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1.  The Global Transmission Network of HIV-1 
The Journal of Infectious Diseases  2013;209(2):304-313.
Human immunodeficiency virus type 1 (HIV-1) is pandemic, but its contemporary global transmission network has not been characterized. A better understanding of the properties and dynamics of this network is essential for surveillance, prevention, and eventual eradication of HIV. Here, we apply a simple and computationally efficient network-based approach to all publicly available HIV polymerase sequences in the global database, revealing a contemporary picture of the spread of HIV-1 within and between countries. This approach automatically recovered well-characterized transmission clusters and extended other clusters thought to be contained within a single country across international borders. In addition, previously undescribed transmission clusters were discovered. Together, these clusters represent all known modes of HIV transmission. The extent of international linkage revealed by our comprehensive approach demonstrates the need to consider the global diversity of HIV, even when describing local epidemics. Finally, the speed of this method allows for near-real-time surveillance of the pandemic's progression.
doi:10.1093/infdis/jit524
PMCID: PMC3873788  PMID: 24151309
human immunodeficiency virus; transmission network; molecular epidemiology
2.  Cytomegalovirus Replication in Semen Is Associated with Higher Levels of Proviral HIV DNA and CD4+ T Cell Activation during Antiretroviral Treatment 
Journal of Virology  2014;88(14):7818-7827.
ABSTRACT
Asymptomatic cytomegalovirus (CMV) replication occurs frequently in the genital tract in untreated HIV-infected men and is associated with increased immune activation and HIV disease progression. To determine the connections between CMV-associated immune activation and the size of the viral reservoir, we evaluated the interactions between (i) asymptomatic seminal CMV replication, (ii) levels of T cell activation and proliferation in blood, and (iii) the size and transcriptional activity of the HIV DNA reservoir in blood from 53 HIV-infected men on long-term antiretroviral therapy (ART) with suppressed HIV RNA in blood plasma. We found that asymptomatic CMV shedding in semen was associated with significantly higher levels of proliferating and activated CD4+ T cells in blood (P < 0.01). Subjects with detectable CMV in semen had approximately five times higher average levels of HIV DNA in blood CD4+ T cells than subjects with no CMV. There was also a trend for CMV shedders to have increased cellular (multiply spliced) HIV RNA transcription (P = 0.068) compared to participants without CMV, but it is unclear if this transcription pattern is associated with residual HIV replication. In multivariate analysis, the presence of seminal plasma CMV (P = 0.04), detectable 2-long terminal repeat (2-LTR), and lower nadir CD4+ (P < 0.01) were independent predictors of higher levels of proviral HIV DNA in blood. Interventions aimed at reducing seminal CMV and associated immune activation may be important for HIV curative strategies. Future studies of anti-CMV therapeutics will help to establish causality and determine the mechanisms underlying these described associations.
IMPORTANCE Almost all individuals infected with HIV are also infected with cytomegalovirus (CMV), and the replication dynamics of the two viruses likely influence each other. This study investigated interactions between asymptomatic CMV replication within the male genital tract, levels of inflammation in blood, and the size of the HIV DNA reservoir in 53 HIV-infected men on long-term antiretroviral therapy (ART) with suppressed HIV RNA in blood plasma. In support of our primary hypothesis, shedding of CMV DNA in semen was associated with increased activation and proliferation of T cells in blood and also significantly higher levels of HIV DNA in blood cells. These results suggest that CMV reactivation might play a role in the maintenance of the HIV DNA reservoir during suppressive ART and that it could be a target of pharmacologic intervention in future studies.
doi:10.1128/JVI.00831-14
PMCID: PMC4097769  PMID: 24789781
3.  Different Plasma Markers of Inflammation Are Influenced by Immune Recovery and cART Composition or Intensification in Treated HIV Infected Individuals 
PLoS ONE  2014;9(12):e114142.
Background
HIV-1 infection increases plasma levels of inflammatory markers. Combination antiretroviral therapy (cART) does not restore inflammatory markers to normal levels. Since intensification of cART with raltegravir reduced CD8 T-cell activation in the Discor-Ral and IntegRal studies, we have evaluated the effect of raltegravir intensification on several soluble inflammation markers in these studies.
Methods
Longitudinal plasma samples (0–48 weeks) from the IntegRal (n = 67, 22 control and 45 intensified individuals) and the Discor-Ral studies (44 individuals with CD4 T-cell counts<350 cells/µl, 14 control and 30 intensified) were assayed for 25 markers. Mann-Whitney, Wilcoxon, Spearman test and linear mixed models were used for analysis.
Results
At baseline, different inflammatory markers were strongly associated with HCV co-infection, lower CD4 counts and with cART regimens (being higher in PI-treated individuals), but poorly correlated with detection of markers of residual viral replication. Although raltegravir intensification reduced inflammation in individuals with lower CD4 T-cell counts, no effect of intensification was observed on plasma markers of inflammation in a global analysis. An association was found, however, between reductions in immune activation and plasma levels of the coagulation marker D-dimer, which exclusively decreased in intensified patients on protease inhibitor (PI)-based cART regimens (P = 0.040).
