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author:("Xu, jiangling")
1.  Willingness to Participate in HIV Therapeutic Vaccine Trials among HIV-Infected Patients on ART in China 
PLoS ONE  2014;9(11):e111321.
More and more HIV therapeutic vaccines will enter clinical trials; however, little is known about the willingness to participate (WTP) in HIV therapeutic vaccine trials among HIV-positive individuals.
To investigate the WTP in HIV therapeutic vaccine trials among Chinese HIV-infected patients.
We conducted a cross-sectional survey on HIV-positive inpatients and outpatients at Shanghai Public Health Center. A total of 447 participants were recruited into this study. Following an introduction with general information on HIV therapeutic vaccine and its potential effectiveness and side effects, each participant completed a questionnaire in a self-administered form. The questionnaires covered demographics, high-risk behaviors, clinical characteristics and willingness to participate in HIV therapeutic vaccine trial.
The overall willingness to participate in HIV therapeutic vaccine trials was 91.5%. Interestingly, multivariate logistic regression analyses demonstrated that the willingness was higher for those sexually infected by HIV (odds ratio [OR]: 4.36; 95% confidence interval [CI]: 1.53–12.41), diagnosed as HIV-1 infection for greater than 5 years (OR: 7.12, 95% CI: 1.83–27.76), and with the presence of infectious complications (OR: 2.75; 95% CI: 1.02–7.45). The primary reason for participation was to delay or reduce antiretroviral treatment (ART) and to avoid ART side effects (76.6%), and then followed by delaying disease progression (74.9%), increasing immune response to suppress opportunistic infections (57.7%) and preventing the development of drug resistance (37.1%). Reasons for unwillingness to participate mainly included concern for safety (37.0%), lack of knowledge on therapeutic vaccine (33.3%), and satisfaction with ART effectiveness (22.2%).
The WTP in HIV therapeutic vaccine trials was high among HIV-infected Chinese patients. HIV+ subjects who acquired infection through sexual contact and who were diagnosed for more than 5 years may represent a good candidate population for enrollment in therapeutic vaccine trials.
PMCID: PMC4221013  PMID: 25372044
2.  Boosting Functional Avidity of CD8+ T Cells by Vaccinia Virus Vaccination Depends on Intrinsic T-Cell MyD88 Expression but Not the Inflammatory Milieu 
Journal of Virology  2014;88(10):5356-5368.
T-cell functional avidity is a crucial determinant for efficient pathogen clearance. Although recombinant DNA priming coupled with a vaccinia-vectored vaccine (VACV) boost has been widely used to mount robust CD8+ T-cell responses, how VACV boost shapes the properties of memory CD8+ T cells remains poorly defined. Here, we characterize the memory CD8+ T cells boosted by VACV and demonstrate that the intrinsic expression of MyD88 is critical for their high functional avidity. Independent of selection of clones with high-affinity T-cell receptor (TCR) or of enhanced proximal TCR signaling, the VACV boost significantly increased T-cell functional avidity through a decrease in the activation threshold. VACV-induced inflammatory milieu is not sufficient for this improvement, as simultaneous administration of the DNA vaccine and mock VACV had no effects on the functional avidity of memory CD8+ T cells. Furthermore, reciprocal adoptive transfer models revealed that the intrinsic MyD88 pathway is required for instructing the functional avidity of CD8+ T cells boosted by VACV. Taking these results together, the intrinsic MyD88 pathway is required for the high functional avidity of VACV-boosted CD8+ T cells independent of TCR selection or the VACV infection-induced MyD88-mediated inflammatory milieu.
IMPORTANCE Functional avidity is one of the crucial determinants of T-cell functionality. Interestingly, although it has been demonstrated that a DNA prime-VACV boost regimen elicits high levels of T-cell functional avidity, how VACV changes the low avidity of CD8+ T cells primed by DNA into higher ones in vivo is less defined. Here, we proved that the enhancement of CD8+ T cell avidity induced by VACV boost is mediated by the intrinsic MyD88 pathway but not the MyD88-mediated inflammatory milieu, which might provide prompts in vaccine design.
