Wei Kong

Jilin University, Yung-chi, Jilin Sheng, China

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Publications (126)360.84 Total impact

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    ABSTRACT: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is capable of selectively inducing apoptosis of cancer cells, is a potential targeted drug for cancer therapy. The TRAIL protein induces apoptosis only in trimeric form. However, the recombinant soluble TRAIL (sTRAIL) trimer has low stability and a short half-life, which is a major obstacle for its advancement into clinical trials. Moreover, a percentage of engineered sTRAIL proteins are produced as dimers which may be toxic to normal human hepatocytes. In this study, we inserted three copies of the same subunit fragment of sTRAIL with a His tag into a polycistronic expression vector (pST39) to explore whether it would increase the proportion of trimers. We also constructed a heterozygous vector containing three subunit fragments of sTRAIL each with a different tag (His, HA, and Cmyc). Hybrid sTRAIL proteins (P-dTags) mainly as heterologous trimers were obtained by elution with a low concentration of imidazole based on different binding affinities of His with a nickel column. Functional analysis demonstrated that heterotrimeric forms of sTRAIL showed more stable activity compared to the P-3H at 4 °C but not at 37 °C without alteration in the native killing capacity. In addition, the heterologous trimers showed decreased toxicity to hepatocytes. These results suggest that the polycistronic expression system may be useful for expression of recombinant sTRAIL and improving its potential in cancer therapeutic applications. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 08/2015; DOI:10.1016/j.pep.2015.08.004 · 1.51 Impact Factor
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    ABSTRACT: The study aims to develop a novel multistep chromatographic purification process for human enterovirus71 virus-like particles (VLPs) produced from insect cells (Sf9) infected with recombinant baculovirus. Sf9 cells were maintained in the Wave Bioreactor system20/50, and harvested when the viability decreased to 75% after infected with Bac-P1-3CD at the multiplicity of infection (MOI) of 1. After sonication and centrifugation, EV71 VLPs were purified with Capto(™) Core 700, Capto(™) adhere and Capto(TM) butyl. The purity was then determined by SDS-PAGE, Western blotting and high-performance liquid chromatography (HPLC), while the diameter of purified EV71 VLPs was analyzed by Dynamic Light Scattering (DLS) and Transmission electron microscopy (TEM). Immunization of BALB/c mice and serum collection were performed after contamination analysis, and neutralization antibodies were then analyzed by pseudovirus-based microneutralization assay. Results showed that these purified EV71 VLPs can be successfully purified with ~31.52% yield and > 95% purity. They could elicit stronger neutralization antibodies in mice compared with those produced from formalin-inactivated EV71 virus. Our results demonstrated that EV71 VLPs can be purified with the multistep chromatographic protocol. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Journal of Applied Microbiology 08/2015; DOI:10.1111/jam.12922 · 2.39 Impact Factor
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    ABSTRACT: Here is reported a novel pneumolysin(Ply) mutant(PlyM2) that addresses a long-standing problem for vaccine development in this field: detoxification of Ply in the premise of retaining antigenic integrity. Structure and function of wild-type Ply(PlyWT) and PlyM2 mutants were detected and compared. Their structures were not significantly different according to the analysis by thermal-dependent fluorescence spectroscopy and circular dichroism spectroscopy. PlyM2 was confirmed to have lost hemolytic activity and yet could induce neutralizing antibodies to prevent in vitro hemolysis by PlyWT and S. Pneumoniae. These results give support to PlyM2 to be a new protein antigen for inclusion in the development of an effective pneumococcal multiprotein vaccine.