Conclusions
The inflammatory profile in treated HIV-infected individuals showed a complex association with HCV co-infection, the levels of CD4 T cells and the cART regimen. Raltegravir intensification specifically reduced D-dimer levels in PI-treated patients, highlighting the link between cART composition and residual viral replication; however, raltegravir had little effect on other inflammatory markers.
doi:10.1371/journal.pone.0114142
PMCID: PMC4252101  PMID: 25462535
4.  Differential Gene Expression in HIV-Infected Individuals Following ART 
Antiviral research  2013;100(2):420-428.
Previous studies of the effect of ART on gene expression in HIV-infected individuals have identified small numbers of modulated genes. Since these studies were underpowered or cross-sectional in design, a paired analysis of peripheral blood mononuclear cells (PBMCs), isolated before and after ART, from a robust number of HIV-infected patients (N=32) was performed. Gene expression was assayed by microarray and 4,157 differentially expressed genes (DEGs) were identified following ART using multivariate permutation tests. Pathways and Gene Ontology (GO) terms over-represented for DEGs reflected the transition from a period of active virus replication before ART to one of viral suppression (e.g., repression of JAK-STAT signaling) and possible prolonged drug exposure (e.g. oxidative phosphorylation pathway) following ART. CMYC was the DEG whose product made the greatest number of interactions at the protein level in protein interaction networks (PINs), which has implications for the increased incidence of Hodgkin’s lymphoma (HL) in HIV-infected patients. The differential expression of multiple genes was confirmed by RT-qPCR including well-known drug metabolism genes (e.g., ALOX12 and CYP2S1). Targets not confirmed by RT-qPCR (i.e., GSTM2 and RPL5) were significantly confirmed by droplet digital (ddPCR), which may represent a superior method when confirming DEGs with low fold changes. In conclusion, a paired design revealed that the number of genes modulated following ART was an order of magnitude higher than previously recognized.
doi:10.1016/j.antiviral.2013.07.017
PMCID: PMC3899918  PMID: 23933117
Antiretroviral therapy; droplet digital PCR; gene expression; HIV; microarray; paired analysis
5.  Increase in 2–Long Terminal Repeat Circles and Decrease in D-dimer After Raltegravir Intensification in Patients With Treated HIV Infection: A Randomized, Placebo-Controlled Trial 
The Journal of Infectious Diseases  2013;208(9):1436-1442.
Background. The degree to which human immunodeficiency virus (HIV) continues to replicate during antiretroviral therapy (ART) is controversial. We conducted a randomized, double-blind, placebo-controlled study to assess whether raltegravir intensification reduces low-level viral replication, as defined by an increase in the level of 2–long terminal repeat (2-LTR) circles.
Methods. Thirty-one subjects with an ART-suppressed plasma HIV RNA level of <40 copies/mL and a CD4+ T-cell count of ≥350 cells/mm3 for ≥1 year were randomly assigned to receive raltegravir 400 mg twice daily or placebo for 24 weeks. 2-LTR circles were analyzed by droplet digital polymerase chain reaction at weeks 0, 1, 2, and 8.
Results. The median duration of ART suppression was 3.8 years. The raltegravir group had a significant increase in the level of 2-LTR circles, compared to the placebo group. The week 1 to 0 ratio was 8.8-fold higher (P = .0025) and the week 2 to 0 ratio was 5.7-fold higher (P = .023) in the raltegravir vs. placebo group. Intensification also led to a statistically significant decrease in the D-dimer level, compared to placebo (P = .045).
Conclusions. Raltegravir intensification resulted in a rapid increase in the level of 2-LTR circles in a proportion of subjects, indicating that low-level viral replication persists in some individuals even after long-term ART. Intensification also reduced the D-dimer level, a coagulation biomarker that is predictive of morbidity and mortality among patients receiving treatment for HIV infection.
doi:10.1093/infdis/jit453
PMCID: PMC3789577  PMID: 23975885
HIV; raltegravir intensification; 2-LTR circles; ongoing viral replication; D-dimer
6.  CD4+ and CD8+ T Cell Activation Are Associated with HIV DNA in Resting CD4+ T Cells 
PLoS ONE  2014;9(10):e110731.