PMCID: PMC4019089  PMID: 24554667
3.  Drug susceptibility profile and pathogenicity of H7N9 influenza virus (Anhui1 lineage) with R292K substitution 
Neuraminidase inhibitors (NAIs) are the only available licensed therapeutics against human H7N9 influenza virus infections. The emergence of NAI-resistant variants of H7N9viruses with an NA R292K mutation poses a therapeutic challenge. A comprehensive understanding of the susceptibility of these viruses to clinically available NAIs, non-NAIs and their combinations is crucial for effective treatment. In this study, by using limited serial passage and plaque purification, an R292K variant of the Anhui1 lineage was isolated from a patient with clinical evidence of resistance to oseltamivir. In vitro and cell-based assays confirmed a high level of resistance conferred by the R292K mutation to oseltamivir carboxylate and a moderate level of resistance to zanamivir and peramivir. Non-NAI antivirals, such as T-705, ribavirin and NT-300, efficiently inhibited both the variant and the wild-type in cell-based assays. A combination of NAIs and non-NAIs did not exhibit a marked synergistic effect against the R292K variant. However, the combination of two non-NAIs (T-705 and ribavirin) exhibited significant synergism against the mutant virus. In experimentally infected mice, the variant showed delayed onset of symptoms, a reduced viral load and attenuated lethality compared with the wild-type. Our study suggested non-NAIs should be tested clinically for H7N9 patients with a sustained high viral load. Possible drug combination regimens, such as T-705 plus ribavirin, should be further tested in animal models. The pathogenicity and transmissibility of the R292K H7N9 variant should be further assessed with genetically well-characterized pairs of viruses and, most-desirably, with competitive fitness experiments.
PMCID: PMC4274890  PMID: 26038501
H7N9; influenza virus; neuraminidase; oseltamivir; peramivir
4.  Glioma-Associated Antigen HEATR1 Induces Functional Cytotoxic T Lymphocytes in Patients with Glioma 
Journal of Immunology Research  2014;2014:131494.
A2B5+ glioblastoma (GBM) cells have glioma stem-like cell (GSC) properties that are crucial to chemotherapy resistance and GBM relapse. T-cell-based antigens derived from A2B5+ GBM cells provide important information for immunotherapy. Here, we show that HEAT repeat containing 1 (HEATR1) expression in GBM tissues was significantly higher than that in control brain tissues. Furthermore, HEATR1 expression in A2B5+ U87 cells was higher than that in A2B5−U87 cells (P = 0.016). Six peptides of HEATR1 presented by HLA-A∗02 were selected for testing of their ability to induce T-cell responses in patients with GBM. When peripheral blood mononuclear cells from healthy donors (n = 6) and patients with glioma (n = 33) were stimulated with the peptide mixture, eight patients with malignant gliomas had positive reactivity with a significantly increased number of responding T-cells. The peptides HEATR1682–690, HEATR11126–1134, and HEATR1757–765 had high affinity for binding to HLA-A∗02:01 and a strong capacity to induce CTL response. CTLs against HEATR1 peptides were capable of recognizing and lysing GBM cells and GSCs. These data are the first to demonstrate that HEATR1 could induce specific CTL responses targeting both GBM cells and GSCs, implicating that HEATR1 peptide-based immunotherapy could be a novel promising strategy for treating patients with GBM.
PMCID: PMC4121097  PMID: 25126583
5.  Structure-based non-canonical amino acid design to covalently crosslink an antibody–antigen complex 
Journal of structural biology  2013;185(2):215-222.
Engineering antibodies to utilize non-canonical amino acids (NCAA) should greatly expand the utility of an already important biological reagent. In particular, introducing crosslinking reagents into antibody complementarity determining regions (CDRs) should provide a means to covalently crosslink residues at the antibody–antigen interface. Unfortunately, finding the optimum position for crosslinking two proteins is often a matter of iterative guessing, even when the interface is known in atomic detail. Computer-aided antibody design can potentially greatly restrict the number of variants that must be explored in order to identify successful crosslinking sites. We have therefore used Rosetta to guide the introduction of an oxidizable crosslinking NCAA, l-3,4-dihydroxyphenylalanine (l-DOPA), into the CDRs of the anti-protective antigen scFv antibody M18, and have measured crosslinking to its cognate antigen, domain 4 of the anthrax protective antigen. Computed crosslinking distance, solvent accessibility, and interface energetics were three factors considered that could impact the efficiency of l-DOPA-mediated crosslinking. In the end, 10 variants were synthesized, and crosslinking efficiencies were generally 10% or higher, with the best variant crosslinking to 52% of the available antigen. The results suggest that computational analysis can be used in a pipeline for engineering crosslinking antibodies. The rules learned from l-DOPA crosslinking of antibodies may also be generalizable to the formation of other crosslinked interfaces and complexes.
