targeting conserved epitope (bCSp) was independent of possession of favorable alleles.

<p>(A) The median CS of epitopes by HLA group: favorable alleles (subjects possessing B*27 and B*57 alleles), unfavorable alleles (subjects possessing B*35Px alleles: B*35Px: B*35∶02, B*35∶03, B*35∶04, and B*53∶01) and neutral alleles (subjects not possessing B*27, B*57 or B*35Px alleles) (Kruskal-Wallis, p = 0.215). Horizontal lines indicate median value. (B) The median plasma VL set point in individuals (subjects not recognizing at least one conserved epitopes were excluded on this analysis) recognizing at least one conserved epitope by possession of favorable allele (Mann Whitney, p = 0.662). (C) The median plasma VL set point in individuals (not possessing favorable alleles, subjects possessing favorable alleles were excluded) who elicited CD8⁺ T cell responses against at least one conserved epitope (Mann Whitney, p = 0.067). (B–C) Subjects possessing B*35Px, B*27 and B*57 alleles are represented by red circles, green triangles and inverted green triangles respectively.</p