The association between the host immune environment and the size of the HIV reservoir during effective antiretroviral therapy is not clear. Progress has also been limited by the lack of a well-accepted assay for quantifying HIV during therapy. We examined the association between multiple measurements of HIV and T cell activation (as defined by markers including CD38, HLA-DR, CCR5 and PD-1) in 30 antiretroviral-treated HIV-infected adults. We found a consistent association between the frequency of CD4+ and CD8+ T cells expressing HLA-DR and the frequency of resting CD4+ T cells containing HIV DNA. This study highlights the need to further examine this relationship and to better characterize the biology of markers commonly used in HIV studies. These results may also have implications for reactivation strategies.
doi:10.1371/journal.pone.0110731
PMCID: PMC4207702  PMID: 25340755
7.  IDEPI: Rapid Prediction of HIV-1 Antibody Epitopes and Other Phenotypic Features from Sequence Data Using a Flexible Machine Learning Platform 
PLoS Computational Biology  2014;10(9):e1003842.
Since its identification in 1983, HIV-1 has been the focus of a research effort unprecedented in scope and difficulty, whose ultimate goals — a cure and a vaccine – remain elusive. One of the fundamental challenges in accomplishing these goals is the tremendous genetic variability of the virus, with some genes differing at as many as 40% of nucleotide positions among circulating strains. Because of this, the genetic bases of many viral phenotypes, most notably the susceptibility to neutralization by a particular antibody, are difficult to identify computationally. Drawing upon open-source general-purpose machine learning algorithms and libraries, we have developed a software package IDEPI (IDentify EPItopes) for learning genotype-to-phenotype predictive models from sequences with known phenotypes. IDEPI can apply learned models to classify sequences of unknown phenotypes, and also identify specific sequence features which contribute to a particular phenotype. We demonstrate that IDEPI achieves performance similar to or better than that of previously published approaches on four well-studied problems: finding the epitopes of broadly neutralizing antibodies (bNab), determining coreceptor tropism of the virus, identifying compartment-specific genetic signatures of the virus, and deducing drug-resistance associated mutations. The cross-platform Python source code (released under the GPL 3.0 license), documentation, issue tracking, and a pre-configured virtual machine for IDEPI can be found at https://github.com/veg/idepi.
doi:10.1371/journal.pcbi.1003842
PMCID: PMC4177671  PMID: 25254639
8.  Shedding of HIV and Human Herpesviruses in the Semen of Effectively Treated HIV-1–Infected Men Who Have Sex With Men 
The study aimed to understand the associations between coinfections and HIV RNA shedding in the genital tract of men receiving antiretroviral therapy with suppressed blood plasma viral load.
Background. Current antiretroviral therapy (ART) suppresses human immunodeficiency virus (HIV) in blood to undetectable levels in most infected individuals; however, some men shed HIV in semen despite suppressed levels in blood.
Methods. This study included 114 chronically HIV type 1–infected men who have sex with men, who were receiving ART with blood plasma HIV <500 copies/mL. Asymptomatic participants were screened for bacterial sexually transmitted infections (STIs) and nonspecific genital inflammation. Levels of HIV and 7 human herpesviruses were measured by real-time polymerase chain reaction in seminal plasma. Predictors of HIV seminal shedding were determined for the entire cohort, and on the subset of 100 subjects with blood plasma HIV <50 copies/mL.
Results. Eleven subjects (9.6%) had detectable levels of seminal HIV (median, 2.1 log10 copies/mL), and 72 (63.2%) had at least 1 herpesvirus detected in their seminal plasma. Detectable levels of seminal HIV were present more often in persons with plasma HIV between 50 and 500 copies/mL compared to those <50 copies/mL (P values adjusted for false discovery rate [FDR] = 0.08). There was a trend for high-level cytomegalovirus (CMV; >4 log10 DNA copies/mL; FDR-adjusted P = .08), and presence of Epstein-Barr virus (FDR-adjusted P = .06) in semen to be associated with detectable seminal HIV levels. In a subanalysis of 100 subjects with blood plasma HIV <50 copies/mL, high levels of CMV in semen was the only significant predictor for seminal HIV shedding.
Conclusions. Low-level HIV replication in blood and high-level seminal CMV shedding, but not presence of asymptomatic STIs, is associated with seminal shedding of HIV in men receiving ART, conferring a potential risk for HIV transmission.
doi:10.1093/cid/cit252
PMCID: PMC3703105  PMID: 23595831
cytomegalovirus; HIV shedding; semen; antiretroviral therapy; HIV transmission
9.  Gut Lactobacillales are associated with higher CD4 and less microbial translocation during HIV infection 
AIDS (London, England)  2013;27(12):1921-1931.
Objective
Early HIV infection is characterized by a dramatic depletion of CD4 T cells in the gastrointestinal tract and translocation of bacterial products from the gut into the blood. In this study, we evaluated if gut bacterial profiles were associated with immune status before and after starting antiretroviral therapy (ART).
Design
We evaluated the gut microbiota of men recently infected with HIV (n = 13) who were participating in a randomized, double-blind controlled trial of combination ART and maraviroc versus placebo and who were followed for 48 weeks.
