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To assess the prevalence of hepatitis B virus (HBV) and hepatitis C virus (HCV) infection and to investigate their mutual influences on infection status among human immunodeficiency virus type 1 (HIV-1)-seropositive injection drug users (IDUs). A cross-sectional study was conducted among HIV infected IDUs in Dali, China. The participants were tested for serological markers of HBV and HCV infection, alanine transaminase (ALT) activity and CD4(+) T cell count. HCV genotype was determined by sequencing. Of 529 patients, 498 (94.1 %) HIV infected IDUs agreed to participate. The overall prevalence of HCV infection (anti-HCV antibody positive) and spontaneous HCV clearance were 90.8 % (452/498) and 21.5 % (97/452), respectively. Of 411 subjects who had not received HBV vaccine, 296 (72.0 %) were positive for antibody against HBV core antigen (HBcAb), while 274 (66.7 %) were positive for both HCV antibody and HBcAb. HBV antigens were detected in 52 of the HBV-infected subjects (17.6 %). HCV clearance was associated with HBV antigenemia (p = 0.0002) and higher CD4(+) T cell count (p = 0.0294). Resolved HBV infection was associated with HCV genotype 3 (p = 0.0365). HBV and HCV infection are highly prevalent and mutually influence infection status in HIV-1 infected IDUs in Dali, China.

Owing to ongoing recognition of pathogen-associated molecular patterns, immune activation and upregulation of IFN-stimulated genes (ISGs) are sustained in the chronically infected host. Albeit most ISGs are important effectors for containing viral replication, some might exert compensatory immune suppression to limit pathological dysfunctions, although the mechanisms are not fully understood. In this study, we report that the ISG lymphocyte Ag 6 complex, locus E (LY6E) is a negative immune regulator of monocytes. LY6E in monocytes negatively modulated CD14 expression and subsequently dampened the responsiveness to LPS stimulation in vitro. In the setting of chronic HIV infection, the upregulation of LY6E was correlated with reduced CD14 level on monocytes; however, the immunosuppressive effect of LY6E was not adequate to remedy the hyperresponsiveness of activated monocytes. Taken together, the regulatory LY6E pathway in monocytes represents one of negative feedback mechanisms that counterbalance monocyte activation, which might be caused by LPS translocation through the compromised gastrointestinal tract during persistent HIV-1 infection and may serve as a potential target for immune intervention., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Ex vivo foreskin models have demonstrated that inner foreskin is more susceptible to HIV-1 infection than outer foreskin. In the present study we characterized the compartition of HIV-1 target cells and quantified these cells in the epidermis and dermis of inner and outer foreskins using immunohistochemistry and flow cytometry. Our data showed that the epidermis of the inner foreskin was more enriched with CD4(+) T cells and Langerhans cells (LCs), with the co-expression of CCR5 and α4β7 receptors, than the outer foreskin. Interestingly, the vast majority of CD4(+) T cells and LCs expressed CCR5, but not CXCR4, indicating that the inner foreskin might capture and transmit R5-tropic HIV strains more efficiently. In addition, lymphoid aggregates, composed of T cells, macrophages and dendritic cells (DCs) in the dermis, were closer to the epithelial surface in the inner foreskin than in the outer foreskin. As dendritic cells are able to capture and pass HIV particles to susceptible target cells, HIV may be able to more efficiently infect the inner foreskin by hijacking the augmented immune communication pathways in this tissue. After the inoculation of HIV-1 particles in a foreskin explant culture model, the level of p24 antigen in the supernatant from the inner foreskin was slightly higher than that from the outer foreskin, although this difference was not significant. The present study is the first to employ both CCR5 and α4β7 to identify HIV target cells in the foreskin. Our data demonstrated that the inner foreskin was more enriched with HIV target immune cells than the outer foreskin, and this tissue was structured for efficient communication among immune cells that may promote HIV transmission and replication. In addition, our data suggests the R5-tropism of HIV sexual transmission is likely shaped through the inherent receptor composition on HIV target cells in the mucosa.

