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1.  Wide variation in susceptibility of transmitted/founder HIV-1 subtype C Isolates to protease inhibitors and association with in vitro replication efficiency 
Scientific Reports  2016;6:38153.
The gag gene is highly polymorphic across HIV-1 subtypes and contributes to susceptibility to protease inhibitors (PI), a critical class of antiretrovirals that will be used in up to 2 million individuals as second-line therapy in sub Saharan Africa by 2020. Given subtype C represents around half of all HIV-1 infections globally, we examined PI susceptibility in subtype C viruses from treatment-naïve individuals. PI susceptibility was measured in a single round infection assay of full-length, replication competent MJ4/gag chimeric viruses, encoding the gag gene and 142 nucleotides of pro derived from viruses in 20 patients in the Zambia-Emory HIV Research Project acute infection cohort. Ten-fold variation in susceptibility to PIs atazanavir and lopinavir was observed across 20 viruses, with EC50s ranging 0.71–6.95 nM for atazanvir and 0.64–8.54 nM for lopinavir. Ten amino acid residues in Gag correlated with lopinavir EC50 (p < 0.01), of which 380 K and 389I showed modest impacts on in vitro drug susceptibility. Finally a significant relationship between drug susceptibility and replication capacity was observed for atazanavir and lopinavir but not darunavir. Our findings demonstrate large variation in susceptibility of PI-naïve subtype C viruses that appears to correlate with replication efficiency and could impact clinical outcomes.
doi:10.1038/srep38153
PMCID: PMC5128871  PMID: 27901085
2.  Impact of Pre-adapted HIV Transmission 
Nature medicine  2016;22(6):606-613.
Human Leukocyte Antigen class I (HLA) restricted CD8+ T lymphocyte (CTL) responses are critical to HIV-1 control. Although HIV can evade these responses, the longer-term impact of viral escape mutants remains unclear, since these variants can also reduce intrinsic viral fitness. To address this question, we here develop a metric to determine the degree of HIV adaptation to an HLA profile. We demonstrate that transmission of viruses pre-adapted to the HLA molecules expressed in the recipient is associated with impaired immunogenicity, elevated viral load and accelerated CD4 decline. Furthermore, the extent of pre-adaptation among circulating viruses explains much of the variation in outcomes attributed to expression of certain HLA alleles. Thus, viral pre-adaptation exploits “holes” in the immune response. Accounting for these holes may be critical for vaccine strategies seeking to elicit functional responses from viral variants, and to HIV cure strategies requiring broad CTL responses to achieve successful eradication of HIV reservoirs.
doi:10.1038/nm.4100
PMCID: PMC4899163  PMID: 27183217
3.  Diversification in the HIV-1 Envelope Hyper-variable Domains V2, V4, and V5 and Higher Probability of Transmitted/Founder Envelope Glycosylation Favor the Development of Heterologous Neutralization Breadth 
PLoS Pathogens  2016;12(11):e1005989.
A recent study of plasma neutralization breadth in HIV-1 infected individuals at nine International AIDS Vaccine Initiative (IAVI) sites reported that viral load, HLA-A*03 genotype, and subtype C infection were strongly associated with the development of neutralization breadth. Here, we refine the findings of that study by analyzing the impact of the transmitted/founder (T/F) envelope (Env), early Env diversification, and autologous neutralization on the development of plasma neutralization breadth in 21 participants identified during recent infection at two of those sites: Kigali, Rwanda (n = 9) and Lusaka, Zambia (n = 12). Single-genome analysis of full-length T/F Env sequences revealed that all 21 individuals were infected with a highly homogeneous population of viral variants, which were categorized as subtype C (n = 12), A1 (n = 7), or recombinant AC (n = 2). An extensive amino acid sequence-based analysis of variable loop lengths and glycosylation patterns in the T/F Envs revealed that a lower ratio of NXS to NXT-encoded glycan motifs correlated with neutralization breadth. Further analysis comparing amino acid sequence changes, insertions/deletions, and glycan motif alterations between the T/F Env and autologous early Env variants revealed that extensive diversification focused in the V2, V4, and V5 regions of gp120, accompanied by contemporaneous viral escape, significantly favored the development of breadth. These results suggest that more efficient glycosylation of subtype A and C T/F Envs through fewer NXS-encoded glycan sites is more likely to elicit antibodies that can transition from autologous to heterologous neutralizing activity following exposure to gp120 diversification. This initiates an Env-antibody co-evolution cycle that increases neutralization breadth, and is further augmented over time by additional viral and host factors. These findings suggest that understanding how variation in the efficiency of site-specific glycosylation influences neutralizing antibody elicitation and targeting could advance the design of immunogens aimed at inducing antibodies that can transition from autologous to heterologous neutralizing activity.