PMCID: PMC4086636  PMID: 23680795
Computer-aided design; Structure-based design; Rosetta; Antibody; Non-canonical amino acid; Crosslinking; Binding affinity; l-DOPA
6.  Extending RosettaDock with water, sugar, and pH for prediction of complex structures and affinities for CAPRI rounds 20–27 
Proteins  2013;81(12):2201-2209.
Rounds 20–27 of the Critical Assessment of PRotein Interactions (CAPRI) provided a testing platform for computational methods designed to address a wide range of challenges. The diverse targets drove the creation of and new combinations of computational tools. In this study, RosettaDock and other novel Rosetta protocols were used to successfully predict four of the 10 blind targets. For example, for DNase domain of Colicin E2–Im2 immunity protein, RosettaDock and RosettaLigand were used to predict the positions of water molecules at the interface, recovering 46% of the native water-mediated contacts. For α-repeat Rep4–Rep2 and g-type lysozyme–PliG inhibitor complexes, homology models were built and standard and pH-sensitive docking algorithms were used to generate structures with interface RMSD values of 3.3 Å and 2.0 Å, respectively. A novel flexible sugar–protein docking protocol was also developed and used for structure prediction of the BT4661–heparin-like saccharide complex, recovering 71% of the native contacts. Challenges remain in the generation of accurate homology models for protein mutants and sampling during global docking. On proteins designed to bind influenza hemagglutinin, only about half of the mutations were identified that affect binding (T55: 54%; T56: 48%). The prediction of the structure of the xylanase complex involving homology modeling and multidomain docking pushed the limits of global conformational sampling and did not result in any successful prediction. The diversity of problems at hand requires computational algorithms to be versatile; the recent additions to the Rosetta suite expand the capabilities to encompass more biologically realistic docking problems.
PMCID: PMC4037910  PMID: 24123494
CAPRI; protein interactions; protein docking; binding
7.  Fusion-Expressed CTB Improves Both Systemic and Mucosal T-Cell Responses Elicited by an Intranasal DNA Priming/Intramuscular Recombinant Vaccinia Boosting Regimen 
Journal of Immunology Research  2014;2014:308732.
Previous study showed that CTB (Cholera toxin subunit B) can be used as a genetic adjuvant to enhance the systemic immune responses. To further investigate whether it can also be used as a genetic adjuvant to improve mucosal immune responses, we constructed DNA and recombinant Tiantan vaccinia (rTTV) vaccines expressing OVA-CTB fusion antigen. Female C57BL/6 mice were immunized with an intranasal DNA priming/intramuscular rTTV boosting regimen. OVA specific T-cell responses were measured by IFN-γ ELISPOT and specific antibody responses were determined by ELISA. Compared to the nonadjuvant group (pSV-OVA intranasal priming/rTTV-OVA intramuscular boosting), pSV-OVA-CTB intranasal priming/rTTV-OVA-CTB intramuscular boosting group significantly improved the magnitudes of T-cell responses at spleen (1562 ± 567 SFCs/106 splenocytes versus 330 ± 182 SFCs/106 splenocytes, P < 0.01), mesenteric LN (96 ± 83 SFCs/106 lymphocytes versus 1 ± 2 SFCs/106 lymphocytes, P < 0.05), draining LNs of respiratory tract (109 ± 60 SFCs/106 lymphocytes versus 2 ± 2 SFCs/106 lymphocytes, P < 0.01) and female genital tract (89 ± 48 SFCs/106 lymphocytes versus 23 ± 21 SFCs/106 lymphocytes, P < 0.01). These results collectively demonstrated that fusion-expressed CTB could act as a potent adjuvant to improve both systemic and mucosal T-cell responses.
PMCID: PMC3988707  PMID: 24741585
8.  Differential Compartmentalization of HIV-Targeting Immune Cells in Inner and Outer Foreskin Tissue 
PLoS ONE  2014;9(1):e85176.
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.