To describe the unique miRNA profiles for HIV seropositive individuals and identify significantly differently expressed miRNAs, we determined the expression level of 754 miRNAs of 10 HIV seropositive individuals and 10 HIV seronegative individuals by using the Taqman low density microRNA array. BRB-Array Tool was used to conduct the significance analysis, and the DIANA online tool was used to perform the miRNA target prediction and pathway analysis. A total of 56 significantly differentially expressed miRNAs were identified by microarray between HIV seropositive and seronegative individuals. Among them, 49 miRNAs were down-regulated and 7 were up-regulated, partially overlapped with reported data. Predicted target genes were mainly involved in MAPK, TGF-beta and Wnt pathways. The results shows that miRNA profile changes in HIV-1 seropositive individuals, and the 56 differentially expressed miRNAs may play important role during HIV infection. Further studies on these miRNAs may be helpful for identify key molecules involved in HIV infection and potential diagnostic markers.

To determine whether CRF07_BC, one of the most predominant strains that accounts for one third HIV-1 prevalence in China, has the ability to infect hematopoietic progenitor cells (HPCs), human Umbilical Cord Blood (UCB) derived CD34+ HPCs isolated with high purity were infected by HIV-1 pseudotyped with CRF07_BC envelope. After HIV-1 infection, ~0.86% CD34+ HPCs were co-stained for CD34 and intracellular HIV Gag. HIV p24 antigen was detectable and reached maximal release between day 2-4 after HIV-1 infection. The data of nested Alu-LTR PCR proved the integration of HIV-1 genome into the host genome occurred in HIV-1-infected HPCs. These data demonstrated that the envelope of CRF07_BC from China has the capability of resulting in infection to CD34+ HPCs, which may serve as a mechanism for long-term latency of HIV-1 infection in vivo.

Objective: To construct and compare the immunogenicities of DNA vaccines expressing pol genes derived from B`/C and A/E recombinant subtypes of HIV-1 in China., Methods: Two DNA vaccines were constructed by inserting the codon optimized pol genes derived from B'/C and A/E subtypes of HIV-1 into mammalian expression vector pSV1.0. In vitro expression efficiencies of the two DNA vaccines were determined by Western blotting and their immunogenicities were compared by i.m. immunizing female BALB/c mice. After immunization, mice splenocytes were isolated sterilely and IFN-γ based enzyme linked immunospot assay (ELISPOT) was employed to read out the specific T cell immunity., Results: The constructed DNA vaccines were validated by restriction enzyme digestion and DNA sequencing. Western blotting result showed both of the two DNA vaccines could be expressed at appreciable levels in vitro. Under the stimulation of Consensus B Pol peptide pools, specific T cell frequency elicited by pSVAE-Pol was (636±178) SFCs/10(6) splenocytes; specific T cell frequency elicited by pSVCN-Pol was (468±265)SFCs/10(6) splenocytes (P=0.412). Under the stimulation of HIV-1 AE2f Pol peptide pools, specific T cell frequency elicited by pSVAE-Pol was (1378±611) SFCs/10(6) splenocytes; specific T cell frequency elicited by pSVCN-Pol was (713±61) SFCs/10(6) splenocytes (P=0.134). Further analysis suggested pSVAE-Pol induced specific T cell responses mainly focused on Pol 1 peptide pool, while, in addition to induce Pol 1 specific T cell responses, pSVCN-Pol could also elicit T cell responses against consensus B Pol 2 peptide pool., Conclusion: Although pSVAE-Pol was more immunogenic, pSVCN-Pol could induce T cell responses against broader epitope spectrum. Rational vaccine design may need combine them together.

In recent years, the prevalence of HIV-1 infection has been rapidly increasing among men who have sex with men (MSM). However, it remains unknown how the host immune system responds to the infection in this population. We assessed the quantity of HIV-specific CD8(+) T-cell responses by using Elispot assay and their functionalities by measuring 5 CD8(+) T-cell evaluations (IL-2, MIP-1β, CD107a, TNF-α, IFN-γ) with flow cytometry assays among 18 primarily and 37 early chronically HIV-infected MSM. Our results demonstrated that subjects at early chronic phase developed HIV-specific CD8(+) T-cell responses with higher magnitudes and more diversified functionalities in comparison with those at primary infection. However, populations with IL-2(+) CD107a(+) or in combination with other functionality failed to develop in parallel. The multifunctional but not monofunctional HIV-specific CD8(+) T cells were associated with higher CD4(+) T -cell counts and lower viral loads. These data revealed that prolonged infection from primary to early chronic infection could selectively increase the functionalities of HIV-specific CD8(+) T cells in HIV-infected MSM population, the failure to develop IL-2 and cytotoxic functionalities in parallel may explain why the increased HIV-specific CD8(+) T cells were unable to enhance the containment of HIV-1 replication at the early chronic stage.