Methods
To evaluate the gut microbiota of participants, we pyrosequenced the bacterial populations from anal swabs collected before and longitudinally after the initiation of ART. Associations of the gut flora with clinical variables (lymphocyte profiles and viral loads), activation and proliferation markers in peripheral blood mononuclear cells and gut biopsies (measured by flow cytometry) and markers of microbial translocation (lipopolysaccharide and soluble CD14) were performed by regression analyses using R statistical software.
Results
Using pyrosequencing, we identified that higher proportions of Lactobacillales in the distal gut of recently HIV-infected individuals were associated with lower markers of microbial translocation, higher CD4% and lower viral loads before ART was started. Similarly, during ART, higher proportions of gut Lactobacillales were associated with higher CD4%, less microbial translocation, less systemic immune activation, less gut T lymphocyte proliferation, and higher CD4% in the gut.
Conclusion
Shaping the gut microbiome, especially proportions of Lactobacillales, could help to preserve immune function during HIV infection.
PMCID: PMC3816380  PMID: 24180001
gut-associated lymphoid tissue; gut microbiome; immune activation; microbial translocation; pyrosequencing
10.  Human APOBEC3 Induced Mutation of Human Immunodeficiency Virus Type-1 Contributes to Adaptation and Evolution in Natural Infection 
PLoS Pathogens  2014;10(7):e1004281.
Human APOBEC3 proteins are cytidine deaminases that contribute broadly to innate immunity through the control of exogenous retrovirus replication and endogenous retroelement retrotransposition. As an intrinsic antiretroviral defense mechanism, APOBEC3 proteins induce extensive guanosine-to-adenosine (G-to-A) mutagenesis and inhibit synthesis of nascent human immunodeficiency virus-type 1 (HIV-1) cDNA. Human APOBEC3 proteins have additionally been proposed to induce infrequent, potentially non-lethal G-to-A mutations that make subtle contributions to sequence diversification of the viral genome and adaptation though acquisition of beneficial mutations. Using single-cycle HIV-1 infections in culture and highly parallel DNA sequencing, we defined trinucleotide contexts of the edited sites for APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H. We then compared these APOBEC3 editing contexts with the patterns of G-to-A mutations in HIV-1 DNA in cells obtained sequentially from ten patients with primary HIV-1 infection. Viral substitutions were highest in the preferred trinucleotide contexts of the edited sites for the APOBEC3 deaminases. Consistent with the effects of immune selection, amino acid changes accumulated at the APOBEC3 editing contexts located within human leukocyte antigen (HLA)-appropriate epitopes that are known or predicted to enable peptide binding. Thus, APOBEC3 activity may induce mutations that influence the genetic diversity and adaptation of the HIV-1 population in natural infection.
Author Summary
Cytidine deaminases of the human APOBEC3 gene family act as an intrinsic defense mechanism against infection with HIV-1 and other viruses. The APOBEC3 proteins introduce mutations into the viral genome by inducing enzymatic modification of nucleotide sequences and inhibiting synthesis of cDNA strands from the viral RNA. Viral Vif counters this impediment to the fidelity of HIV-1 replication by targeting the APOBEC3 proteins for degradation. Low-level APOBEC3 activity that outlasts blockade by viral Vif may foster infrequent mutations that provide a source of genetic variation upon which natural selection acts. Here, we defined the APOBEC3 nucleotide contexts of the edited sites by titration of the wild type and non-editing APOBEC3 mutant in cultured cells. We then followed the patterns of G-to-A mutations we identified in viral DNA in cells obtained from ten patients with acute infection. Our deep sequencing analyses demonstrate an association between sub-lethal APOBEC3 editing and HIV-1 diversification. Mutations at APOBEC3 editing contexts that occurred at particular positions within specific known or predicted epitopes could disrupt peptide binding critical for immune control. Our findings reveal a role for human APOBEC3 in HIV-1 sequence diversification that may influence fitness and evolution of beneficial variants and phenotypes in the population.
doi:10.1371/journal.ppat.1004281
PMCID: PMC4117599  PMID: 25080100
11.  Using Ultradeep Pyrosequencing to Study HIV-1 Coreceptor Usage in Primary and Dual Infection 
The Journal of Infectious Diseases  2013;208(2):271-274.