Author Summary
HIV-1 has proven difficult to vaccinate against due to its ability to generate high levels of genetic diversity, particularly in the envelope glycoproteins. An ideal prophylactic vaccine would therefore elicit immune responses capable of blocking the full range of HIV-1 variants to which a population might be exposed. An essential component of protective immunity against HIV-1 is likely to be an antibody response that is capable of neutralizing genetically diverse HIV-1 viral variants. In an effort to understand how this type of ‘broad’ antibody response develops during natural HIV-1 infection, a large cohort study recently found that certain factors, such as high viral load, HLA-A*03 genotype, and subtype C infection, were correlated with the development of greater neutralization breadth. Here we investigated the viral envelope proteins and antibody responses in early infection in a small subset of individuals from two of the African sites included in the larger cohort study. We found that a marker for the efficiency of envelope glycosylation in the infecting viral variant was strongly correlated with the development of antibodies with greater neutralization breadth. We also found that extensive viral changes in the V2, V4, and V5 regions of the envelope gp120 protein, were strongly associated with the development of antibodies with greater neutralization breadth. Based on these results, we propose that more efficient glycosylation of the envelope protein of the infecting viral variant elicits neutralizing antibodies that drive early and complex amino acid changes in gp120, which triggers the development of antibodies whose neutralization breadth can be augmented over time by additional viral and host factors. These findings suggest that a better understanding of the efficiency of envelope glycosylation in HIV-1 could inform current vaccine strategies aimed at eliciting antibodies with neutralization breadth.
doi:10.1371/journal.ppat.1005989
PMCID: PMC5112890  PMID: 27851829
4.  Cepstral peak sensitivity: A theoretic analysis and comparison of several implementations 
Summary
Objective
The aim of this study was to develop a theoretic analysis of the cepstral peak, to compare several cepstral peak software programs, and to propose methods for reducing variability in cepstral peak estimation.
Study Design
Descriptive, experimental study.
Methods
The theoretic cepstral peak value of a pulse train was derived and compared to estimates computed for pulse train WAV files using available cepstral peak software programs: 1) Hillenbrand’s cepstral peak prominence (CPP) software, 2) KayPENTAX Multi-Speech implementation of CPP, and 3) a MATLAB implementation using cepstral interpolation. Cepstral peak variation was also investigated for synthetic breathy vowels.
Results
For pulse trains with period T samples, the theoretic cepstral peak is 1/2+ε/T, |ε|<0.1 for all pulse trains (ε=0 for integer T). For fundamental frequencies between 70 and 230 Hz, cepstral peak mean ± st. dev. was 0.496±0.002 using cepstral interpolation and 0.29±0.03 using Hillenbrand’s software, whereas CPP was 35.0±3.8 dB using Hillenbrand’s software and 20.5±2.7 dB using KayPENTAX’s software. CP and CPP vs. signal-to-noise ratio for synthetic breathy vowels were fit to a logistic model for the Hillenbrand (R2 = 0.92) and KayPENTAX (R2 = 0.82) estimators as well as an ideal estimator (R2 = 0.98) which used a period-synchronous analysis.
Conclusions
The findings indicate that several variables unrelated to the signal itself impact cepstral peak values, with some factors introducing large variability in cepstral peak values that would otherwise be attributed to the signal (e.g., voice quality). Variability may be reduced by using a period-synchronous analysis with Hann windows.
doi:10.1016/j.jvoice.2014.11.005
PMCID: PMC4630216  PMID: 25944288
5.  Teachers and Teaching: Speech Production Accommodations Due to Changes in the Acoustic Environment 
Energy procedia  2015;78:3102-3107.
School teachers have an elevated risk of voice problems due to the vocal demands in the workplace. This manuscript presents the results of three studies investigating teachers’ voice use at work. In the first study, 57 teachers were observed for 2 weeks (waking hours) to compare how they used their voice in the school environment and in non-school environments. In a second study, 45 participants performed a short vocal task in two different rooms: a variable acoustic room and an anechoic chamber. Subjects were taken back and forth between the two rooms. Each time they entered the variable acoustics room, the reverberation time and/or the background noise condition had been modified. In this latter study, subjects responded to questions about their vocal comfort and their perception of changes in the acoustic environment. In a third study, 20 untrained vocalists performed a simple vocal task in the following conditions: with and without background babble and with and without transparent plexiglass shields to increase the first reflection. Relationships were examined between [1] the results for the room acoustic parameters; [2] the subjects’ perception of the room; and [3] the recorded speech acoustic. Several differences between male and female subjects were found; some of those differences held for each room condition (at school vs. not at school, reverberation level, noise level, and early reflection).
doi:10.1016/j.egypro.2015.11.764
PMCID: PMC4774899  PMID: 26949426
6.  HIV-1-specific CD8 T cells exhibit limited cross-reactivity during acute infection 
Prior work has demonstrated that HIV-1-specific CD8 T cells can cross-recognize variant epitopes. However, the majority of these studies were performed in the context of chronic infection, where the presence of viral quasispecies makes it difficult to ascertain the true nature of the original antigenic stimulus. To overcome this limitation, we evaluated the extent of CD8 T-cell cross-reactivity in patients with acute HIV-1 clade B infection. In each case, we determined the transmitted founder virus sequence to identify the autologous epitopes restricted by individual HLA class I molecules. Our data show that cross-reactive CD8 T cells are infrequent during the acute phase of HIV-1 infection. Moreover, in the uncommon instances where cross-reactive responses were detected, the variant epitopes were poorly recognized in cytotoxicity assays. Molecular analysis revealed that similar antigenic structures could be cross-recognized by identical CD8 T-cell clonotypes mobilized in vivo, yet even subtle differences in a single TCR-accessible peptide residue were sufficient to disrupt variant-specific reactivity. These findings demonstrate that CD8 T cells are highly specific for autologous epitopes during acute HIV-1 infection. Polyvalent vaccines may therefore be required to provide optimal immune cover against this genetically labile pathogen.