Objective: Conflicting data have been generated from previous studies to determine which kind of relationship exists between HIV-1 specific CD8 Tcell responses and HIV-1 viral load or CD4 count over the course of infection. In this study, 153 HIV-1 infected LTNPs were enrolled to investigate the role of HIV-1 specific CD8 T-cell responses in chronic HIV-1 infection among HIV-1 infected former blood donors., Methods: The patients were stratified into three groups according to CD4 count: CD4≥500 cells/μL; 350 cells/μL≤CD4<500 cells/μL; CD4<350 cells/μL. PBMCs were isolated from the patients' anticoagulated blood samples. IL-2 and IFN-γ secretions of CD 8 T cells against 17 HIV-1 consensus B full peptide pools were analyzed by using ICS assay., Results: An overall inverse correlation were observed between CD4 count and plasma viral load. Although no significant difference was observed during the comparisons of frequency/breadth of HIV-1 specific CD8 T cell responses, CD4 count stratification analysis showed that different correlation pattern existed in three strata: as for patients whose CD4 counts were less than 350 cells/μL, no significant correlations were identified between frequency/breadth of HIV-1 specific CD8 T cell responses and CD4 count/viral load; as for patients whose CD4 counts ranged from 350 cells/μL to 500 cells/μL, significant correlation was only observed between the response breadth of IL-2+IFN-γ+ CD8 T cells and CD4 count; however, as for patients whose CD4 counts were more than 500 cells/μL, direct correlations were identified between IL-2+IFN-γ+/IL-2+/IFN-γ+ CD8 T cells and viral load or CD4 count., Conclusions: Universal consistent inverse correlation was only indentified between CD4 count and viral load. The relationship between HIV-1 specific CD8 T cell responses and CD4 count/viral load varied in different CD4 strata, which showed that better preserved CD4 T cells were correlated with better CD8 T cell functions., (Copyright © 2010 The Editorial Board of Biomedical and Environmental Sciences. Published by Elsevier B.V. All rights reserved.)

The aim of this study was to test whether deglycosylation of an HIV-1 AE recombinant-derived gp140 could enhance the induction of neutralizing antibodies. N-to-Q mutations were introduced in the V1/V2 (m157/161) or V4 (m382/388) loops by using overlapping PCR. BALB/c mice were inoculated with different DNA vaccines at weeks 0, 2, 4, and 7. The Elispot assay was used to quantify Env-specific T-cell immunity, and the TZM-bl cell-based in vitro neutralizing assay with primary isolates was used to assess humoral immune responses. Our data showed that two mutant DNA vaccines, designated m157/161 and m382/388, mounted total T-cell responses that were at levels similar those of the unmutated vaccine. Although the levels of binding antibodies elicited by the two mutants were significantly lower than the levels elicited by the unmutated vaccine, cross-reactive neutralizing antibodies were observed only in the sera that received the mutant DNA vaccines. These data demonstrate that deglycosylation of HIV-1 Env could enhance the capacity to elicit cross-reactive neutralizing antibodies.

To minimize the cytotoxicity of poly (2-(dimethylamino) ethyl methacrylate) (PDMAEMA) as a gene delivery vector, we synthesized PEGylated PDMAEMA by atom transfer radical polymerization (ATRP). Here we report its effects on transfection efficiency in vitro delivered with a GFP expression plasmid and immunogenicity in vivo after complexed with a HIV gag gene DNA vaccine. mPEG(113)-b-PDMAEMA(94) was efficient in condensing DNA and formed polyplexes with an average diameter of about 150 nm. The in vitro transfection experiments demonstrated that PEGylation dramatically decreased the cytotoxicity at the N/P ratios above 30, although the transfection efficiency in vitro was reduced. Interestingly, mice in vivo vaccination study clearly showed that PEGylated PDMAEMA used as DNA delivery vector significantly improved the prime effect of DNA vaccine through intranasal administration. Importantly, PEGylated PDMAEMA was further proved its ability to induce cytokines production by murine macrophages. Overall, mPEG-b-PDMAEMA can be used as an efficient DNA vaccine vector which enhances adaptive immune responses by activating innate immunity.