    Chemical Research in Chinese Universities 08/2015; 31(4). DOI:10.1007/s40242-015-5105-3 · 1.12 Impact Factor
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    ABSTRACT: In this study, a novel glycol chitosan (GCS)-bestatin conjugate was synthesized and evaluated to demonstrate its efficacy in protecting thymopoietin oligopeptides from aminopeptidase-mediated degradation. Moreover, the mechanism and relative susceptibility of three thymopoietin oligopeptides, thymocartin (TP4), thymopentin (TP5), and thymotrinan (TP3), to enzymatic degradation were investigated and compared at the molecular level. Initial investigations indicated that formation of the GCS-bestatin conjugate, with a substitution degree of 7.0% (moles of bestatin per mole of glycol glucosamine unit), could significantly protect all three peptides from aminopeptidase-mediated degradation in a concentration-dependent manner. The space hindrance and loss of one pair of hydrogen bonds, resulting from the covalent conjugation of chitosan with bestatin, did not affect the specific interaction between bestatin and aminopeptidase. Moreover, TP4 displayed a higher degradation clearance compared with those of TP5 and TP3 under the same experimental conditions. The varying levels of susceptibility of these three peptides to aminopeptidase (TP4 > TP5 > TP3) were closely related to differences in their binding energies to enzyme, which mainly involved Van der Waals forces and electrostatic interactions, as supported by the results of molecular dynamics simulations. These results suggest that GCS-bestatin conjugate might be useful in the delivery of thymopoietin oligopeptides by mucosal routes, and that TP3 and TP5 are better alternatives to TP4 for delivery because of their robust resistance against enzymatic degradation. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.
    Journal of Pharmaceutical Sciences 07/2015; DOI:10.1002/jps.24567 · 3.01 Impact Factor
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    ABSTRACT: The absence of a productive cell culture system hampered detailed analysis of the structure and protein composition of the hepatitis E virion. In this study, hepatitis E virus from a robust HEV cell culture system and from the feces of infected monkeys at the peak of virus excretion was purified by ultra-centrifugation. The common feature of the two samples after ultracentrifugation was that the ORF2 protein mainly remained in the top fractions. The ORF2 protein from cell culture system was glycosylated, with an apparent molecular weight of 88 kDa, and was not infectious in PLC/PRF/5 cells. The ORF2 protein in this fraction can bind to and protect HEV RNA from digestion by RNase A. The RNA-ORF2 product has a similar sedimentation coefficient to the virus from feces. The viral RNA in the cell culture supernatant was mainly in the fraction of 1.15g/cm3 but that from the feces was mainly in the fraction of 1.21 g/cm3. Both were infectious in PLC/PRF/5 cells. And the fraction in the middle of the gradient (1.06g/cm3) from the cell culture supernatant,but not that from the feces, also has ORF2 protein and HEV RNA but was not infectious in PLC/PRF/5.The infectious RNA-rich fraction from the cell culture contained ORF3 protein and lipid but the corresponding fraction from feces had no lipid and little ORF3 protein. The lipid on the surface of the virus has no effect on its binding to cells but the ORF3 protein interferes with binding. The result suggests that most of the secreted ORF2 protein is not associated with HEV RNA and that hepatitis E virus produced in cell culture differs in structure from the virus found in feces in that it has a lipid envelope.
    PLoS ONE 07/2015; 10(7):e0132503. DOI:10.1371/journal.pone.0132503 · 3.23 Impact Factor
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    ABSTRACT: Plant proteins have been drawing increasing attention owing to their safety, abundance and relatively low cost in comparison with animal proteins. The development of plant protein-based delivery vehicles may lead to the provision of novel pharmaceutical products to patients. Zein is a class of alcohol-soluble prolamine proteins present in maize endosperm that was approved as a generally recognised as safe excipient in 1985 by the US FDA for use in pharmaceutical film coatings. Over the past few decades, numerous studies have been carried out to illustrate zein’s potential for novel applications in the biomedical field. This paper reviews the present status of zein-based nanofibres, with emphasis on their fabrication and biomedical applications, particularly for drug delivery. Their benefits and limitations are also discussed to provide further insight into zein’s potential as a promising biomaterial.