HIV-1 dual infection (DI) and CXCR4 (X4) coreceptor usage are associated with accelerated disease progression but frequency and dynamics of coreceptor usage during DI is unknown. Ultradeep sequencing was used to interrogate for DI and infer coreceptor usage in longitudinal blood samples of 102 subjects. At baseline, X4 usage was high (23 subjects harbored X4 variants) and was not associated with infection duration or DI. Coreceptor usage changed over time in 12 of 47 participants, and X4 usage emerged in 4 of 41 monoinfections vs 2 of 5 superinfections (P = .12), suggesting a weak statistical trend toward occurrence of superinfection and acquiring X4 usage.
doi:10.1093/infdis/jit168
PMCID: PMC3685231  PMID: 23599311
HIV-1 dual infection; HIV-1 coinfection; HIV-1 superinfection; coreceptor tropism; coreceptor usage; ultradeep pyrosequencing; next-generation sequencing; genotypic tropism prediction; genotypic coreceptor usage prediction
12.  Antibody Response to Achromobacter xylosoxidans during HIV Infection Is Associated with Lower CD4 Levels and Increased Lymphocyte Activation 
Inflammation during HIV infection is associated with worse disease outcomes and progression. Many mechanisms have been indicted, including HIV itself, coinfections, and gut microbial translocation. Concerning microbial translocation, we hypothesized that adaptive immune responses to a specific bacterial species known to be present in gut-associated lymphoid tissue are higher among HIV-infected individuals than among HIV-uninfected controls and are associated with T cell activation and lower CD4 T cell counts. By characterizing the IgG response to Achromobacter xylosoxidans, we found that HIV-infected participants who were immunoresponsive (n = 48) had significantly lower CD4 percentages (P = 0.01), greater CD4 activation (percentages of RA− CD38+) (P = 0.03), and higher soluble CD14 (P = 0.01). HIV-positive individuals had higher anti-A. xylosoxidans IgG titers than HIV-uninfected individuals (P = 0.04). The results suggest an abnormal adaptive immune activation to gut microflora during HIV infection.
doi:10.1128/CVI.00553-13
PMCID: PMC3910916  PMID: 24173027
13.  Barriers to a Cure: New concepts in targeting and eradicating HIV-1 reservoirs 
Lancet  2013;381(9883):10.1016/S0140-6736(13)60104-X.
Antiretroviral therapy for HIV infection requires life-long access and strict adherence to regimens that are both expensive and associated with toxicities. There is growing recognition that a curative intervention will be needed to fully stop the epidemic. The failure to eradicate HIV infection during long-term antiretroviral therapy reflects the intrinsic stability of the viral genome in latently infected CD4+ T cells and other cells and perhaps ongoing low-level viral replication. Heterogeneity in latently infected cell populations and homeostatic proliferation of infected cells may influence the dynamics of virus production and persistence. Chronic immune activation, inflammation and immune dysfunction persist despite potent antiretroviral therapy and likely have important effects on the size and distribution of the viral reservoir. The inability of the immune system to recognize cells harboring latent virus and to eliminate cells actively producing virus represents the biggest challenge to finding a cure. In this perspective, we highlight new approaches toward unraveling the complex virus-host interactions that lead to persistent infection and latency and discuss the rationale for combining novel therapeutic strategies with current antiretroviral treatment options with the goal of curing HIV disease.
doi:10.1016/S0140-6736(13)60104-X
PMCID: PMC3815451  PMID: 23541541
Histone Deacetylases; Epigenetics; Gene Silencing; HIV-1 /immunology
14.  A Synthetic Bivalent Ligand of CXCR4 Inhibits HIV Infection 
G-protein-coupled receptors (GPCRs) are cell membrane protein receptors that transduce signals across the cell membrane and are important targets for therapeutic interventions. As members of the GPCR superfamily, chemokine receptors such as CXCR4 play critical roles in normal physiology as well as the pathology of many human diseases including cancer, inflammation, autoimmune diseases, and human immunodeficiency virus (HIV) infection. Here we report the discovery and study of a novel peptide ligand of CXCR4 using D-amino acids and bivalent ligand approach. This peptide, DV1-K-(DV3), shows very high affinity for CXCR4 with an IC50 of 4 nM in anti-CXCR4 monoclonal antibody (mAb) 12G5 competitive assay, which is more potent than full length natural ligand SDF-1α, even though the peptide is less than half of the number of residues of SDF-1α. This peptide can block the calcium influx stimulated by SDF-1α and inhibit cancer cell migration in vitro via CXCR4, thus functioning as a CXCR4 antagonist. Furthermore, DV1-K-(DV3) peptide displayed anti-HIV activity by inhibiting HIV-1 infection mediated by CXCR4. With its high receptor affinity and stability from D-amino acids, this peptide may be a new probe of CXCR4 functions in physiology and pathology and promising lead for therapeutic development.
doi:10.1016/j.bbrc.2013.05.038
PMCID: PMC3752463  PMID: 23688427
CXCR4; Chemokine receptors; SDF-1α; Peptide antagonist; Bivalent ligand; Protein-protein interaction; Cell migration; HIV infection
15.  Predictors of virologic response in persons who start antiretroviral therapy during recent HIV infection 
AIDS (London, England)  2014;28(6):841-849.
Objective
Despite evidence supporting antiretroviral therapy (ART) in recent HIV infection, little is known about factors that are associated with successful ART. We assessed demographic, virologic, and immunologic parameters to identify predictors of virologic response.