Background: Developing an effective vaccine against human immunodeficiency virus type 1 (HIV-1) remains a grand challenge after more than two decades of intensive effort. It is partially due to the lack of suitable animal models for screening and prioritizing vaccine candidates. In this study, we aim to develop a mice model to test HIV-1 vaccine efficacy., Methods: We constructed a recombinant vaccinia expressing firefly luciferase and HIV-1 Gag fusion protein based on Tiantan strain, an attenuated but replication-competent poxvirus (rTTV-lucgag). By quantifying the luciferase activity as its read out, we defined the biodistribution of Tiantan strain poxvirus in mice inoculated intraperitoneally and attempted to apply this model to evaluate the HIV-1 vaccine efficacy., Results: Our data demonstrated that the rTTV-lucgag was able to express high level of luciferase (< or = 10(6) relative luciferase units (RLU)/mg protein) and HIV-1 Gag (> 3 folds increase comparing to the control). After intraperitoneal inoculation, this virus had dominant replication in the ovary, uterus, and cervix of mice and the luciferase activities in those organs are significantly correlated with viral titers (r(2) = 0.71, P < 0.01). Pre-immunization with an HIV gag DNA vaccine reduced the luciferase activity in ovary from (6006 +/- 3141) RLU/mg protein in control group to (1538 +/- 463) RLU/mg protein in vaccine group (P = 0.1969)., Conclusions: The luciferase activity in ovary could represent viral replication in vivo; this rTTV-lucgag/mice model may be suitable to assess the protective efficacy of cytotoxic T-cell responses to HIV Gag with less tedious work and high through-put.

An effective vaccine strategy for HIV-1 will probably require the induction and maintenance of both humoral and cellular immunity at mucosal and systemic sites. We tested a new prime-boost approach of intranasal priming with 3 x 10(6) PFU of replicative recombinant Tiantan vaccinia virus (rTTV) and intramuscular boosting with 100 microg DNA plasmid expressing HIV-1 Gag in BALB/c mice along with other strategies. Our data demonstrated that intranasal priming with replicative recombinant Tiantan vaccinia and intramuscular boosting with DNA vaccine raised the highest vaginal IgA and systemic T-cell responses, and modest lung IgA and sera IgG responses among all vaccination regimens; each vaccination regimen generated its own imprint of the most preferential T-cell receptor usage of Vbeta. These results demonstrate that the combination of intranasal priming with replicative recombinant Tiantan vaccinia and intramuscular boosting with DNA vaccine is a preferable regimen for induction of both T-cell and humoral immune responses at mucosal and systemic sites.

An effective vaccine strategy for HIV-1 will probably requires the induction and maintenance of both humoral and cellular immunity. We tested a new prime-boost approach of intranasal priming with 10 microg DNA plasmid in the PEI/DNA complexes and boosting with 10(7)PFU of replicative recombinant TianTan vaccinia virus (rTTV) expressing HIV-1 Gag in BALB/c mice. Intranasal priming with PEI/DNA complexes elicited strikingly stronger HIV-specific T-cell (p=0.0358) and IgA immune responses at mucosal sites of lung (p=0.0445) and vaginal tract (p=0.0469) than intranasal priming with naked DNA, though both are followed by the same rTTV boosting. Furthermore, an intramuscular boosting with rTTV could profoundly enhance both T-cell and antibody immune responses raised by intranasal priming. These results demonstrate that the combination of intranasal priming with PEI/DNA complexes and systemic boosting with rTTV is a preferable regimen for induction of both T-cell and humoral immune responses.

Background: The effort to develop an effective preventive vaccine against HIV-1 infection is challenged by the wide genetic diversity of HIV-1 among different isolates., Objectives: To explore a new vaccination strategy by using heterologous HIV immunogens derived from different clades for sequential priming and boosting., Methods: HIV Env and Gag immunogens derived from Thailand B (B'), C/B' recombinant and A/E recombinant were selected as these three clades account for 29%, 45% and 15% of HIV-1 prevalence in China, respectively. Three humanized fusion genes of env and gag derived from those three clades were synthesized and inserted into DNA and recombinant Tiantan vaccinia vectors as model vaccines. C57BL/6 and Balb/c mice were used as animal model. Peptides spanning the entire Env and Gag were used as stimuli and Elispot assay was used to assess the T cell immunity., Results: Sequential priming and boosting was observed with heterologous HIV immunogens predominantly stimulated T cell immunity against conserved epitopes, whereas a single vaccine derived from one clade or the mixture of multiple vaccines from different clades primarily raised T cells against less conservative or non-conservative epitopes., Conclusions: This is the first demonstration of a practical strategy to raise immune responses against conserved epitopes. This strategy has important implications for vaccine development against HIV and other pathogens that have high genetic diversity, such as influenza.