    Current Pharmaceutical Design 07/2015; 21(22). DOI:10.2174/1381612821666150531170448 · 3.29 Impact Factor
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    ABSTRACT: Despite significant efforts directed toward research on HIV-1 vaccines, a truly effective immunogen has not been achieved. However, the broadly neutralizing antibodies (BnAbs) 2F5 and 4E10, targeting the highly conserved membrane proximal external region (MPER) of HIV-1, are two promising tools for vaccine development. Here we engrafted the MPER into the linker domain between the trimeric core structure and the transmembrane domain of influenza A virus HA2 to investigate the potential of such chimeric viruses to elicit HIV-1 neutralizing antibodies. In the context of proliferating attenuated influenza A viruses, these HIV-1 neutralizing antibody epitopes could be continuously expressed and mimicked their native conformation to induce humoral immune responses. While MPER-specific antibodies could be detected in serum of guinea pigs vaccinated with the chimeric viruses, they exhibited only weakly neutralizing activities. These antisera from vaccinated animals neutralized viruses of clades B and BC (tier 1), but not of clades AE (tier 1) and C (tier 2). These results suggest that influenza A virus can be used as a vehicle for displaying MPER and inducing BnAbs, but it provides limited protection against HIV-1 infection. In the future development of HIV-1 vaccines by rational design, a more effective live virus vector or multiple antigens should be chosen to facilitate the process of neutralizing antibody maturation. Copyright © 2015. Published by Elsevier Ltd.
    Vaccine 06/2015; DOI:10.1016/j.vaccine.2015.06.072 · 3.49 Impact Factor
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    ABSTRACT: Streptococcus pneumoniae is an important pathogen accounting for a large number of deaths worldwide. Due to drawbacks of the current polysaccharide-based vaccine, the most promising way to generate an improved vaccine may be to utilize protection-eliciting pneumococcal proteins. Pneumococcal surface adhesin A (PsaA) and pneumococcal surface protein A (PspA) are two vaccine candidates which have been evaluated against S. pneumoniae infection in animal models or human clinical trials with encouraging results. In this study, the efficacy of the fusion protein PsaA-PspA, which includes PsaA part and PspA part, in inducing immunoprotective effects against fatal pneumococcal challenge was evaluated in an animal model. PspA part of PsaA-PspA fusion protein contains both family1 N-terminal region and family 2 N-terminal clade-defining region of PspA. Immunization with the PsaA-PspA fusion protein induced high levels of antibodies against both PsaA and PspA, which could bind to intact S. pneumoniae strains bearing different PspAs. Ex vivo stimulation of splenocytes from mice immunized with PsaA-PspA induced IL-17A secretion. Mice immunized with PsaA-PspA showed reduced S. pneumoniae levels in the blood and lungs compared with the PBS group after intranasal infection. Finally, mice immunized with PsaA-PspA fusion proteins were protected against fatal challenge with pneumococcal strains expressing different PspAs regardless of the challenge route. These results support the PsaA-PspA fusion protein as a promising vaccine strategy, as demonstrated by its ability to enhance the immune response and stimulate production of high titer antibodies against S. pneumoniae strains bearing heterologous PspAs, as well as confer protection against fatal challenge with PspA family 1 and family 2 strains.
    Immunological Investigations 06/2015; 44(5):482-96. DOI:10.3109/08820139.2015.1037956 · 1.90 Impact Factor
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    ABSTRACT: Enterovirus 71(EV71) has caused severe epidemics of hand, foot and mouth disease (HFMD) in the Asia Pacific in recent years, particularly in infants and pre-school children. It has become a serious public health threat, as currently there are no approved vaccines or antiviral drugs for EV71 infection. Many EV71 vaccines have been under development worldwide, however the main focus is inactivated EV71 vaccines. For example, the inactivated EV71 vaccine has recently finished phase III clinical trial in Mainland China. There have been very few studies on EV71 virus like particles (VLPs). In this study, the immunogenicity and protective potency of the EV71 VLPs produced in insect cells were evaluated in mice with different dosages. Our results showed that EV71 VLPs could elicit high titers of neutralizing antibodies (NTAbs) in a dose-dependent manner and NTAbs were sustained after the second injection with an average GMT (geometric mean titer) level from 19 to 2960 in immunized mice. Survival rates were 100%, 100%, 85%, and 40% after challenge with 15 LD50 (median lethal dose) of EV71 in these newborn mice, respectively. ED50 (50% effective dose) of VLPs was 0.20 μg/dose in newborn mice, while NTAb titer under this dosage was about 50. Passive protection was determined with two methods and demonstrated that the survival rates were positively correlated with NTAb titers, which at 24 and 54 induced 50% survival rates in experimental animals. The ED50 of VLP vaccines and the passive NTAb titers were also analyzed. The maternal NTAb titer was similar as the passive NTAb titer in the mouse model challenged with our lethal mouse EV71 strain. Hence, our work has provided preliminary data on the protection potency of VLPs as a vaccine candidate and would facilitate future VLP vaccine development.