Design
A 24-week observational study of ART on persons enrolled within 6 months of their estimated date of infection (EDI) evaluated baseline demographics and the collection of blood and gut specimens.
Methods
Flow cytometry analyses of blood and gut lymphocytes allowed characterization of CD4+ and CD8+ T cells at study entry and end. Additional assessments included soluble CD14 (sCD14), lipopolysaccharide, CD4+ T-cell counts, and HIV RNA levels.
Results
Twenty nine participants initiated ART, and 17 achieved undetectable HIV RNA by study end. A longer time from EDI to ART, older age, higher sCD14, lower proportions of central memory CD4+ T cells, and higher proportions of activated CD8+ T cells were associated with detectable viremia. Multivariable logistic regression found only older age and elevated sCD14 were independently associated with persistent viremia. Additionally, we observed that ART in recent infection did not result in discernible recovery of CD4+ T cells in the gut.
Conclusion
In persons who started ART within 3–33 weeks from EDI, age and microbial translocation were associated with detectable HIV RNA. As observed in other cohorts, ART in recent infection did not improve proportions of total CD4+ T cells in gut-associated lymphoid tissue (GALT). This lends support to further evaluate the use of more potent ART or regimens that protect the GALT in recent HIV infection.
doi:10.1097/QAD.0000000000000149
PMCID: PMC4049563  PMID: 24401640
antiretroviral therapy; gut-associated lymphoid tissue; microbial translocation; recent HIV; virologic response
16.  Dynamics of Viral Evolution and Neutralizing Antibody Response after HIV-1 Superinfection 
Journal of Virology  2013;87(23):12737-12744.
Investigating the incidence and prevalence of HIV-1 superinfection is challenging due to the complex dynamics of two infecting strains. The superinfecting strain can replace the initial strain, be transiently expressed, or persist along with the initial strain in distinct or in recombined forms. Various selective pressures influence these alternative scenarios in different HIV-1 coding regions. We hypothesized that the potency of the neutralizing antibody (NAb) response to autologous viruses would modulate viral dynamics in env following superinfection in a limited set of superinfection cases. HIV-1 env pyrosequencing data were generated from blood plasma collected from 7 individuals with evidence of superinfection. Viral variants within each patient were screened for recombination, and viral dynamics were evaluated using nucleotide diversity. NAb responses to autologous viruses were evaluated before and after superinfection. In 4 individuals, the superinfecting strain replaced the original strain. In 2 individuals, both initial and superinfecting strains continued to cocirculate. In the final individual, the surviving lineage was the product of interstrain recombination. NAb responses to autologous viruses that were detected within the first 2 years of HIV-1 infection were weak or absent for 6 of the 7 recently infected individuals at the time of and shortly following superinfection. These 6 individuals had detectable on-going viral replication of distinct superinfecting virus in the env coding region. In the remaining case, there was an early and strong autologous NAb response, which was associated with extensive recombination in env between initial and superinfecting strains. This extensive recombination made superinfection more difficult to identify and may explain why the detection of superinfection has typically been associated with low autologous NAb titers.
doi:10.1128/JVI.02260-13
PMCID: PMC3838120  PMID: 24049166
17.  Absence of Detectable HIV-1 Viremia after Treatment Cessation in an Infant 
The New England journal of medicine  2013;369(19):1828-1835.
SUMMARY
An infant born to a woman with human immunodeficiency virus type 1 (HIV-1) infection began receiving antiretroviral therapy (ART) 30 hours after birth owing to high-risk exposure. ART was continued when detection of HIV-1 DNA and RNA on repeat testing met the standard diagnostic criteria for infection. After therapy was discontinued (when the child was 18 months of age), levels of plasma HIV-1 RNA, proviral DNA in peripheral-blood mononuclear cells, and HIV-1 antibodies, as assessed by means of clinical assays, remained undetectable in the child through 30 months of age. This case suggests that very early ART in infants may alter the establishment and long-term persistence of HIV-1 infection.
doi:10.1056/NEJMoa1302976
PMCID: PMC3954754  PMID: 24152233
18.  Cytomegalovirus DNA in Semen and Blood Is Associated With Higher Levels of Proviral HIV DNA 
The Journal of Infectious Diseases  2012;207(6):898-902.
