Therapeutic prediction of HIV-1 DNA decay: a multicenter longitudinal cohort study.

BACKGROUND: Factors predicting peripheral blood total HIV-1 DNA size in chronically infected patients with successfully suppressed viremia remain unclear. Prognostic power of such factors are of clinical significance for making clinical decisions. METHODS: Two sets of study populations were included: 490 China AIDS Clinical Trial (CACT) participants (Training cohort, followed up for 144 to 288 weeks) and 117 outpatients from Peking Union Medical College Hospital (PUMCH) (Validation cohort, followed up for more than 96 weeks). All patients were chronically HIV-1-infected and achieved successful HIV-1 plasma RNA suppression within week 48. Total HIV-1 DNA in blood at baseline, 12, 24, 48, 96, 144 and 288 weeks after combined antiretroviral therapy (cART) initiation were quantified. Generalized estimating equations and logistic regression methods were used to derive and validate a predictive model of total HIV-1 DNA after 96 weeks of cART. RESULTS: The total HIV-1 DNA rapidly decreased from baseline [median = 3.00 log10 copies/106 peripheral blood mononuclear cells (PBMCs)] to week 24 (median = 2.55 log10 copies/106 PBMCs), and leveled off afterwards. Of the 490 patients who had successful HIV-1 plasma RNA suppression by 96 w post-cART, 92 (18.8%) had a low total HIV-1 DNA count (< 100 copies/106 PBMCs) at week 96. In the predictive model, lower baseline total HIV-1 DNA [risk ratio (RR) = 0.08, per 1 log10 copies/106 PBMCs, P < 0.001] and higher baseline CD4+ T cell count (RR = 1.72, per 100 cells/µL, P < 0.001) were significantly associated with a low total HIV-1 DNA count at week 96. In an independent cohort of 117 patients, this model achieved a sensitivity of 75.00% and specificity of 69.52%. CONCLUSIONS: Baseline total HIV-1 DNA and CD4+ T cell count are two independent predictors of total HIV-1 DNA after treatment. The derived model based on these two baseline factors provides a useful prognostic tool in predicting HIV-1 DNA reservoir control during cART.

Whole blood as an alternative to peripheral blood mononuclear cell for detection of total HIV-1 DNA.

BACKGROUND: A more time saving, convenient, reproducible, and scalable method is needed to assess total HIV-1 DNA levels. METHODS: Frozen whole blood and peripheral blood mononuclear cell (PBMC) samples both 200 µl at the same point were used to detect total HIV-1 DNA. Automatic extraction of total HIV-1 DNA was used to ensure the consistency of sample extraction efficiency. The detection reagent was HIV-1 DNA quantitative detection kit and real-time quantitative PCR was utilized. RESULTS: Of the 44 included patients, 42 were male and 2 were female, with a median age of 33 years. Thirty-three cases were collected after receiving antiviral treatment, with a median duration of treatment of 3 months, and the other 11 cases were collected before antiviral treatment. The median viral load was 1.83 log10 copies/mL, the median CD4 and CD8 count were 94 and 680 cells/µL, and the median CD4/CD8 ratio was 0.18. The results of the two samples were 3.02 ± 0.39 log10 copies/106 PBMCs in PBMC samples and 3.05 ± 0.40 log10 copies/106 PBMCs in whole blood samples. The detection results of the two methods were highly correlated and consistent by using paired t test (P = 0.370), pearson correlation (r = 0.887, P < 0.0001) and intra-group correlation coefficient (ICC = 0.887, P < 0.0001) and bland-altman [4.55% points were outside the 95% limits of agreement (- 0.340 ~ 0.390)]. CONCLUSIONS: The results of the whole blood sample test for total HIV-1 DNA are consistent with those of PBMC samples. In a clinical setting it is recommended to use whole blood samples directly for the evaluation of the HIV reservoir.

The effects of antiretroviral therapy initiation time on HIV reservoir size in Chinese chronically HIV infected patients: a prospective, multi-site cohort study.