It remains poorly defined whether any human miRNAs play protective roles during HIV infection. Here, focusing on a unique cohort of HIV-infected former blood donors, we identified miR-31 (hsa-miR-31) by comparative miRNA profiling as the only miRNA inversely correlating with disease progression. We further validated this association in two prospective cohort studies. Despite conservation during evolution, hsa-miR-31, unlike its mouse counterpart (mmu-miR-31), was downregulated in human T cell upon activation. Our ex vivo studies showed that inhibiting miR-31 in naïve CD4+ T cells promoted a transcriptional profile with activation signature. Consistent with this skewing effect, miR-31 inhibition led to remarkably increased susceptibility to HIV infection. The suppressive nature of miR-31 in CD4+ T cell activation was pinpointed to its ability to decrease T-bet, the key molecule governing IFN-γ production and activation of CD4+ T cells, by directly targeting the upstream STAT1 transcriptional factor for downregulation, thus blunting Th1 response. Our results implicated miR-31 as a useful biomarker for tracking HIV disease progression and, by demonstrating its importance in tuning the activation of CD4+ T cells, suggested that miR-31 may play critical roles in other physiological contexts where the CD4+ T cell homeostasis needs to be deliberately controlled. [ABSTRACT FROM AUTHOR]

The understanding of protective immunity during HIV infection remains elusive. Here we showed that CD160 defines a polyfunctional and proliferative CD8+ T cell subset with a protective role during chronic HIV-1 infection. CD160+ CD8+ T cells derived from HIV+ patients correlated with slow progressions both in a cross-sectional study and in a 60-month longitudinal cohort, displaying enhanced cytotoxicity and proliferative capacity in response to HIV Gag stimulation; triggering CD160 promoted their functionalities through MEK–ERK and PI3K–AKT pathways. These observations were corroborated by studying chronic lymphocytic choriomeningitis virus (LCMV) infection in mice. The genetic ablation of CD160 severely impaired LCMV-specific CD8+ T cell functionalities and thereby resulted in loss of virus control. Interestingly, transcriptional profiling showed multiple costimulatory and survival pathways likely to be involved in CD160+ T cell development. Our data demonstrated that CD160 acts as a costimulatory molecule positively regulating CD8+ T cells during chronic viral infections, thus representing a potential target for immune intervention. [ABSTRACT FROM AUTHOR]

Objective: Conflicting data have been generated from previous studies to determine which kind of relationship exists between HIV-1 specific CD8 Tcell responses and HIV-1 viral load or CD4 count over the course of infection. In this study, 153 HIV-1 infected LTNPs were enrolled to investigate the role of HIV-1 specific CD8 T-cell responses in chronic HIV-1 infection among HIV-1 infected former blood donors. Methods: The patients were stratified into three groups according to CD4 count: CD4⩾500 cells/μL; 350 cells/μL⩽CD4<500 cells/μL; CD4<350 cells/μL. PBMCs were isolated from the patients'' anticoagulated blood samples. IL-2 and IFN-γ secretions of CD 8 T cells against 17 HIV-1 consensus B full peptide pools were analyzed by using ICS assay. Results: An overall inverse correlation were observed between CD4 count and plasma viral load. Although no significant difference was observed during the comparisons of frequency/breadth of HIV-1 specific CD8 T cell responses, CD4 count stratification analysis showed that different correlation pattern existed in three strata: as for patients whose CD4 counts were less than 350 cells/μL, no significant correlations were identified between frequency/breadth of HIV-1 specific CD8 T cell responses and CD4 count/viral load; as for patients whose CD4 counts ranged from 350 cells/μL to 500 cells/μL, significant correlation was only observed between the response breadth of IL-2+IFN-γ+ CD8 T cells and CD4 count; however, as for patients whose CD4 counts were more than 500 cells/μL, direct correlations were identified between IL-2+IFN-γ+/IL-2+/IFN-γ+ CD8 T cells and viral load or CD4 count. Conclusions: Universal consistent inverse correlation was only indentified between CD4 count and viral load. The relationship between HIV-1 specific CD8 T cell responses and CD4 count/viral load varied in different CD4 strata, which showed that better preserved CD4 T cells were correlated with better CD8 T cell functions. [Copyright &y& Elsevier]