doi:10.4049/jimmunol.1502411
PMCID: PMC4821763  PMID: 26983786
7.  Intraoral pressures produced by thirteen semi-occluded vocal tract gestures 
The use of semi-occluded vocal tract (SOVT) exercises as habilitative and rehabilitative tools has grown substantially in the past two decades. As the use or these exercises has grown, so too has the number of variations of the phonatory gestures used to create oral semi-occlusions. While much of the research on SOVT exercises to this point has been conducted using straw phonation, there has been little discussion or investigation regarding how other phonatory gestures that are considered to be SOVT compare to one another. The current study sought to measure the intraoral pressure produced by 13 phonatory gestures generally thought of as oral semi-occlusions. Twenty subjects (10 male, 10 female) produced three tokens of each gesture, and intraoral pressure was recorded via a thin, flexible-cannula pressure transducer. Pressures ranged between 0.1 and 1.0 kPa, but varied significantly between gestures and between subjects.
doi:10.3109/14015439.2014.913074
PMCID: PMC4970513  PMID: 24865621
Manometry; oral pressures; semi-occlusion; vocal tract
8.  Signatures in Simian Immunodeficiency Virus SIVsmE660 Envelope gp120 Are Associated with Mucosal Transmission but Not Vaccination Breakthrough in Rhesus Macaques 
Journal of Virology  2016;90(4):1880-1887.
ABSTRACT
Mucosal surfaces are vulnerable to human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection and thus are key sites for eliciting vaccine-mediated protection. Vaccine protocols carried out at the Yerkes Primate Research Center utilized SIVmac239-based immunization strategies with intrarectal and intravaginal SIVsmE660 challenge of rhesus macaques. We investigated whether there were genetic signatures associated with SIVsmE660 intrarectal and intravaginal transmissions in vaccinated and unvaccinated monkeys. When transmitted/founder (T/F) envelope (Env) sequences from 49 vaccinated and 15 unvaccinated macaques were compared to each other, we were unable to identify any vaccine breakthrough signatures. In contrast, when the vaccinated and control T/F Envs were combined and compared to the challenge stock, residues at gp120 positions 23, 45, 47, and 70 (Ile-Ala-Lys-Asn [I-A-K-N]) emerged as signatures of mucosal transmission. However, T/F Envs derived from intrarectal and intravaginal infections were not different. Our data suggest that the vaginal and rectal mucosal environments both imposed a strong selection bias for SIVsmE660 variants carrying I-A-K-N that was not further enhanced by immunization. These findings, combined with the strong conservation of A-K-N in most HIV-2/SIVsmm isolates and the analogous residues in HIV-1/SIVcpz isolates, suggest that these residues confer increased transmission fitness to SIVsmE660.
IMPORTANCE Most HIV-1 infections occur across a mucosal barrier, and it is therefore important to understand why these sites are vulnerable and how to protect them with a vaccine. To gain insight into these questions, we studied rhesus macaques that were vaccinated with SIVmac239 and unvaccinated controls to determine whether the SIVsmE660 viral variants that infected these two groups were different. We did not find differences between viral variants in the absence versus presence of vaccination-induced immunity, but we did find that the SIVsmE660 viral variants that infected the monkeys, regardless of vaccination, were different from the dominant population found in the viral challenge inoculum. Our data suggest that the mucosal environments of the vagina and rectum both impose a strong selection for the SIVsmE660 variants in the challenge inoculum that are most like SIV and HIVs that circulate in nature.
doi:10.1128/JVI.02711-15
PMCID: PMC4734005  PMID: 26676777
9.  Comparison of Vocal Vibration-Dose Measures for Potential-Damage Risk Criteria 
Purpose
Schoolteachers have become a benchmark population for the study of occupational voice use. A decade of vibration-dose studies on the teacher population allows a comparison to be made between specific dose measures for eventual assessment of damage risk.
Method
Vibration dosimetry is reformulated with the inclusion of collision stress. Two methods of estimating amplitude of vocal-fold vibration are compared to capture variations in vocal intensity. Energy loss from collision is added to the energy-dissipation dose. An equal-energy-dissipation criterion is defined and used on the teacher corpus as a potential-damage risk criterion.
Results
Comparison of time-, cycle-, distance-, and energy-dose calculations for 57 teachers reveals a progression in information content in the ability to capture variations in duration, speaking pitch, and vocal intensity. The energy-dissipation dose carries the greatest promise in capturing excessive tissue stress and collision but also the greatest liability, due to uncertainty in parameters. Cycle dose is least correlated with the other doses.