BACKGROUND: The effect of ART initiation time on HIV-1 DNA reservoir in chronically infected individuals is not well understood. Determining the potential influencing factors associated with a low HIV-1 DNA level in chronic infection is an important step toward drug-free control. METHODS: A prospective study included 444 chronically HIV-infected adults was performed. Participants were divided into two groups: early initiation group (EIG) or delayed initiation group (DIG) based on their baseline CD4 count; 350 to 500 and < 350 cells/mm3, respectively. Total HIV-1 DNA was measured by quantitative PCR. Using the Mann-Whitney U test, the HIV-1 DNA level at week 48 was compared between the two groups. The influencing factors of the HIV-1 DNA and factors associated with achieving a low HIV-1 level at week 48 were analyzed. RESULTS: The HIV-1 DNA at week 48 in EIG was significantly lower than in DIG [2.12 (1.80-2.51) vs 2.58 (2.21-2.87) log10 copies/106peripheral blood mononuclear cells (PBMCs); p = 0.001]. Early ART initiation was positively associated with lower HIV-1 DNA at week 48 (p = 0.025). Similarly, baseline HIV-1DNA (p = 0.001) was positively associated with HIV-1DNA at week 48 and baseline CD4/CD8 ratio (p = 0.001) was inversely associated with HIV-1DNA at week 48. Early ART initiation (p = 0.003) and baseline HIV-1 DNA level (p < 0.001) were positively associated with achieving HIV-1 DNA < 100 copies/106 PBMCs at week 48. CONCLUSION: Early ART initiation is positively associated with a smaller size of viral reservoir and a higher possibility of achieving a low HIV-1DNA level at week 48 in Chinese chronically HIV-1 infected adult. TRIAL REGISTRATION: NCT01844297 ; Registered 1 May, 2013.

HIV sequence diversity during the early phase of infection is associated with HIV DNA reductions during antiretroviral therapy.

The association between baseline human immunodeficiency virus (HIV) sequence diversity and HIV DNA decay after the initiation of antiretroviral therapy (ART) remains uncharacterized during the early stages of HIV infection. Samples were obtained from a cohort of 17 patients with early HIV infection (<6 months after infection) who initiated ART, and the C2V5 region of the HIV-1 envelope (env) gene was amplified via single genome amplification (SGA) to determine the peripheral plasma HIV quasispecies. We categorized HIV quasispecies into two groups according to baseline viral sequence genetic distance, which was determined by the Poisson-Fitter tool. Total HIV DNA in peripheral blood mononuclear cells (PBMCs), viral load, and T cell subsets were measured prior to and after the initiation of ART. The median SGA sequence number was 17 (range 6-28). At baseline, we identified 7 patients with homogeneous viral populations (designated the Homogeneous group) and 10 patients with heterogeneous viral populations (designated the Heterogeneous group) based on SGA sequences. Both groups exhibited similar HIV DNA decay rates during the first 6 months of ART (P > 0.99), but the Homogenous group experienced more prominent decay than the Heterogeneous group after 6 months (P = 0.037). The Heterogeneous group had higher CD4 cell counts after ART initiation; however, both groups had comparable recovery in terms of CD4/CD8 ratios and CD8 T cell activation levels. Viral population homogeneity upon the initiation of ART is associated with a decrease in HIV DNA levels during ART. J. Med. Virol. 89:982-988, 2017. © 2017 Wiley Periodicals, Inc.

A higher CD4/CD8 ratio correlates with an ultralow cell-associated HIV-1 DNA level in chronically infected patients on antiretroviral therapy: a case control study.