Over three-fourths of human immunodeficiency virus (HIV)–infected men who have sex with men (MSM) have at least one herpesvirus detected in their semen, and cytomegalovirus (CMV) is the most prevalent. The presence of CMV is associated with higher T-cell immune activation and with HIV disease progression in treated and untreated individuals. In this study of 113 antiretroviral (ART)–naive HIV-infected MSM, we found that CMV replication in blood and semen was associated with higher levels of HIV DNA in peripheral blood mononuclear cells. These observations suggest that interventions aimed to reduce CMV replication and, thus, systemic immune activation could decrease the size of the latent HIV reservoir.
doi:10.1093/infdis/jis777
PMCID: PMC3571441  PMID: 23275608
cytomegalovirus; HIV DNA; latent reservoir; immune activation; early HIV infection
19.  HIV Downregulates Interferon-Stimulated Genes in Primary Macrophages 
HIV is able to outpace the innate immune response, including that mediated by interferon (IFN), to establish a productive infection. Primary macrophages, however, may be protected from HIV infection by treatment with type I IFN before virus exposure. The ability of HIV to modulate the type I IFN-mediated innate immune response when it encounters a cell that has already been exposed to IFN remains poorly defined. The optimal pretreatment time (12 h) and the most potent HIV-inhibitors (e.g., IFN-α2 and -ω) were identified to investigate the ability of HIV to modulate an established type I IFN response. Gene expression at the level of the entire transcriptome was then compared between primary macrophages treated with type I IFNs, as opposed to treated with IFNs and then infected with HIV. Although HIV was not able to establish a robust infection, the virus was able to downregulate a number of IFN-stimulated genes (ISGs) with a fold change greater than 1.5 (i.e., AXL, IFI27, IFI44, IFI44L, ISG15, OAS1, OAS3, and XAF1). The downregulation of OAS1 by the presence of HIV was confirmed by real-time quantitative polymerase chain reaction. In conclusion, even though HIV replication is significantly inhibited by IFN pretreatment, the virus is able to downregulate the transcription of known antiviral ISGs (e.g., IFI44, ISG15, and OAS1).
doi:10.1089/jir.2012.0052
PMCID: PMC3573720  PMID: 23276142
20.  Role of Seminal Shedding of Herpesviruses in HIV Type 1 Transmission 
The Journal of Infectious Diseases  2012;207(2):257-261.
To investigate the role of genital shedding of herpesviruses in human immunodeficiency virus type 1 (HIV) transmission, we compared 20 HIV-infected men who did and 26 who did not transmit HIV to their sex partners. As described previously, HIV transmission was associated with the potential source partner having higher levels of HIV RNA in blood and semen, having lower CD4+ T cell counts, having bacterial coinfections in the genital tract, and not using antiretroviral therapy. This study extended these findings by observing significant associations between HIV transmission and the following characteristics, especially among therapy-naive potential source partners: seminal cytomegalovirus (CMV) shedding, seminal Epstein-Barr virus shedding, and levels of anti CMV immunoglobulin in blood plasma.
doi:10.1093/infdis/jis683
PMCID: PMC3532824  PMID: 23148284
Herpes viruses; HIV-1 transmission; genital coinfections; men who have sex with men; antiretroviral therapy
21.  The Use of High Performance Liquid Chromatography to Speciate and Characterize the Epidemiology of Mycobacteria 
Laboratory medicine  2011;42(10):612-617.
Objective
We evaluated high-performance liquid chromatography (HPLC) for species identification of mycobacteria from various clinical specimens in an urban hospital in South Korea between January 2005 and December 2009.
Methods
In the study period 24,774 cultures were completed, yielding the 3215 clinical isolates cultivated for mycobacteria and positive cultures that had mycolic acid investigated by HPLC. For species identification, we compared HPLC patterns of clinical isolates with 33 standard Mycobacterium species.
Results
There were 3 different HPLC groups with single, double, and triple-cluster patterns representing 9, 20, and 4 mycobacterial species, respectively. Species identification rates of HPLC for Mycobacterium tuberculosis and nontuberculous mycobacteria (NTM) were found to be 100% and 95.6%, respectively. Among mycobacterial isolates, 12.1% were NTM-positive. There were 20 different NTM species with frequencies of 0.3%~15.5%.
Conclusion
The HPLC method was highly sensitive identifying NTM isolated from clinical specimens.
doi:10.1309/LMDDEHPSYE6ZDM3C
PMCID: PMC3892985  PMID: 24443588
mycobacteria; Mycobacterium tuberculosis; nontuberculous mycobacteria; HPLC
22.  Herpesviruses and HIV-1 drug resistance mutations influence the virologic and immunologic milieu of the male genital tract 
AIDS (London, England)  2013;27(1):39-47.
Objectives
To further understand the role that chronic viral infections of the male genital tract play on HIV-1 dynamics and replication.
Design
Retrospective, observational study including236 paired semen and blood samples collected from 115 recently HIV-1 infected antiretroviral naïve men who have sex with men (MSM).
Methods
In this study, we evaluated the association ofseminal HIV-1 shedding to coinfections with seven herpesviruses, blood plasma HIV-1 RNA levels, CD4+ T-cell counts, presence of transmitted drug resistance mutations (DRM) in HIV-1 pol, participants’ age and stage of HIV-infection using multivariate generalized estimating equation methods (GEE). Associations between herpesvirus shedding, seminal HIV-1 levels, number and immune activation of seminal T-cells was also investigated (Mann Whitney).