Conclusion
As a first guide to damage risk in excessive voice use, the equal-energy-dissipation dose criterion can be used to structure trade-off relations between loudness, adduction, and duration of speech.
doi:10.1044/2015_JSLHR-S-13-0128
PMCID: PMC4686305  PMID: 26172434
10.  COMPARISON OF THE PRODUCED AND PERCEIVED VOICE RANGE PROFILES IN UNTRAINED AND TRAINED CLASSICAL SINGERS 
Frequency and intensity ranges (in true dB SPL re 20 μPa at 1 meter) of voice production in trained and untrained vocalists were compared to the perceived dynamic range (phons) and units of loudness (sones) of the ear. Results were reported in terms of standard Voice Range Profiles (VRPs), perceived VRPs (as predicted by accepted measures of auditory sensitivities), and a new metric labeled as an Overall Perceptual Level Construct. Trained classical singers made use of the most sensitive part of the hearing range (around 3–4 KHz) through the use of the singer’s formant. When mapped onto the contours of equal-loudness (depicting non-uniform spectral and dynamic sensitivities of the auditory system), the formant is perceived at an even higher sound level, as measured in phons, than a flat or A-weighted spectrum would indicate. The contributions of effects like the singer’s formant and the sensitivities of the auditory system helped the trained singers produce 20–40 percent more units of loudness, as measured in sones, than the untrained singers. Trained male vocalists had a maximum Overall Perceptual Level Construct that was 40% higher than the untrained male vocalists. While the A-weighted spectrum (commonly used in VRP measurement) is a reasonable first order approximation of auditory sensitivities, it misrepresents the most salient part of the sensitivities (where the singer’s formant is found) by nearly 10 dB.
doi:10.1016/j.jvoice.2005.08.009
PMCID: PMC4782147  PMID: 16325373
Voice Range Profile; long-term average spectrum; Singer’s formant; equal loudness; sone; phon; perception
11.  Objective Measurement of Vocal Fatigue in Classically Trained Singers: A Pilot Study of Vocal Dosimetry Data 
Objectives
To evaluate vocal fatigue by using objective and subjective measurements of dose recorded by the National Center for Voice and Speech (NCVS) Dosimeter™ (Dosimeter).
Study Design and Setting
Seven subjects completed a two-week study period. The Dosimeter recorded vocal load, soft phonation tasks and subjective soft voice ratings. Three vocal doses (time, distance, and cycle) were measured in classical singers' larynges during an intensive practice period.
Results
Spikes in vocal load are reflected as harsher subjective ratings on the same day as well as 24–72 hours later. When at least 48 hours of vocal rest occurred before a vocal load, improved subjective evaluations were seen after the load.
Conclusions
The NCVS Dosimeter appears to be an effective tool for data collection on prolonged use of the voice.
Significance
This is the first multi-day study comparing objective and subjective data on vocal fatigue in a group of professional singers.
doi:10.1016/j.otohns.2006.06.1268
PMCID: PMC4782153  PMID: 17011424
12.  SENSITIVITY OF ELASTIC PROPERTIES TO MEASUREMENT UNCERTAINTIES IN LARYNGEAL MUSCLES WITH IMPLICATIONS FOR VOICE FUNDAMENTAL FREQUENCY PREDICTION 
Objectives/Hypothesis
This paper discusses the effects of measurement uncertainties when calculating elastic moduli of laryngeal tissue.
Methods
Small dimensions coupled with highly nonlinear elastic properties exacerbate the uncertainties. The sensitivity of both tangent and secant Young’s Modulus was quantified in terms of the coefficient of variation, which depended on measurement of reference length and cross-sectional area.
Results
Uncertainties in the measurement of mass, used to calculate cross-sectional area of a small tissue sample, affected Young’s Modulus calculations when tissue absorption of the hydrating solution was not accounted for. Uncertainty in reference length had twice the effect on elasticity than other measures.
Conclusions
The implication of these measurement errors on predicted fundamental frequency of vocalization is discussed. Refinements on isolated muscle experimental protocols are proposed that pay greatest attention to measures of highest sensitivity.
doi:10.1016/j.jvoice.2006.06.004
PMCID: PMC4778974  PMID: 16904867
elasticity; fundamental frequency; larynx; sensitivity analysis
13.  Characterization and Implementation of a Diverse Simian Immunodeficiency Virus SIVsm Envelope Panel in the Assessment of Neutralizing Antibody Breadth Elicited in Rhesus Macaques by Multimodal Vaccines Expressing the SIVmac239 Envelope 
Journal of Virology  2015;89(16):8130-8151.