BACKGROUND: The HIV-1 DNA reservoir is an important marker that reflects viro-immunological status and can be affected by multiple viral or cellular factors. Determining the potential factors associated with the size of the HIV-1 DNA reservoir benefits the surveillance of disease progression and antiretroviral treatments. METHODS: We conducted a case control study to explore the factors that may affect the level of HIV-1 DNA. The level of HIV-1 total DNA in peripheral blood at 5 time points was quantified by quantitative PCR. Chronically HIV-1-infected patients whose cell-associated HIV-1 DNA levels were below the detection limit after receiving antiretroviral therapy (ART) for 96 weeks were identified (group 1), and patients who still had detectable levels of cell-associated HIV-1 DNA after ATR treatment were used as the control (group 2). RESULTS: Twenty-one patients with ultralow levels of cell-associated HIV-1 DNA [<20 copies/106 peripheral blood mononuclear cells (PBMCs)] presented with a lower CD8+ T-cell count (average: 511 ± 191 versus 715 ± 256 cells/µL, p = 0.013) and a higher CD4/CD8 ratio (average: 1.04 ± 0.37 versus 0.72 ± 0.32, respectively, p = 0.002) at week 96. In the multivariate analysis, patients with a higher CD4/CD8 ratio at week 96 were more likely to have levels of HIV-1 DNA below the detection limit (per 0.1 increase, OR = 1.29, 95% CI, 1.05-1.59, p = 0.017). CONCLUSION: After matching baseline HIV-1 DNA levels, a higher CD4/CD8 ratio at week 96 was the only factor associated with an ultralow level of HIV-1 DNA. The CD4/CD8 ratio can be used as an easy biomarker to help monitor patients on ART who will be selected to participate in eradication studies.

Factors Associated with the Size of HIV DNA Reservoir.

OBJECTIVE: To review the recent literatures related to the factors associated with the size of the HIV reservoir and their clinical significance. DATA SOURCES: Literatures related to the size of HIV DNA was collected from PubMed published from 1999 to June 2016. STUDY SELECTION: All relevant articles on the HIV DNA and reservoir were collected and reviewed, with no limitation of study design. RESULTS: The composition and development of the HIV-1 DNA reservoir in either treated or untreated patients is determined by integrated mechanism comprising viral characteristics, immune system, and treatment strategies. The HIV DNA reservoir is a combination of latency and activity. The residual viremia from the stochastic activation of the reservoir acts as the fuse, continuing to stimulate the immune system to maintain the activated microenvironment for the rebound of competent virus once treatment with antiretroviral therapy is discontinued. CONCLUSION: The size of the HIV-1 DNA pool and its composition has great significance in clinical treatment and disease progression.

Elevated pre-treatment IL-18 level is associated with HBeAg seroconversion in HIV-HBV coinfection.

BACKGROUND: In HBV-infected patients, hepatitis B e antigen (HBeAg) seroconversion is associated with better outcomes. Interleukin-18 (IL-18) controls hepatitis B replication in a mouse model. However, its role in treatment response in HIV-HBV-coinfected patients is unknown. METHODS: We enrolled 35 treatment-naive, HBeAg-positive, HIV-HBV-coinfected patients. HBV DNA, HIV RNA, CD4+ T-cell count, HBV surface antigen (HBsAg) quantification (qHBsAg), HBeAg quantification (qHBeAg) and IL-18 levels were measured prior to, at 24 and 48 weeks of HBV-active combination antiretroviral therapy (cART). Multivariate Poisson regression models with robust standard errors were used to determine factors associated with HBeAg seroconversion. RESULTS: Twenty-one patients received tenofovir (TDF) + lamivudine (3TC) based cART while 14 patients received 3TC-based cART. After 48 weeks of treatment, 10 patients experienced HBeAg seroconversion. Compared with non-seroconverters, seroconverters had higher median HIV RNA (5.22 versus 4.58 log copies/ml; P=0.030), lower median qHBsAg (3.97 versus 4.76 log IU/ml; P=0.011), lower median qHBeAg (1.61 versus 3.01 log PEIU/ml; P=0.004) and marginally higher median IL-18 (2.70 versus 2.53 log pg/ml; P=0.068) prior to ART. In the multivariate regression, higher baseline IL-18 (adjusted relative risk [aRR] 2.99 per 1 log pg/ml increase; P=0.035), high HIV RNA (aRR 1.84 per 1 log copies/ml; P=0.029) and low qHBeAg (aRR 0.71 per 1 log PEIU/ml; P=0.029) were significantly associated with HBeAg seroconversion. CONCLUSIONS: In HIV-HBV-coinfected patients with HBeAg positivity, higher IL-18 levels, HIV RNA load, as well as low qHBeAg prior to cART were associated with HBeAg seroconversion.