PMCID: PMC3893184  PMID: 24454812
9.  Five New CRF07_BC Near Full-Length Sequences Isolated from Sichuan, China 
The main heroine traffic from Yunnan province to the Xinjiang Autonomous Region is believed to initiate the transmission of CRF07_BC which is the predominant strain in intravenous drug users (IDUs) in China. However, the great distances between Yunnan and Xinjiang lead to an unclear and elusive diffusion process of CRF07_BC due to the absence of an important middle site such as Sichuan province. Moreover, in recent years the rapidly increasing infection rate among IDUs in the Liangshan region of Sichuan made it necessary to characterize the genetic character of the circulating strain of Sichuan IDUs. In this study, we characterized the genetic character of seven newly isolated CRF07_BC genomes (five from Sichuan and two from Xinjiang) and analyzed the transmission linkage among strains from IDUs in different regions. By conducting Markov chain Monte Carlo (MCMC) analysis and reconstruction of neighbor-joining trees and maximum-likelihood trees, our results revealed the genetic variation and important role of Sichuan-derived CRF07_BC strains during the transmission of CRF07_BC.
PMCID: PMC3537321  PMID: 22931113
10.  Multiple Independent Introductions of HIV-1 CRF01_AE Identified in China: What Are the Implications for Prevention? 
PLoS ONE  2013;8(11):e80487.
HIV-1 CRF01_AE accounts for an important fraction of HIV infections in Asia including China, but little is known about the phylogenetic and evolutionary history of this CRF (circulating recombinant form). In the current study, we collected a large number of 1,957 CRF01_AE gag p17 sequences with known sampling year (1990-2010) from 5 global regions representing 15 countries to better understand the phylogenetic relationships and epidemic history of CRF01_AE strains in China.
Methodology/Principal Findings
CRF01_AE gag p17 sequences were retrieved from public databases to explore phylogenetic relationships and phylogeographic dynamics of CRF01_AE in Asia by using maximum-likelihood phylogenetics and Bayesian coalescent-based analyses. We found close phylogenetic relationships between sequences from Thailand, Vietnam and China. Moreover, at least 5 independent introductions and 5 independent autochthonous clades of CRF01_AE, which descended from Thailand or Vietnam were identified in China from 1991 through 2003.
The current study not only defines the migration of CRF01_AE clades to/in Asia, but also demonstrates the criticalness of identifying the circulating strains in the population for the development of vaccine and microbicides.
PMCID: PMC3839914  PMID: 24282546
11.  Identification of Dominant Antibody-Dependent Cell-Mediated Cytotoxicity Epitopes on the Hemagglutinin Antigen of Pandemic H1N1 Influenza Virus 
Journal of Virology  2013;87(10):5831-5840.
Antibody-dependent cell-mediated cytotoxicity (ADCC) bridges innate and adaptive immunity, and it involves both humoral and cellular immune responses. ADCC has been found to be a main route of immune protection against viral infections in vivo. Hemagglutinin (HA) of influenza virus is highly immunogenic and considered the most important target for immune protection. Several potent cross-reactive HA-specific neutralizing monoclonal antibodies (MAbs) have been reported, and their conserved neutralizing epitopes have been revealed, but there has been no report so far about ADCC epitopes on HA. Here we identified two dominant ADCC epitopes, designated E1 (amino acids [aa] 92 to 117) and E2 (aa 124 to 159), on HA of pandemic H1N1 influenza virus by epitope mapping of convalescent-phase plasma IgG antibodies from six H1N1-infected human subjects in China that exhibited different levels of ADCC activity. The E1 and E2 ADCC epitopes overlapped with immunodominant epitopes of HA. Depletion of purified patient plasma IgG antibodies with EBY100 yeast cells expressing E1 or E2 decreased the ADCC activity of the IgG antibodies. E1 and E2 sequences were found to be highly conserved in H1N1 strains but less so in other subtypes of influenza A viruses. Our study may aid in designing immunogens that can elicit antibodies with high ADCC activity. Vaccine immunogens designed to include the structural determinants of potent broadly neutralizing antibodies and ADCC epitopes may confer comprehensive immune protection against influenza virus infection.
PMCID: PMC3648183  PMID: 23487456
12.  Safe Pseudovirus-based Assay for Neutralization Antibodies against Influenza A(H7N9) Virus 
Emerging Infectious Diseases  2013;19(10):1685-1687.
Serologic studies are urgently needed to assist in understanding an outbreak of influenza A(H7N9) virus. However, a biosafety level 3 laboratory is required for conventional serologic assays with live lethal virus. We describe a safe pseudovirus–based neutralization assay with preliminary assessment using subtype H7N9–infected samples and controls.
