Haitao Hu

Preferential infection of human Ad5-specific CD4 T cells by HIV in Ad5 naturally exposed and recombinant Ad5-HIV vaccinated individuals

Significance Efforts to develop an efficacious HIV vaccine have been unsuccessful to date. Efficacy trials have reported that recombinant Ad5 (rAd5)-HIV vaccines were not efficacious and unexpectedly associated with excess HIV infection in vaccine recipients. Understanding the underlying mechanisms is urgent and will further HIV vaccine design. By comparing human CD4 T cells specific to Ad5 and CMV, we report that natural exposure- or vaccine-induced Ad5-specific CD4 T cells are highly susceptible to HIV compared with CMV-specific CD4 T cells and selectively manifest a Th17-like proinflammatory phenotype. Our findings suggest a potential mechanism for rAd5-associated excess HIV infections in vaccine recipients and highlight that testing HIV susceptibility of vaccine-generated CD4 T cells may have utility before vaccine evaluation in human trials. Efficacy trials of adenovirus 5-vectored candidate HIV vaccines [recombinant Ad5 (rAd5)-HIV] were halted for futility due to lack of vaccine efficacy and unexpected excess HIV infections in the vaccine recipients. The potential immunologic basis for these observations is unclear. We comparatively evaluated the HIV susceptibility and phenotypes of human CD4 T cells specific to Ad5 and CMV, two viruses that have been used as HIV vaccine vectors. We show that Ad5-specific CD4 T cells, either induced by natural Ad5 exposure or expanded by rAd5 vaccination, are highly susceptible to HIV in vitro and are preferentially lost in HIV-infected individuals compared with CMV-specific CD4 T cells. Further investigation demonstrated that Ad5-specific CD4 T cells selectively display a proinflammatory Th17-like phenotype and express macrophage inflammatory protein 3α and α4β7 integrin, suggestive of gut mucosa homing potential of these cells. Analysis of HIV p24 and cytokine coexpression using flow cytometry revealed preferential infection of IL-17- and IL-2-producing, Ad5-specific CD4 T cells by HIV in vitro. Our data suggest a potential mechanism explaining the excess HIV infections in vaccine recipients after rAd5-HIV vaccination and highlight the importance of testing the HIV susceptibility of vaccine-generated, vector and insert-specific CD4 T cells in future HIV vaccine studies.

Distinct gene-expression profiles associated with the susceptibility of pathogen-specific CD4 T cells to HIV-1 infection.

In HIV infection, CD4 responses to opportunistic pathogens such as Candida albicans are lost early, but CMV-specific CD4 response persists. Little is currently known about HIV infection of CD4 T cells of different pathogen/antigen specificities. CFSE-labeled PBMCs were stimulated with CMV, tetanus toxoid (TT), and C albicans antigens and subsequently exposed to HIV. HIV infection was monitored by intracellular p24 in CFSE(low) population. We found that although TT- and C albicans-specific CD4 T cells were permissive, CMV-specific CD4 T cells were highly resistant to both R5 and X4 HIV. Quantification of HIV DNA in CFSE(low) cells showed a reduction of strong-stop and full-length DNA in CMV-specific cells compared with TT- and C albicans-specific cells. β-Chemokine neutralization enhanced HIV infection in TT- and C albicans-specific cells, whereas HIV infection in CMV-specific cells remained low despite increased entry by β-chemokine neutralization, suggesting postentry HIV restriction by CMV-specific cells. Microarray analysis (Gene Expression Omnibus accession number: GSE42853) revealed distinct transcriptional profiles that involved selective up-regulation of comprehensive innate antiviral genes in CMV-specific cells, whereas TT- and C albicans-specific cells mainly up-regulated Th17 inflammatory response. Our data suggest a mechanism for the persistence of CMV-specific CD4 response and earlier loss of mucosal Th17-associated TT- and C albicans-specific CD4 response in AIDS.