Rapid purification of human albumin with immunomagnetic microsphere technology.

AIM: To develop a rapid and inexpensive method for purification of human albumin, a method of immunomagnetic microspheres (IMMS) based on enzyme-linked immunosorbent assay (ELISA) for the purification of human albumin from human serum. METHODS: Polystyrene magnetic microspheres with carboxyl groups as carriers were prepared, and then the carboxyl groups on the surface of the microspheres were activated by ethylcarbodiimide (EDC). Finally rabbit anti-human serum albumin (HSA) antibodies were covalently bound to it and the complex can specifically capture HSA. After the procedure of capturing HSA, through taking rabbit anti-human albumin protein antibodies as a capture antibody, and goat anti-human albumin protein antibodies as a detection antibody, an ELISA on IMMS was developed, which can determine the recovery yield of HSA from the human serum. RESULTS: The result of the experiment was that the recovery of human albumin with IMMS was (86 +/- 4)%, and IMMS were reused for two other purifying cycles, the results of which were (69.0 +/- 0.6)% and (40.8 +/- 0.8)%, and the purity of the product was about 90%. CONCLUSION: The results above prove that the immunomagnetic purifiying strategy was shown to be efficient and offers an new thought for a large scale production of high-purity HSA.

Molecular linkage tracing of HIV-1 transmission events in seroconcordant couples in Guangxi Province, Southeastern China.

BACKGROUND: Guangxi Province in Southeastern China has one of the highest HIV-1 infection and transmission rates in stable couples. However, the mode of transmission at the molecular level has seldom been reported amongst this group. It is important to investigate this issue to support the treatment-as-prevention approach and for efficient interventions. METHODS: HIV-1 subgenomic regions (1.2 kb of pol and a 660-bp env C2V5 fragment) were sequenced in 42 couples. A couple linkage assessment was performed by phylogenetic analysis of sequences and Bayesian analysis of genetic distances. A subset of pairs was selected for single-genome amplification. RESULTS: Thirty-five pairs (83.3 %, 35/42) were identified as linked, 3 pairs (7.1 %, 3/42) were identified as indeterminate, and 4 pairs (9.5 %) were identified as unlinked. The predominant intra-couple-transmitted HIV-1 subtype was CRF01_AE (80 %, 28/35). The median genetic distance of linked couples was 0.5 %. CONCLUSION: The majority of HIV-1 transmission events in this study occurred within the partnership, and the predominant HIV-1 subtype was CRF01_AE. Further research on the mode of HIV transmission in other locations is needed.

Lamivudine Monotherapy-Based cART Is Efficacious for HBV Treatment in HIV/HBV Coinfection When Baseline HBV DNA <20,000 IU/mL.