Results
Seminal herpesvirus shedding was observed in 75.7% of subjects. Blood HIV-1 RNA levels (p<0.01), and seminal CMV and HHV-8 levels (p<0.05) were independent predictors of detectable seminal HIV-1 RNA, and higher seminal HIV-1 levelswere associated with CMV and EBV seminal shedding,and absence of DRM (p < 0.05). CMV and EBV seminal shedding was associated with higher number of seminalT-lymphocytes, but onlypresence of seminal CMV DNA was associated withincreasedimmune activation of T-lymphocytes in semen and blood.
Conclusions
Despite high median CD4cells number, we found a high frequency of herpesviruses seminal shedding in our cohort. Shedding of CMV, EBV and HHV-8 and absence of DRM were associated with increased frequency of HIV-1 shedding and/or higher levels of HIV-1 RNA in semen, which are likely important co-factors for HIV-1 transmission.
doi:10.1097/QAD.0b013e3283573305
PMCID: PMC3769229  PMID: 22739399
cytomegalovirus; genital tract; herpesvirus; HIV; HIV drug resistance; men who have sex with men; seminal shedding
23.  Sexual Transmission of predicted CXCR4-tropic HIV-1 likely originating from the Source Partner’s Seminal Cells 
Virology  2012;434(1):2-4.
We present a case of sexual transmission of HIV-1 predicted to have CXCR4-tropism during male-to-male sexual exposure. Phylogenetic analyses exclude cell-free virus in the seminal plasma of the source partner and possibly point to the seminal cells as the origin of the transmission event.
doi:10.1016/j.virol.2012.09.010
PMCID: PMC3485073  PMID: 23040890
24.  Higher HIV-1 Genetic Diversity is Associated with AIDS and Neuropsychological Impairment 
Virology  2012;433(2):498-505.
Standard methods used to estimate HIV-1 population diversity are often resource intensive (e.g., single genome amplification, clonal amplification and pyrosequencing) and not well suited for large study cohorts. Additional approaches are needed to address the relationships between intraindividual HIV-1 genetic diversity and disease. With a small cohort of individuals, we validated three methods for measuring diversity: Shannon entropy and average pairwise distance (APD) using single genome sequences, and counts of mixed bases (i.e. ambiguous nucleotides) from population-based sequences. In a large cohort, we then used the mixed base approach to determine associations between measure HIV-1 diversity and HIV associated disease. Normalized counts of mixed bases correlated with Shannon Entropy at both the nucleotide (rho=0.72, p=0.002) and amino acid level (rho=0.59, p=0.015), and APD (rho=0.75, p=0.001). Among participants who underwent neuropsychological and clinical assessments (n=187), increased HIV-1 population diversity was associated with both a diagnosis of AIDS and neuropsychological impairment.
doi:10.1016/j.virol.2012.08.028
PMCID: PMC3466337  PMID: 22999095
HIV; AIDS; genetic diversity; neuropsychological impairment; viral population dynamics
25.  Impact of Seminal Cytomegalovirus Replication on HIV-1 Dynamics between Blood and Semen 
Journal of medical virology  2012;84(11):1703-1709.
The genital tract of individuals infected with HIV-1 is an anatomic compartment that supports local HIV-1 and CMV replication. This study investigated the association of seminal CMV replication with changes in HIV-1 clonal expansion, evolution and phylogenetic compartmentalization between blood and semen. Fourteen paired blood and semen samples were analyzed from four untreated subjects. Clonal sequences (n=607) were generated from extracted HIV-1 RNA (env C2-V3 region), and HIV-1 and CMV levels were measured in the seminal plasma by real-time PCR. Sequence alignments were evaluated for: (i) viral compartmentalization between semen and blood samples using Slatkin-Maddison and FST methods, (ii) different nucleotide substitution rates in semen and blood, and (iii) association between proportions of clonal HIV-1 sequences in each compartment and seminal CMV levels. Half of the semen samples had detectable CMV DNA, with at least one CMV positive sample for each patient. Seminal CMV DNA levels correlated positively with seminal HIV-1 RNA levels (Spearman p=0.05). A trend towards an association between compartmentalization of HIV-1 sequences sampled from blood and semen and presence of seminal CMV was observed (Cochran Q test p=0.12). Evolutionary rates between semen and blood HIV-1 populations did not differ significantly, and there was no significant association between seminal CMV DNA levels and the frequency of non-unique clonal HIV-1 sequences in the semen. In conclusion, the effects of CMV replication on HIV-1 viral and immunologic dynamics within the male genital tract are not significant enough to perturb evolution or disrupt compartmentalization in the genital tract.
doi:10.1002/jmv.23398
PMCID: PMC3472447  PMID: 22997072
HIV-1; Cytomegalovirus; compartmentalization; evolution; semen

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