IL-33-Dependent Endothelial Activation Contributes to Apoptosis and Renal Injury in Orientia tsutsugamushi-Infected Mice

Endothelial cells (EC) are the main target for Orientia tsutsugamushi infection and EC dysfunction is a hallmark of severe scrub typhus in patients. However, the molecular basis of EC dysfunction and its impact on infection outcome are poorly understood. We found that C57BL/6 mice that received a lethal dose of O. tsutsugamushi Karp strain had a significant increase in the expression of IL-33 and its receptor ST2L in the kidneys and liver, but a rapid reduction of IL-33 in the lungs. We also found exacerbated EC stress and activation in the kidneys of infected mice, as evidenced by elevated angiopoietin (Ang) 2/Ang1 ratio, increased endothelin 1 (ET-1) and endothelial nitric oxide synthase (eNOS) expression. Such responses were significantly attenuated in the IL-33-/- mice. Importantly, IL-33-/- mice also had markedly attenuated disease due to reduced EC stress and cellular apoptosis. To confirm the biological role of IL-33, we challenged wild-type (WT) mice with a sub-lethal dose of O. tsutsugamushi and gave mice recombinant IL-33 (rIL-33) every 2 days for 10 days. Exogenous IL-33 significantly increased disease severity and lethality, which correlated with increased EC stress and activation, increased CXCL1 and CXCL2 chemokines, but decreased anti-apoptotic gene BCL-2 in the kidneys. To further examine the role of EC stress, we infected human umbilical vein endothelial cells (HUVEC) in vitro. We found an infection dose-dependent increase in the expression of IL-33, ST2L soluble ST2 (sST2), and the Ang2/Ang1 ratio at 24 and 48 hours post-infection. This study indicates a pathogenic role of alarmin IL-33 in a murine model of scrub typhus and highlights infection-triggered EC damage and IL-33-mediated pathological changes during the course of Orientia infection.

Non-High-Density Lipoprotein Cholesterol on the Risks of Stroke: A Result from the Kailuan Study

Aims To prospectively explore the association between non-high-density lipoprotein cholesterol (non-HDLC) and the risks of stroke and its subtypes. Methods A total of 95,916 participants (18-98 years old; 76,354 men and 19,562 women) from a Chinese urban community who were free of myocardial infarction and stroke at baseline time point (2006-2007) were eligible and enrolled in the study. The serum non-HDLC levels of participants were determined by subtracting the high-density lipoprotein cholesterol (HDLC) from total serum cholesterol. The primary outcome was the first occurrence of stroke, which was diagnosed according to the World Health Organization criteria and classified into three subtypes: ischemic stroke, intracerebral hemorrhage, or subarachnoid hemorrhage. The Cox proportional hazards models were used to estimate risk of stroke and its subtypes. Results During the four-year follow-up, we identified 1614 stroke events (1,156 ischemic, 416 intracerebral hemorrhagic and 42 subarachnoid hemorrhagic). Statistical analyses showed that hazard ratios (HR) (95% Confidence Interval: CI) of serum Non-HDLC level for total and subtypes of stroke were: 1.08 (1.03-1.12) (total), 1.10 (1.05-1.16) (ischemic), 1.03 (0.96-1.10) (intracerebral hemorrhage) and 0.83 (0.66-1.05) (subarachnoid hemorrhage). HR for non-HDLC refers to the increase per each 20 mg/dl. For total and ischemic stroke, the risks were significantly higher in the fourth and fifth quintiles of non-HDLC concentrations compared to the first quintile after adjusting the confounding factors (total stroke: 4th quintile HR=1.33 (1.12-1.59); 5th quintile HR = 1.36 (1.15-1.62); ischemic stroke: 4th quintile HR =1.34 (1.09-1.66); 5th quintile HR = 1.53 (1.24-1.88)). Conclusions Our data suggest that serum non-HDLC level is an independent risk factor for total and ischemic stroke, and that higher serum non-HDLC concentrations are associated with increased risks for total stroke and ischemic stroke, but not for intracerebral and subarachnoid hemorrhage.