BACKGROUND: Although combination antiretroviral therapy (cART) including tenofovir (TDF)+lamivudine (3TC) or emtricitabine (FTC) is recommended for treatment of HIV/HBV coinfected patients, TDF is unavailable in some resource-limited areas. Some data suggest that 3TC monotherapy-based cART may be effective in patients with low pretreatment HBV DNA. METHODS: Prospective study of 151 Chinese HIV/HBV coinfected subjects of whom 60 received 3TC-based cART and 91 received TDF+3TC-based cART. Factors associated with HBV DNA suppression at 24 and 48 weeks, including anti-HBV drugs, baseline HBV DNA, and baseline CD4 cell count, were evaluated overall and stratified by baseline HBV DNA using Poisson regression with a robust error variance. RESULTS: Baseline HBV DNA ≥20,000 IU/mL was present in 48.3% and 44.0% of subjects in the 3TC and TDF groups, respectively (P = 0.60). After 48 weeks of treatment, HBV DNA suppression rates were similar between these 2 groups (96.8% vs. 98.0% for 3TC and TDF+3TC, P > 0.999) in subjects with baseline HBV DNA <20,000 IU/mL; whereas in those with baseline HBV DNA ≥20,000 IU/mL, TDF+3TC was associated with higher suppression rates (34.5% vs. 72.5% in 3TC and TDF+3TC groups, respectively, P = 0.002). In stratified multivariate regression, TDF use (RR 1.98, P = 0.010) and baseline HBV DNA (per 1 log increase in International Units Per Milliliter, RR 0.74, P < 0.001) were associated with HBV DNA suppression only when baseline HBV DNA ≥20,000 IU/mL. CONCLUSION: This study suggests that 3TC monotherapy-based cART is efficacious for HBV treatment through 48 weeks in HIV/HBV coinfection when baseline HBV DNA <20,000 IU/mL. Studies with long-term follow-up are warranted to determine if this finding persists.

Genome sequence of a novel HIV-1 circulating recombinant form (CRF64_BC) identified from Yunnan, China.

We report a novel HIV-1 circulating recombinant form (CRF64_BC) that was isolated from five epidemiologically unlinked HIV-infected persons in Yunnan province. CRF64_BC was composed of subtype B and subtype C, with five short subtype B segments inserted into the subtype C backbone. Phylogenetic analysis demonstrated that the C subregion was correlated with the India C lineage, which was transmitted into China in the early 1990s. The evolutionary history of the B subregion was not as clear as the C subregion, as the short length of this region yielded poor phylogenetic results. Dehong is considered the epicenter of HIV-1 in China, and recombinant strains such as CRF07_BC and CRF08_BC, which also originated from this region, have spread widely in China. The newly emerged CRF64_BC increases the complexity of the HIV epidemic in China and complicates the development of subtype-specific tools against HIV transmission.

Identification of a novel HIV Type 1 circulating recombinant form (CRF65_cpx) composed of CRF01_AE and subtypes B and C in Western Yunnan, China.

A novel HIV-1 circulating recombinant form (CRF) designated CRF65_cpx was recently characterized from three epidemiologically unlinked individuals infected through heterosexual contact in western Yunnan province of China. This is the first complex mosaic HIV-1 CRF, consisting of contributions from three or more different subtypes, identified in China. An additional full-length genome sequence with identical recombinant breakpoints was found among a previously reported recombinant strain from a man who had sex with a man in Anhui province of East Central China. The breakpoint analysis of the recombinants showed a complex genome organization composed of parental subtypes B' (Thailand variant of subtype B), C, and CRF01_AE, with 13 recombination breakpoints observed in almost all structure genes of HIV-1. The generation of complex recombinant forms is likely due to cocirculation of multiple lineages of HIV-1 strains in high-risk populations in western Yunnan.

Near full-length genome characterization of a new CRF01_AE/CRF08_BC recombinant transmitted between a heterosexual couple in Guangxi, China.

Human immunodeficiency virus type 1 (HIV-1) subtype CRF01_AE is transmitted mainly by sexual activity in Guangxi, southwestern China. Other subtypes, including CRF07_BC, CRF08_BC, and subtype B, are also prevalent in this region. Cocirculation of multiple subtypes, as well as a high rate of drug use, creates favorable conditions for the emergence of recombinant viruses in Guangxi. In the present study, we identified a new HIV-1 unique recombinant form (CRF01_AE /08BC) transmitted from the infected index patient to his seronegative sexual partner. This is the first near full-length genome characterization of a CRF01_AE /08BC recombinant virus in Guangxi, and provides an important basis for future analysis on potential new recombinant transmission events.

Identification of a novel HIV-1 circulating recombinant form (CRF62_BC) in western Yunnan of China.