ABSTRACT
Antibodies that can neutralize diverse viral strains are likely to be an important component of a protective human immunodeficiency virus type 1 (HIV-1) vaccine. To this end, preclinical simian immunodeficiency virus (SIV)-based nonhuman primate immunization regimens have been designed to evaluate and enhance antibody-mediated protection. However, these trials often rely on a limited selection of SIV strains with extreme neutralization phenotypes to assess vaccine-elicited antibody activity. To mirror the viral panels used to assess HIV-1 antibody breadth, we created and characterized a novel panel of 14 genetically and phenotypically diverse SIVsm envelope (Env) glycoproteins. To assess the utility of this panel, we characterized the neutralizing activity elicited by four SIVmac239 envelope-expressing DNA/modified vaccinia virus Ankara vector- and protein-based vaccination regimens that included the immunomodulatory adjuvants granulocyte-macrophage colony-stimulating factor, Toll-like receptor (TLR) ligands, and CD40 ligand. The SIVsm Env panel exhibited a spectrum of neutralization sensitivity to SIV-infected plasma pools and monoclonal antibodies, allowing categorization into three tiers. Pooled sera from 91 rhesus macaques immunized in the four trials consistently neutralized only the highly sensitive tier 1a SIVsm Envs, regardless of the immunization regimen. The inability of vaccine-mediated antibodies to neutralize the moderately resistant tier 1b and tier 2 SIVsm Envs defined here suggests that those antibodies were directed toward epitopes that are not accessible on most SIVsm Envs. To achieve a broader and more effective neutralization profile in preclinical vaccine studies that is relevant to known features of HIV-1 neutralization, more emphasis should be placed on optimizing the Env immunogen, as the neutralization profile achieved by the addition of adjuvants does not appear to supersede the neutralizing antibody profile determined by the immunogen.
IMPORTANCE Many in the HIV/AIDS vaccine field believe that the ability to elicit broadly neutralizing antibodies capable of blocking genetically diverse HIV-1 variants is a critical component of a protective vaccine. Various SIV-based nonhuman primate vaccine studies have investigated ways to improve antibody-mediated protection against a heterologous SIV challenge, including administering adjuvants that might stimulate a greater neutralization breadth. Using a novel SIV neutralization panel and samples from four rhesus macaque vaccine trials designed for cross comparison, we show that different regimens expressing the same SIV envelope immunogen consistently elicit antibodies that neutralize only the very sensitive tier 1a SIV variants. The results argue that the neutralizing antibody profile elicited by a vaccine is primarily determined by the envelope immunogen and is not substantially broadened by including adjuvants, resulting in the conclusion that the envelope immunogen itself should be the primary consideration in efforts to elicit antibodies with greater neutralization breadth.
doi:10.1128/JVI.01221-14
PMCID: PMC4524250  PMID: 26018167
14.  A Randomized Controlled Trial of Two Semi-Occluded Vocal Tract Voice Therapy Protocols 
Purpose
Although there is a long history of use of semi-occluded vocal tract gestures in voice therapy, including phonation through thin tubes or straws, the efficacy of phonation through tubes has not been established. This study compares results from a therapy program on the basis of phonation through a flow-resistant tube (FRT) with Vocal Function Exercises (VFE), an established set of exercises that utilize oral semi-occlusions.
Method
Twenty subjects (16 women, 4 men) with dysphonia and/or vocal fatigue were randomly assigned to 1 of 4 treatment conditions: (a) immediate FRT therapy, (b) immediate VFE therapy, (c) delayed FRT therapy, or (d) delayed VFE therapy. Subjects receiving delayed therapy served as a no-treatment control group.
Results
Voice Handicap Index (Jacobson et al., 1997) scores showed significant improvement for both treatment groups relative to the no-treatment group. Comparison of the effect sizes suggests FRT therapy is noninferior to VFE in terms of reduction in Voice Handicap Index scores. Significant reductions in Roughness on the Consensus Auditory-Perceptual Evaluation of Voice (Kempster, Gerratt, Verdolini Abbott, Barkmeier-Kraemer, & Hillman, 2009) were found for the FRT subjects, with no other significant voice quality findings.
Conclusions
VFE and FRT therapy may improve voice quality of life in some individuals with dysphonia. FRT therapy was noninferior to VFE in improving voice quality of life in this study.
doi:10.1044/2015_JSLHR-S-13-0231
PMCID: PMC4610291  PMID: 25675335
15.  Whole-body immunoPET reveals active SIV dynamics in viremic and antiretroviral therapy-treated macaques 
Nature methods  2015;12(5):427-432.
The detection of viral dynamics and localization in the context of controlled HIV infection remains a challenge and is limited to blood and biopsies. We developed a method to capture total-body simian immunodeficiency virus (SIV) replication using immunoPET (antibody-targeted positron emission tomography). The administration of a poly(ethylene glycol)-modified, 64Cu-labeled SIV Gp120–specifc antibody led to readily detectable signals in the gastrointestinal and respiratory tract, lymphoid tissues and reproductive organs of viremic monkeys. Viral signals were reduced in aviremic antiretroviral-treated monkeys but detectable in colon, select lymph nodes, small bowel, nasal turbinates, the genital tract and lung. In elite controllers, virus was detected primarily in foci in the small bowel, select lymphoid areas and the male reproductive tract, as confirmed by quantitative reverse-transcription PCR (qRT-PCR) and immunohistochemistry. This real-time, in vivo viral imaging method has broad applications to the study of immunodeficiency virus pathogenesis, drug and vaccine development, and the potential for clinical translation.
doi:10.1038/nmeth.3320
PMCID: PMC4425449  PMID: 25751144
16.  Particle infectivity of HIV-1 full-length genome infectious molecular clones in a subtype C heterosexual transmission pair following high fidelity amplification and unbiased cloning 
Virology  2014;0:454-461.