Increased fasting glucose and the prevalence of arterial stiffness: A cross-sectional study in Chinese adults

Abstract Objective: Previous studies have shown that diabetes increases the prevalence of arterial stiffness. However, it remains controversial whether impaired fasting glucose (IFG), a key pre-diabetes condition, is associated with increased risk of arterial stiffness. This study aimed to investigate the relationship between increased fasting plasma glucose (FPG) and the prevalence of arterial stiffness in a Chinese adult population. Methods: A random sample of 5039 participants aged 40 years or older (40·0% female) were enrolled in this study. Information on potential risk factors for cardiovascular disease was collected, and the presence of arterial stiffness was assessed by measuring brachial-ankle pulse wave velocity (baPWV). Participants were stratified into three groups: normal fasting glucose (NFG), IFG, and diabetes mellitus (DM). The IFG group was further stratified by quartiles based on the level of FPG into Q1, Q2, Q3, and Q4. Results: Fasting plasma glucose level was found to be independently and positively associated with baPWV. The adjusted odds ratios (ORs) (95% confidence interval (CI)) for arterial stiffness were 1·09 (0·80–1·48), 1·33 (0·98–1·81), 1·27 (0·93–1·73), 1·82 (1·31–2·53), and 2·15 (1·66–2·79) for those in IFG Q1, Q2, Q3, Q4, and DM groups compared with NFG group (P < 0·001), respectively, after adjusting for age, sex, and other potential confounders. Moreover, male participants and participants younger than 60 years were closely associated with the presence and severity of arterial stiffness (P < 0·001). Conclusion: Our study reports a previously unidentified positive association between increased FPG and the prevalence of arterial stiffness, suggesting the importance of FPG control in the prevention of arterial stiffness.

HIV Envelope Suppresses CD4+ T Cell Activation Independent of T Regulatory Cells

We previously demonstrated that HIV envelope glycoprotein (Env), delivered in the form of a vaccine and expressed by dendritic cells or 293T cells, could suppress Ag-stimulated CD4+ T cell proliferation. The mechanism remains to be identified but is dependent on CD4 and independent of coreceptor binding. Recently, CD4+ regulatory T (Treg) cells were found to inhibit protective anti-HIV CD4+ and CD8+ T cell responses. However, the role of Tregs in HIV remains highly controversial. HIV Env is a potent immune inhibitory molecule that interacts with host CD4+ cells, including Treg cells. Using an in vitro model, we investigated whether Treg cells are involved in Env-induced suppression of CD4+ T cell proliferation, and whether Env directly affects the functional activity of Treg cells. Our data shows that exposure of human CD4+ T cells to Env neither induced a higher frequency nor a more activated phenotype of Treg cells. Depletion of CD25+ Treg cells from PBMC did not overcome the Env-induced suppression of CD4+ T cell proliferation, demonstrating that CD25+FoxP3+ Treg cells are not involved in Env-induced suppression of CD4+ T cell proliferation. In addition, we extend our observation that similar to Env expressed on cells, Env present on virions also suppresses CD4+ T cell proliferation.

Sequential Dysfunction and Progressive Depletion of Candida albicans -Specific CD4 T Cell Response in HIV-1 Infection