PMCID: PMC3810762  PMID: 24047684
Influenza; subtype H7N9; serology; neutralization tests; pseudovirus; viruses
13.  Enzymatic Excision of Uracil Residues in Nucleosomes Depends on Local DNA Structure and Dynamics† 
Biochemistry  2012;51(30):6028-6038.
The excision of uracil bases from DNA is accomplished by the enzyme uracil DNA glycosylase (UNG). Recognition of uracil bases in free DNA is facilitated by uracil base pair dynamics, but it is not known whether this same mechanistic feature is relevant for detection and excision of uracil residues embedded in nucleosomes. Here we investigate this question using nucleosome core particles (NCPs) generated from X. laevis histones and the high-affinity “Widom 601” positioning sequence. The reactivity of uracil residues in NCPs under steady-state multiple turnover conditions was generally decreased as compared to free 601 DNA, mostly due to anticipated steric effects of histones. However, some sites in NCPs had equal or even greater reactivity than free DNA, and the observed reactivities were not readily explained by simple steric considerations, or by global DNA unwrapping models for nucleosome invasion. In particular, some reactive uracils were found in occluded positions, while some unreactive uracils were found in exposed positions. One feature of many exposed reactive sites is a wide DNA minor groove, which allows penetration of a key active site loop of the enzyme. In single-turnover kinetic measurements, multi-phasic reaction kinetics were observed for several uracil sites, where each kinetic transient was independent of the UNG concentration. These kinetic measurements, and supporting structural analyses, support a mechanism where some uracils are transiently exposed to UNG by local, rate-limiting nucleosome conformational dynamics, followed by rapid trapping of the exposed state by the enzyme. We present structural models and plausible reaction mechanisms for the reaction of UNG at three distinct uracil sites in the NCP.
PMCID: PMC3448002  PMID: 22784353
14.  Recruitment of HIV-1 target cells at topical mucosal sites: a sensitive and early marker for determining the safety of microbicide candidates 
To explore early biomarkers for establishing more sensitive safety evaluation assays in preclinical settings that determine the potential risks during the application of microbicide candidates, three representative microbicide candidates (cellulose sulphate, nonoxynol-9 and tenofovir), whose safety profiles have been well established in clinical trials, were included to gauge the sensitivities of different assays. Both mouse models and cell lines were employed to determine the sensitivities. The recruitment of immune cells at topical mucosal sites and the upregulation of HIV receptor/coreceptors in vitro were identified as highly sensitive biomarkers of the impact of microbicide candidates. Our data suggest that different evaluations/assays have their inherent sensitivities, and at least one assay from each sensitivity level should be included in the safety evaluation algorithm.
PMCID: PMC3820983  PMID: 26038476
HIV; microbicides; coreceptor; recruitment; receptor; safety; sensitivity
15.  Serverification of Molecular Modeling Applications: The Rosetta Online Server That Includes Everyone (ROSIE) 
PLoS ONE  2013;8(5):e63906.
The Rosetta molecular modeling software package provides experimentally tested and rapidly evolving tools for the 3D structure prediction and high-resolution design of proteins, nucleic acids, and a growing number of non-natural polymers. Despite its free availability to academic users and improving documentation, use of Rosetta has largely remained confined to developers and their immediate collaborators due to the code’s difficulty of use, the requirement for large computational resources, and the unavailability of servers for most of the Rosetta applications. Here, we present a unified web framework for Rosetta applications called ROSIE (Rosetta Online Server that Includes Everyone). ROSIE provides (a) a common user interface for Rosetta protocols, (b) a stable application programming interface for developers to add additional protocols, (c) a flexible back-end to allow leveraging of computer cluster resources shared by RosettaCommons member institutions, and (d) centralized administration by the RosettaCommons to ensure continuous maintenance. This paper describes the ROSIE server infrastructure, a step-by-step ‘serverification’ protocol for use by Rosetta developers, and the deployment of the first nine ROSIE applications by six separate developer teams: Docking, RNA de novo, ERRASER, Antibody, Sequence Tolerance, Supercharge, Beta peptide design, NCBB design, and VIP redesign. As illustrated by the number and diversity of these applications, ROSIE offers a general and speedy paradigm for serverification of Rosetta applications that incurs negligible cost to developers and lowers barriers to Rosetta use for the broader biological community. ROSIE is available at
PMCID: PMC3661552  PMID: 23717507
16.  New Emerging Recombinant HIV-1 Strains and Close Transmission Linkage of HIV-1 Strains in the Chinese MSM Population Indicate a New Epidemic Risk 
PLoS ONE  2013;8(1):e54322.