    Human Vaccines & Immunotherapeutics 06/2015; DOI:10.1080/21645515.2015.1053675 · 3.64 Impact Factor
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    ABSTRACT: Plant proteins have been drawing increasing attention owing to their safety, abundance and relatively low cost in comparison with animal proteins. The development of plant protein-based delivery vehicles may lead to the provision of novel pharmaceutical products to patients. Zein is a class of alcohol-soluble prolamine proteins present in maize endosperm that was approved as a generally recognised as safe excipient in 1985 by the US FDA for use in pharmaceutical film coatings. Over the past few decades, numerous studies have been carried out to illustrate zein's potential for novel applications in the biomedical field. This paper reviews the present status of zein-based nanofibres, with emphasis on their fabrication and biomedical applications, particularly for drug delivery. Their benefits and limitations are also discussed to provide further insight into zein's potential as a promising biomaterial.
    Current pharmaceutical design 05/2015; · 3.29 Impact Factor
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    PLoS ONE 04/2015; 10(4):e0125701. DOI:10.1371/journal.pone.0125701 · 3.23 Impact Factor
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    ABSTRACT: Rbx1 and Rbx2 are essential components of Cullin-RING E3 Ligases. Vif is generally believed to preferentially recruit the Cul5-Rbx2 module to induce proteasomal degradation of antiretroviral enzyme APOBEC3G, although some investigators have found that the Cul5-Rbx1 module is recruited. Here, to investigate the function of the two Rbx proteins in the Vif-Cul5 complex, we analyzed the performance of Cul5-Rbx1/Cul5-Rbx2 module in the activity of Vif E3 ligase and evaluated the interactions between Rbx1/Rbx2 and Cul5. We found that either Rbx1 or Rbx2 could promote ubiquitination of APOBE3G (A3G) in vitro. We also found that both Rbx1 and Rbx2 could bind Cul5 in cells and Rbx2 could dose-dependently inhibit the interaction of Rbx1 with Cul5. Furthermore, only the decrease of endogenous Rbx2 but not Rbx1 could impair the Vif-induced A3G degradation in cells. These findings indicate that Rbx1 and Rbx2 can both activate Cul5-Vif E3 ligase in vitro, but they may undergo a more delicate selection mechanism in vivo. Copyright © 2015. Published by Elsevier Inc.
    Biochemical and Biophysical Research Communications 04/2015; 461(4). DOI:10.1016/j.bbrc.2015.04.077 · 2.28 Impact Factor
  • Viruses 04/2015; 7(4):1558-1577. DOI:10.3390/v7041558 · 3.28 Impact Factor
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    ABSTRACT: The hepatitis E virus (HEV) capsid antigen expressed in insect cell has been proposed as a candidate subunit vaccine for the prevention of hepatitis E. However, the expression and purification of HEV virus-like particles (VLPs) from insect cells have not been explored. We aimed to optimize the procedure to obtain HEV VLPs. In this study, two conformations of the HEV capsid proteins were expressed in insect cells, virus-like particles (VLPs) and non-VLPs, and they were purified separately. The physicochemical properties and the humoral immune responses induced by the two forms were analyzed and compared. We found that HEV VLPs were more immunogenic in mice than HEV non-VLPs. Therefore, we optimized the conditions that yielded high VLPs expression in insect cell cultures and developed an efficient purification method. The results suggest that the distinction and isolation of VLPs from non-VLPs are essential to generate a more immunogenic vaccine. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Biotechnology and Applied Biochemistry 03/2015; DOI:10.1002/bab.1379 · 1.32 Impact Factor
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    ABSTRACT: Zein is a class of alcohol-soluble prolamine proteins present in maize endosperm, which was approved as a generally recognized as safe (GRAS) excipient in 1985 by the United States Food and Drug Administration (US-FDA) for film coating of pharmaceuticals, e.g., tablets. Despite its long-term application in tablet production, effects of zein coating on tablet properties are still not fully understood. Moreover, many studies have also been conducted to illustrate its potential as an active ingredient of direct compressed tablets and film-based delivery carriers. In addition, the use of zein as a functional film coating material for new biomedical applications was also widely investigated in recent reports, which involved medical devices, nanoparticles, quantum dots and nanofibers. In this review, the present status of zein in the form of a thin film and uniform layer for use as a biomedical material is discussed. In addition, studies related to the behaviors and properties of zein films are also summarized and analyzed based on published works to gain mechanistic insights into the relationship between zein film and various improved profiles. This review will benefit future prospects of the use of zein film in drug delivery and biomedical applications. Copyright © 2015. Published by Elsevier B.V.