The high genetic diversity of HIV-1 impedes high throughput, large-scale sequencing and full-length genome cloning by common restriction enzyme based methods. Applying novel methods that employ a high-fidelity polymerase for amplification and an unbiased fusion-based cloning strategy, we have generated several HIV-1 full-length genome infectious molecular clones from an epidemiologically linked transmission pair. These clones represent the transmitted/founder virus and phylogenetically diverse non-transmitted variants from the chronically infected individual's diverse quasispecies near the time of transmission. We demonstrate that, using this approach, PCR-induced mutations in full-length clones derived from their cognate single genome amplicons are rare. Furthermore, all eight non-transmitted genomes tested produced functional virus with a range of infectivities, belying the previous assumption that a majority of circulating viruses in chronic HIV-1 infection are defective. Thus, these methods provide important tools to update protocols in molecular biology that could be universally applied to the study of human viral pathogens.
doi:10.1016/j.virol.2014.08.018
PMCID: PMC4252503  PMID: 25243334
HIV-1; Transmission; Cloning; Amplification; Fitness; RNA Virus; Quasispecies; Infectious molecular clones
17.  Immunogenetic influences on acquisition of HIV-1 infection: Consensus findings from two African cohorts point to an enhancer element in IL19 (1q32.2) 
Genes and immunity  2015;16(3):213-220.
Numerous reports have suggested that immunogenetic factors may influence HIV-1 acquisition, yet replicated findings that translate between study cohorts remain elusive. Our work aimed to test several hypotheses about genetic variants within the IL10-IL24 gene cluster that encodes interleukin (IL)-10, IL-19, IL-20, and IL-24. In aggregated data from 515 Rwandans and 762 Zambians with up to 12 years of follow-up, 190 single nucleotide polymorphisms (SNPs) passed quality control procedures. When HIV-1-exposed seronegative subjects (n = 486) were compared with newly seroconverted individuals (n = 313) and seroprevalent subjects (n = 478) who were already infected at enrollment, rs12407485 (G>A) in IL19 showed a robust association signal in adjusted logistic regression models (odds ratio = 0.64, P = 1.7 × 10−4, and q = 0.033). Sensitivity analyses demonstrated that (i) results from both cohorts and subgroups within each cohort were highly consistent; (ii) verification of HIV-1 infection status after enrollment was critical; and (iii) supporting evidence was readily obtained from Cox proportional hazards models. Data from public databases indicate that rs12407485 is part of an enhancer element for three transcription factors. Overall, these findings suggest that molecular features at the IL19 locus may modestly alter the establishment of HIV-1 infection.
doi:10.1038/gene.2014.84
PMCID: PMC4409473  PMID: 25633979
Africa; HIV-1 infection; IL19; SNP; association; validation
18.  Heterosexual Transmission of Subtype C HIV-1 Selects Consensus-Like Variants without Increased Replicative Capacity or Interferon-α Resistance 
PLoS Pathogens  2015;11(9):e1005154.
Heterosexual transmission of HIV-1 is characterized by a genetic bottleneck that selects a single viral variant, the transmitted/founder (TF), during most transmission events. To assess viral characteristics influencing HIV-1 transmission, we sequenced 167 near full-length viral genomes and generated 40 infectious molecular clones (IMC) including TF variants and multiple non-transmitted (NT) HIV-1 subtype C variants from six linked heterosexual transmission pairs near the time of transmission. Consensus-like genomes sensitive to donor antibodies were selected for during transmission in these six transmission pairs. However, TF variants did not demonstrate increased viral fitness in terms of particle infectivity or viral replicative capacity in activated peripheral blood mononuclear cells (PBMC) and monocyte-derived dendritic cells (MDDC). In addition, resistance of the TF variant to the antiviral effects of interferon-α (IFN-α) was not significantly different from that of non-transmitted variants from the same transmission pair. Thus neither in vitro viral replicative capacity nor IFN-α resistance discriminated the transmission potential of viruses in the quasispecies of these chronically infected individuals. However, our findings support the hypothesis that within-host evolution of HIV-1 in response to adaptive immune responses reduces viral transmission potential.