In recent years, the population of men who have sex with men (MSM) have become the most significant increasing group of HIV-1 transmission in China. To identify new recombinant strains and transmission patterns of HIV-1 in Chinese MSM population, a cross-sectional investigation of MSM in Anhui Province (in south-eastern China) was performed in 2011. The diagnosed AIDS case rate, CD4 T-cell counts, HIV subtypes, and origin of the recombinant strains were investigated in 138 collected samples. The phylogenetic and bootscan analyses demonstrated that, apart from three previously reported circulating strains (CRF07_BC, CRF01_AE, subtype B), various recombinant strains among subtype B, subtype C, CRF01_AE, and CRF07_BC were simultaneously identified in Chinese MSM for the first time. The introducing time of B subtype in Chinese MSM populations was estimated in 1985, CRF01_AE in 2000, and CRF07_BC in 2003; the latter two account for more than 85% of MSM infections. Notably, in comparison with B subtype infections in Anhui MSM, CRF01_AE, with the highest prevalence rate, may accelerate AIDS progression. Over half of patients (56%) infected with new recombinant strains infection are diagnosed as progression into AIDS. Both Bayes and phylogenetic analyses indicated that there was active HIV transmission among MSM nationwide, which may facilitate the transmission of the new 01B recombinant strains in MSM. In conclusion, new recombinant strains and active transmission were identified in the Chinese MSM population, which may lead to a new alarming HIV pandemic in this population due to the increased pathogenesis of the newly emerging strains.
PMCID: PMC3553145  PMID: 23372706
17.  A New Migration Map of HIV-1 CRF07_BC in China: Analysis of Sequences from 12 Provinces over a Decade 
PLoS ONE  2012;7(12):e52373.
As one of prevalence HIV-1 strains in IDUs in Asia, the origination and full transmission map of CRF07_BC is of great interested and remains unclear. In the study, we collected 769 CRF07_BC derived sequences (including 45 sequences generated in our laboratory) from 12 provinces in China for reconstructing transmission map. Meanwhile, ample historic epidemic evidences were also reviewed to assist sequences analysis.
Methodology/Principal Findings
In the study, we collected 769 CRF07_BC derived sequences and identified 138 independent sequences from 12 provinces in China for subsequent phylogeographic tree analysis, Bayes Factor test and the estimation of state tMRCA. The analyses demonstrated that CRF07_BC was originated in 1993 in IDU in Yunnan province and then initially spread to Guangxi (eastern neighbor to Yunnan) in 1994, to Xinjiang (northwest) in 1995 and to Sichuan (northern neighbor to Yunnan) in 1996. The subsequent transmissions occurred from Yunnan to Liaoning (northeast) in 1997 and to Jiangsu in 1998. Interestingly, after the early introduction of CRF07_BC into Guangxi, Xinjiang and Sichuan, these three regions served as secondary epicenters for further spreading into Gansu, Ningxia, Qinghai, Beijing and Hunan during 1999–2001. These analyzed results are in accordance with early epidemic investigations.
Our data not just reconstructed the migration map of CRF07_BC, but also firstly revealed the active roles of these secondary epicenters in the dynamic migration of CRF07_BC in China.
PMCID: PMC3530601  PMID: 23300654
18.  The Average IFN-γ Secreting Capacity of Specific CD8+ T Cells Is Compromised While Increasing Copies of a Single T Cell Epitope Encoded by DNA Vaccine 
Previous studies suggested that both the frequency and the mean fluorescence intensity (MFI) of cytokine secreting T cells could be of great value for immunogenicity evaluation of a vaccine. In this study, by constructing epitope-based DNA vaccines encoding a previously identified CD8+ T cell epitope, we investigated the influence of multiplying epitope copies on both the frequency and the MFI of specific IFN-γ secreting CD8+ T cells. We found that frequencies of specific CD8+ T cell could be improved by multiplying epitope copies, while the MFI of IFN-γ secreted by epitope-specific CD8+ T cells decreased synchronously. And further analysis showed that the decrease of MFI was not caused by the functional avidity variation of CD8+ T cell receptor.
PMCID: PMC3509377  PMID: 23251217
19.  Development of Skewed Functionality of HIV-1-Specific Cytotoxic CD8+ T Cells from Primary to Early Chronic Phase of HIV Infection 
PLoS ONE  2012;7(9):e44983.