Loss of immune control over opportunistic infections can occur at different stages of HIV-1 (HIV) disease, among which mucosal candidiasis caused by the fungal pathogen Candida albicans (C. albicans) is one of the early and common manifestations in HIV-infected human subjects. The underlying immunological basis is not well defined. We have previously shown that compared to cytomegalovirus (CMV)-specific CD4 cells, C. albicans-specific CD4 T cells are highly permissive to HIV in vitro. Here, based on an antiretroviral treatment (ART) naïve HIV infection cohort (RV21), we investigated longitudinally the impact of HIV on C. albicans- and CMV-specific CD4 T-cell immunity in vivo. We found a sequential dysfunction and preferential depletion for C. albicans-specific CD4 T cell response during progressive HIV infection. Compared to Th1 (IFN-γ, MIP-1β) functional subsets, the Th17 functional subsets (IL-17, IL-22) of C. albicans-specific CD4 T cells were more permissive to HIV in vitro and impaired earlier in HIV-infected subjects. Infection history analysis showed that C. albicans-specific CD4 T cells were more susceptible to HIV in vivo, harboring modestly but significantly higher levels of HIV DNA, than CMV-specific CD4 T cells. Longitudinal analysis of HIV-infected individuals with ongoing CD4 depletion demonstrated that C. albicans-specific CD4 T-cell response was preferentially and progressively depleted. Taken together, these data suggest a potential mechanism for earlier loss of immune control over mucosal candidiasis in HIV-infected patients and provide new insights into pathogen-specific immune failure in AIDS pathogenesis.

Expression of HIV-encoded microRNA-TAR and its inhibitory effect on viral replication in human primary macrophages

A number of virus-encoded microRNAs have been shown to play important roles in virus replication and virus-host interactions, although the expression and function of miR-TAR-3p derived from the human immunodeficiency virus type 1 (HIV-1) TAR element remain controversial. In this study, miR-TAR-3p was detected in human peripheral blood monocyte-derived macrophages (MDMs) infected by HIV-1. Overexpression of miR-TAR-3p impaired viral replication, while inhibition of miR-TAR-3p enhanced it. Additionally, miR-TAR-3p repressed viral transcription and replication by targeting the TAR element in the HIV-1 5’-LTR in a sequence-specific manner. These results confirm the presence of miR-TAR-3p in HIV-1-infected MDMs and suggest that its function might be used as a mechanism to modulate HIV-1 replication through the expression of a negative regulatory factor.

Priming and Activation of Inflammasome by Canarypox Virus Vector ALVAC via the cGAS/IFI16–STING–Type I IFN Pathway and AIM2 Sensor

Viral vectors derived from different virus families, including poxvirus (canarypox virus vector ALVAC) and adenovirus (human Ad5 vector), have been widely used in vaccine development for a range of human diseases including HIV/AIDS. Less is known about the mechanisms underlying the host innate response to these vectors. Increasing evidence from clinical vaccine trials testing different viral vectors has suggested the importance of understanding basic elements of host–viral vector interactions. In this study, we investigated the innate interactions of APCs with two commonly used HIV vaccine vectors, ALVAC and Ad5, and identified AIM2 as an innate sensor for ALVAC, triggering strong inflammasome activation in both human and mouse APCs. Microarray and comprehensive gene-knockout analyses (CRISPR/Cas9) identified that ALVAC stimulated the cGAS/IFI16–STING–type I IFN pathway to prime AIM2, which was functionally required for ALVAC-induced inflammasome activation. We also provided evidence that, in contrast to ALVAC, the Ad5 vector itself was unable to induce inflammasome activation, which was related to its inability to stimulate the STING–type I IFN pathway and to provide inflammasome-priming signals. In preconditioned APCs, the Ad5 vector could stimulate inflammasome activation through an AIM2-independent mechanism. Therefore, our study identifies the AIM2 inflammasome and cGAS/IFI16–STING–type I IFN pathway as a novel mechanism for host innate immunity to the ALVAC vaccine vector.

Distinct gene expression profiles associated with the susceptibility of pathogen-specific CD4+ T cells to HIV-1 infection

Background HIV infection causes the progressive depletion of CD4+ T cells. Contrary to the early loss of CD4 response to opportunistic pathogens like Candida albicans, cytomegalovirus (CMV)-specific CD4 response is persistent when total CD4+ T cell number is low. The mechanism is less clear. Despite considerable knowledge for the impact of HIV infection on total CD4+ T cells and their subsets, little is known about HIV infection of CD4+ T cells of different pathogen/antigen (Ag) specificity.