Author Summary
Despite the available HIV-1 diversity present in a chronically infected individual, single viral variants are transmitted in 80–90% of heterosexual transmission events. These breakthrough viruses may have unique properties that confer a higher capacity to transmit. Determining these properties could help inform the rational design of vaccines and enhance our understanding of viral transmission. We isolated the transmitted variant and a set of related non-transmitted variants from the transmitting partner near the estimated date of transmission from six epidemiologically linked transmission pairs to investigate viral correlates of transmission. The simplest explanation that transmitted variants are inherently more infectious or faster replicators in vitro did not hold true. In addition, transmitted variants did not replicate more efficiently than their non-transmitted counterparts in dendritic cells or in the presence of interferon-alpha in vitro, suggesting that they are not uniquely adapted to these components of the innate immune system. More ancestral genomes that were relatively sensitive to antibody neutralization tended to transmit, supporting previous reports that mutational escape away from the adaptive immune response likely reduces the ability to transmit. Our investigation into the traits of transmitted HIV-1 variants adds to the understanding of viral determinants of transmission.
doi:10.1371/journal.ppat.1005154
PMCID: PMC4574710  PMID: 26378795
19.  HLA Class-II Associated HIV Polymorphisms Predict Escape from CD4+ T Cell Responses 
PLoS Pathogens  2015;11(8):e1005111.
Antiretroviral therapy, antibody and CD8+ T cell-mediated responses targeting human immunodeficiency virus-1 (HIV-1) exert selection pressure on the virus necessitating escape; however, the ability of CD4+ T cells to exert selective pressure remains unclear. Using a computational approach on HIV gag/pol/nef sequences and HLA-II allelic data, we identified 29 HLA-II associated HIV sequence polymorphisms or adaptations (HLA-AP) in an African cohort of chronically HIV-infected individuals. Epitopes encompassing the predicted adaptation (AE) or its non-adapted (NAE) version were evaluated for immunogenicity. Using a CD8-depleted IFN-γ ELISpot assay, we determined that the magnitude of CD4+ T cell responses to the predicted epitopes in controllers was higher compared to non-controllers (p<0.0001). However, regardless of the group, the magnitude of responses to AE was lower as compared to NAE (p<0.0001). CD4+ T cell responses in patients with acute HIV infection (AHI) demonstrated poor immunogenicity towards AE as compared to NAE encoded by their transmitted founder virus. Longitudinal data in AHI off antiretroviral therapy demonstrated sequence changes that were biologically confirmed to represent CD4+ escape mutations. These data demonstrate an innovative application of HLA-associated polymorphisms to identify biologically relevant CD4+ epitopes and suggests CD4+ T cells are active participants in driving HIV evolution.
Author Summary
In HIV, CD4+ T cells are best known as the primary targets of infection. Although emerging data has suggested a more active role in viral pathogenesis, the CD4+ T cell population remains relatively understudied. Using a novel computational approach, we predicted 29 different epitopes with mutations that potentially represent escape from CD4+ T cell responses. The predicted escaped epitopes were found to be less immunogenic than the wild type forms, suggesting that the identified escapes allow HIV to reduce its visibility to the immune system. Using longitudinal samples, we were able to show CD4+ T cells driving viral escape following acute infection. Overall, these findings significantly expand our knowledge of how CD4+ T cells can exert HIV control and influence HIV evolution, providing important implications to future vaccine development strategies.
doi:10.1371/journal.ppat.1005111
PMCID: PMC4547780  PMID: 26302050
20.  High Transmitter CD4+ T-Cell Count Shortly after the Time of Transmission in a Study of African Serodiscordant Couples 
PLoS ONE  2015;10(8):e0134438.
Background
2013 WHO guidelines recommend starting ART at CD4+ T-cell counts ≤500 cells/μL. We present the T-cell counts from adult Africans with HIV shortly following transmission to their sexual partners.
Methods
HIV-discordant couples in Zambia, Uganda and Rwanda were followed prospectively and received couples counseling and condoms. HIV uninfected partners were tested for HIV at least quarterly and HIV-infected partners received HIV care and referral for ART per national guidelines. Upon diagnosis of incident HIV infection in the previously HIV-uninfected partner, a blood sample was collected from both partners to measure CD4+ T-cells and perform viral linkage. The estimated date of infection (EDI) of the incident case was calculated based on testing history. EDI was unknown for suspected transmitting partners.
Results
From 2006–2011, 4,705 HIV-discordant couples were enrolled in this cohort, and 443 cases of incident HIV infection were documented. Virus linkage analysis was performed in 374 transmission pairs, and 273 (73%) transmissions were linked genetically. CD4 counts in the transmitting partner were measured a median of 56 days after EDI (mean:90.5, min:10, max:396). The median CD4 count was 339 cells/μl (mean:386.4, min:15, max:1,434), and the proportion of partners with a CD4+ T-cell count above 500/μl was 25% (95% CI:21, 31).
Conclusions
In our cohort of discordant couples, 73% of HIV transmissions occurred within the relationship, and the transmitter CD4+ T cell count shortly after the transmission event was frequently higher than the WHO 2013 ART-initiation guidelines.
doi:10.1371/journal.pone.0134438
PMCID: PMC4546189  PMID: 26291456
21.  Low Antibody-Dependent Cellular Cytotoxicity Responses in Zambians Prior to HIV-1 Intrasubtype C Superinfection 
Virology  2014;0:295-298.