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.
PMCID: PMC3441698  PMID: 23028721
20.  Minocycline Down-Regulates Topical Mucosal Inflammation during the Application of Microbicide Candidates 
PLoS ONE  2012;7(8):e43211.
An effective anti-human immunodeficiency virus-1 (HIV-1) microbicide should exert its action in the absence of causing aberrant activation of topical immunity that will increase the risk of HIV acquisition. In the present study, we demonstrated that the vaginal application of cellulose sulfate (CS) gel induced topical mucosal inflammatory responses; the addition of minocycline to CS gel could significantly attenuate the inflammation in a mice model. The combined gel of CS plus minocycline not only reduced the production of inflammatory cytokines in cervicovaginal lavages (CVLs), also down-regulated the activation of CD4+ T cells and the recruitment of other immune cells including HIV target cells into vaginal tissues. Furthermore, an In vitro HIV-1 pseudovirus infection inhibition assay showed that the combined gel decreased the infection efficacy of different subtypes of HIV-1 pseudoviruses compared with that of CS gel alone. These results implicate that minocycline could be integrated into microbicide formulation to suppress the aberrant activation of topical mucosal immunity and enhance the safety profile during the application of microbicides.
PMCID: PMC3419165  PMID: 22905236
21.  Efficacy, Stability, and Biosafety of Sifuvirtide Gel as a Microbicide Candidate against HIV-1 
PLoS ONE  2012;7(5):e37381.
Sifuvirtide is a proven effective HIV-1 entry inhibitor and its safety profile has been established for systemic administration. The present study evaluated the potential of sifuvirtide formulated in a universal gel for topical use as a microbicide candidate for preventing sexual transmission of HIV. Our data showed that sifuvirtide formulated in HEC gel is effective against HIV-1 B, C subtypes, CRF07_BC and CRF01_AE, the latter two recombinants represents the most prevalent strains in China. In addition, we demonstrated that sifuvirtide in gel is stable for at least 8 weeks even at 40°C, and did not cause the disruption of integrity of mucosal epithelial surface, or the up-regulation of inflammatory cytokines both in vitro or in vivo. These results suggest that sifuvirtide gel is an effective, safe and stable product, and should be further tested as a vaginal or rectal microbicide in pre-clinical model or clinical trial for preventing HIV sexual transmission.
PMCID: PMC3353890  PMID: 22615996
22.  Boosting Heterosubtypic Neutralization Antibodies in Recipients of 2009 Pandemic H1N1 Influenza Vaccine 
Our data demonstrated that the inoculation with vaccine derived from the 2009 pandemic influenza raised vigorous neutralization antibodies against both cognate H1N1 and heterotypic influenza viruses. This observation has important implication for vaccine development.
Background. A mass vaccination has been implemented to prevent the spread of 2009 pandemic influenza virus in China. Highly limited information is available on whether this vaccine induces cross-reactive neutralization antibodies against other subtypes of influenza viruses.
Methods. We employed pseudovirus-based assays to analyze heterosubtypic neutralization responses in serum samples of 23 recipients of 2009 pandemic influenza vaccine.
Results. One dose of pandemic vaccine not only stimulated good neutralization antibodies against cognate influenza virus 2009 influenza A (H1N1), but also raised broad cross-reactive neutralization activities against seasonal H3N2 and highly pathogenic avian influenza virus H5N1 and lesser to H2N2. The cross-reactive neutralization activities were completely abolished after the removal of immunoglobin G (IgG). In contrast, H1N1 vaccination alone in influenza-naive mice elicited only vigorous homologous neutralizing activities but not cross-reactive neutralization activities.
Conclusions. Our data suggest that the cross-reactive neutralization epitopes do exist in this vaccine and could elicit significant cross-reactive neutralizing IgG antibodies in the presence of preexisting responses. The exposure to H1N1 vaccine is likely to modify the hierarchical order of preexisting immune responses to influenza viruses. These findings provide insights into the evolution of human immunity to influenza viruses after experiencing multiple influenza virus infections and vaccinations.
PMCID: PMC3243653  PMID: 22052887
23.  Early Adaptive Humoral Immune Responses and Virus Clearance in Humans Recently Infected with Pandemic 2009 H1N1 Influenza Virus 
PLoS ONE  2011;6(8):e22603.