We report here a novel HIV-1 circulating recombinant form (CRF62_BC) that was isolated from three epidemiologically unlinked individuals [one from an injecting drug user (IDU); two from heterosexuals] in Dehong prefecture of western Yunnan province. CRF62_BC harbored two subtype B segments in the pol and vpu-env regions in a subtype C backbone. Subregion tree analysis demonstrated that subtype B regions originated from a Thai-B (subtype B') lineage and the subtype C region was from an India C lineage. CRF62_BC is the fourth CRF composed of subtypes B' and C known to date after CRF07_BC, CRF08_BC, and CRF57_BC, which were originally found among IDUs in China. The emergence of CRF62_BC may indicate the continual generation of new recombinant strains in various high-risk populations in western Yunnan. This may complicate the development of effective vaccines to limit the HIV-1 epidemic and increase the difficulty of AIDS prevention and control in China.

Compromised NK cell-mediated antibody-dependent cellular cytotoxicity in chronic SIV/SHIV infection.

Increasing evidence indicates that antibody-dependent cellular cytotoxicity (ADCC) contributes to the control of HIV/SIV infection. However, little is known about the ADCC function of natural killer (NK) cells in non-human primate model. Here we demonstrated that ADCC function of NK cells was significantly compromised in chronic SIV/SHIV infection, correlating closely with the expression of FcγRIIIa receptor (CD16) on NK cells. CD32, another class of IgG Fc receptors, was identified on NK cells with higher expression in the infected macaques and the blockade of CD32 impacted the ability of NK cells to respond to antibody-coated target cells. The inhibition of matrix metalloproteases (MMPs), a group of enzymes normally involved in tissue/receptor remodeling, could restore NK cell-mediated ADCC with increased CD16 expression on macaque NK cells. These data offer a clearer understanding of NK cell-mediated ADCC in rhesus macaques, which will allow us to evaluate the ADCC repertoire arising from preclinical vaccination studies in non-human primates and inform us in the future design of effective HIV vaccination strategies.

Lipid rafts uncouple surface expression of transmembrane TNF-alpha from its cytotoxicity associated with ICAM-1 clustering in Raji cells.

Since transmembrane tumor necrosis factor-alpha (tmTNF-alpha) has been reported to have a palmitoylated site at Cys(-47), and therefore its functions may be linked to lipid raft membrane microdomains. The present study tested a hypothesis that lipid rafts may serve as a signaling platform to mediate the bioactivity of tmTNF-alpha. We found that destruction of lipid rafts with methyl-beta-cyclodextrin (MCD) in Raji cells almost completely blocked the cytotoxicity of tmTNF-alpha, as did an anti-TNF-alpha antibody. Although a proportion of tmTNF-alpha was colocated with lipid rafts, either the replacement of Cys at -47 by Ala, destructing its possible lipid rafts-attaching site or the displacement of its cytoplasmic domain by the C-terminal sequence (131-157) of caveolin-1, making all tmTNF-alpha target to lipid rafts, had no effect on tmTNF-alpha cytotoxicity. The data suggest that the cytotoxicity of tmTNF-alpha is not associated with its lipid rafts location. Unparallel to decreased cytotoxicity, moreover, MCD significantly increased tmTNF-alpha expression on the cell surface, and these increased tmTNF-alpha molecules were capable of binding to sTNFR1. To further explore the mechanism of lipid rafts-mediated cytotoxicity of tmTNF-alpha, we demonstrated that MCD led to a marked decrease in adhesion of Raji cells to T24 cells, which was due to dissociation of adhesion molecule ICAM-1 from lipid rafts. These results indicate that lipid rafts importantly participate in the cytotoxicity of tmTNF-alpha through ICAM-1 clustering and consequent enhancement of the cell-cell contact. The data suggest that lipid rafts are essential for the killing of tmTNF-alpha through the cell-cell contact mediated by ICAM-1. However, lipid rafts may limit exposure of tmTNF-alpha to the cell surface.