We have previously shown that HIV-1 superinfected Zambian seroconverters mount low binding and neutralizing antibody responses to their primary HIV-1 infecting virus, which could increase susceptibility to re-infection. Here, we investigated if antibody-dependent cellular cytotoxicity (ADCC), a process by which virus-infected cells are killed, was also reduced. Superinfected individuals exhibited low ADCC activity compared to non-superinfected individuals, but similar levels of CMV-reactive binding antibodies, suggesting superinfected individuals are capable of generating and maintaining virus-specific antibodies.
doi:10.1016/j.virol.2014.06.016
PMCID: PMC4125417  PMID: 25004405
HIV-1 superinfection; ADCC; HIV-1 ADCC; HIV dual infection
22.  CD4:CD8 lymphocyte ratio as a quantitative measure of immunologic health in HIV-1 infection: findings from an African cohort with prospective data 
In individuals with human immunodeficiency virus type 1 (HIV-1) infection, CD4:CD8 lymphocyte ratio is often recognized as a quantitative outcome that reflects the critical role of both CD4+ and CD8+ T-cells in HIV-1 pathogenesis or disease progression. Our work aimed to first establish the dynamics and clinical relevance of CD4:CD8 ratio in a cohort of native Africans and then to examine its association with viral and host factors, including: (i) length of infection, (ii) demographics, (iii) HIV-1 viral load (VL), (iv) change in CD4+ T-lymphocyte count (CD4 slope), (v) HIV-1 subtype, and (vi) host genetics, especially human leukocyte antigen (HLA) variants. Data from 499 HIV-1 seroconverters with frequent (monthly to quarterly) follow-up revealed that CD4:CD8 ratio was stable in the first 3 years of infection, with a modest correlation with VL and CD4 slope. A relatively normal CD4:CD8 ratio (>1.0) in early infection was associated with a substantial delay in disease progression to severe immunodeficiency (<350 CD4 cells/μl), regardless of other correlates of HIV-1 pathogenesis (adjusted hazards ratio (HR) = 0.43, 95% confidence interval (CI) = 0.29-0.63, P < 0.0001). Low VL (<10,000 copies/ml) and HLA-A*74:01 were the main predictors of CD4:CD8 ratio >1.0, but HLA variants (e.g., HLA-B*57 and HLA-B*81) previously associated with VL and/or CD4 trajectories in eastern and southern Africans had no obvious impact on CD4:CD8 ratio. Collectively, these findings suggest that CD4:CD8 ratio is a robust measure of immunologic health with both clinical and epidemiological implications.
doi:10.3389/fmicb.2015.00670
PMCID: PMC4486831  PMID: 26191056
Africa; CD4:CD8 ratio; HIV-1; subtype; HLA; statistical models; viral load
23.  Multiplexed highly-accurate DNA sequencing of closely-related HIV-1 variants using continuous long reads from single molecule, real-time sequencing 
Nucleic Acids Research  2015;43(20):e129.
Single Molecule, Real-Time (SMRT®) Sequencing (Pacific Biosciences, Menlo Park, CA, USA) provides the longest continuous DNA sequencing reads currently available. However, the relatively high error rate in the raw read data requires novel analysis methods to deconvolute sequences derived from complex samples. Here, we present a workflow of novel computer algorithms able to reconstruct viral variant genomes present in mixtures with an accuracy of >QV50. This approach relies exclusively on Continuous Long Reads (CLR), which are the raw reads generated during SMRT Sequencing. We successfully implement this workflow for simultaneous sequencing of mixtures containing up to forty different >9 kb HIV-1 full genomes. This was achieved using a single SMRT Cell for each mixture and desktop computing power. This novel approach opens the possibility of solving complex sequencing tasks that currently lack a solution.
doi:10.1093/nar/gkv630
PMCID: PMC4787755  PMID: 26101252
25.  Dynamics of Viremia in Primary HIV-1 infection in Africans: Insights from Analyses of Host and Viral Correlates 
Virology  2013;449:254-262.
In HIV-1 infection, plasma viral load (VL) has dual implications for pathogenesis and public health. Based on well-known patterns of HIV-1 evolution and immune escape, we hypothesized that VL is an evolving quantitative trait that depends heavily on duration of infection (DOI), demographic features, human leukocyte antigen (HLA) genotypes and viral characteristics. Prospective data from 421 African seroconverters with at least four eligible visits did show relatively steady VL beyond 3 months of untreated infection, but host and viral factors independently associated with cross-sectional and longitudinal VL often varied by analytical approaches and sliding time windows. Specifically, the effects of age, HLA-B*53 and infecting HIV-1 subtypes (A1, C and others) on VL were either sporadic or highly sensitive to time windows. These observations were strengthened by the addition of 111 seroconverters with 2–3 eligible VL results, suggesting that DOI should be a critical parameter in epidemiological and clinical studies.
doi:10.1016/j.virol.2013.11.024
PMCID: PMC3931417  PMID: 24418560
Africa; HIV-1; subtype; HLA; statistical models; viral load

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