Few studies on the humoral immune responses in human during natural influenza infection have been reported. Here, we used serum samples from pandemic 2009 H1N1 influenza infected patients to characterize the humoral immune responses to influenza during natural infection in humans. We observed for the first time that the pandemic 2009 H1N1 influenza induced influenza A-specific IgM within days after symptoms onset, whereas the unit of IgG did not changed. The magnitude of influenza A-specific IgM antibodies might have a value in predicting the rate of virus clearance to some degree. However, the newly developed IgM was not associated with hemagglutination inhibition (HI) activities in the same samples but correlated with HI activities of subsequently collected sera which were mediated by IgG antibodies, indicating that IgM was critical for influenza infection and influences subsequent IgG antibody responses. These findings provide new important insights on the human immunity to natural influenza infection.
PMCID: PMC3160288  PMID: 21886767
24.  HCV Coinfection Associated with Slower Disease Progression in HIV-Infected Former Plasma Donors Naïve to ART 
PLoS ONE  2008;3(12):e3992.
It remains controversial how HCV coinfection influences the disease progression during HIV-1 infection. This study aims to define the influence of HCV infection on the replication of HIV-1 and the disease progression in HIV-infected former plasma donors (FPDs) naïve to ART.
Methodology/Principal Findings
168 HIV-1-infected FPDs were enrolled into a cohort study from Anhui province in central China, and thereafter monitored at month 3, 9, 15, 21 and 33. Fresh whole blood samples were used for CD4+ T-cell counting. Their plasma samples were collected and stored for quantification of HIV-1 viral loads and for determination of HCV and Toxoplasma. Out of 168 HIV-infected FBDs, 11.9% (20 cases), 80.4% (135 cases) and 7.7% (13 cases) were infected with HIV-1 alone, HIV-1/HCV and HIV/HCV/Toxoplasma, respectively. During the 33-month follow-up, only 35% (7 out of 20 cases) HIV-1 mono-infected subjects remained their CD4+ T-cell counts above 200 cells/µl and retained on the cohort study, which was significantly lower than 56% (75 out of 135 cases) for HIV/HCV group and 69% (9 out of 13 cases) for HIV/HCV/Toxoplasma group (p<0.05). CD4+ T cells in HIV mono infection group were consistently lower than that in HIV/HCV group (p = 0.04, 0.18, 0.03 and 0.04 for baseline, month 9, month 21 and month 33 visit, respectively). In accordance with those observations, HIV viral loads in HIV mono-infection group were consistently higher than that in HIV/HCV group though statistical significances were only reached at baseline (p = 0.04).
These data indicated HCV coinfection with HIV-1 is associated with the slower disease progression at the very late stage when comparing with HIV-1 mono-infection. The coinfection of Toxoplasma with HIV and HCV did not exert additional influence on the disease progression. It will be highly interesting to further explore the underlying mechanism for this observation in the future.
PMCID: PMC2602976  PMID: 19098990
25.  Comparison of Immunogenicity between Codon Optimized HIV-1 Thailand Subtype B gp140 and gp145 Vaccines 
Vaccine  2007;25(26):4949-4959.
HIV-1 pandemic posed an unprecedent challenge to the global health and it is believed that an effective vaccine will be the final solution against HIV-1. HIV-1 envelope is the primary immunogen in developing neutralization antibody based HIV vaccine. To define the suitable Env derived immunogen, we systemically compared the immunogenicity of gp140 and gp145 in a DNA vaccination alone and a prime-boost modalities. 2 DNA vaccines and 2 recombinant Tiantan vaccinia vaccines (rTTV) were constructed for vaccination of female Balb/c mice. Elispot assay was used to read out the T cell immunity and ELISA assay was used to quantify antibody immunity. PLL (poly-L-Leucine)-ELISA assay was used in linear antibody epitope mapping. Mice primed with gp145 tended to elicit more Env-specific T cells responses than those primed with gp140, significant difference was observed in DNA immunization alone. The ultimate T cell responses in prime-boost regimen tends to be determined mainly by the priming efficacy. Linear antibody epitope mapping displayed that sera raised by gp145 priming were vigorously reactive to more peptides than that by gp140. Our data demonstrated HIV-1 Thailand B-derived gp145 may raise higher T-cell responses and broader linear peptide-specific antibody responses than gp140 does. However, it remains to be determined that how these observations are relevant to neutralization antibody activities.
PMCID: PMC1961633  PMID: 17350736
HIV-1; Vaccine; Envelope; Immunogen design

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