    Journal of Controlled Release 03/2015; 206. DOI:10.1016/j.jconrel.2015.03.030 · 7.26 Impact Factor
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    ABSTRACT: The objective of this study was to find common immune mechanism across different kinds of vaccines. A meta-analysis of microarray datasets was performed using publicly available microarray Gene Expression Omnibus (GEO) and Array Express data sets of vaccination records. Seven studies (out of 35) were selected for this meta-analysis. A total of 447 chips (145 pre-vaccination and 302 post-vaccination) were included. Significance analysis of microarrays (SAM) program was used for screening differentially expressed genes (DEGs). Functional pathway enrichment for the DEGs was conducted in DAVID Gene Ontology (GO) database. Twenty DEGs were identified, of which 10 up-regulated genes involved immune response. Six of which were type I interferon (IFN) related genes, including LY6E, MX1, OAS3, IFI44L, IFI6 and IFITM3. Ten down-regulated genes mainly mediated negative regulation of cell proliferation and cell motion. Results of a subgroup analysis showed that although the kinds of genes varied widely between days 3 and 7 post vaccination, the pathways between them are basically the same, such as immune response and response to viruses, etc. For an independent verification of these 6 type I IFN related genes, peripheral blood mononuclear cells (PBMCs) were collected at baseline and day 3 after the vaccination from 8 Enterovirus 71(EV71) vaccinees and were assayed by RT-PCR. Results showed that the 6 DEGs were also upregulated in EV71 vaccinees. In summary, meta-analysis methods were used to explore the immune mechanism of vaccines and results indicated that the type I IFN related genes and corresponding pathways were common in early immune responses for different kinds of vaccines.
    Human Vaccines & Immunotherapeutics 03/2015; 11(3):739-745. DOI:10.1080/21645515.2015.1008884 · 3.64 Impact Factor
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    ABSTRACT: HIV-1 gp120/gp41 is heavily modified by n-linked carbohydrates that play important roles either in correct folding or in shielding vulnerable viral protein surfaces from antibody recognition. In our previous work, 25 potential N-linked glycosylation sites (PNGS) of a CRF07_BC isolate of HIV-1 were individually mutated, and the resulting effects on infectivity and antibody-mediated neutralization were evaluated. In order to further understand the functional role of these PNGS, we generated double and multiple mutants from selected individual PNGS mutants. The effects were then evaluated by examining infectivity and sensitivity to antibody-mediated neutralization by neutralizing monoclonal antibodies (nMAbs) and serum antibodies from HIV-1 positive donors. Infectivity results showed that, among the twelve combined PNGS mutants, only 197M.1 (N197D/N301Q) lost infectivity completely, while all others (except for 197M.6) showed reduced viral infectivity. In terms of neutralization sensitivity to known nMAbs, we found that adding N463Q mutation to all the gp120 mutants containing N197D significantly increased neutralization sensitivity to VRC01 and VRC03, suggesting N197 and N463 have a strong synergistic effect in regulating the neutralizing sensitivity of HIV-1 to the anti-CD4bs nMAbs VRC01/VRC03. Structural analysis based on the available structures of gp120 alone and in complex with CD4 and various nMAbs elucidates a molecular rationale for this experimental observation. The data indicate that N463 plays an important role in regulating the CD4bs MAbs VRC01/VRC03 sensitivity in the genetic background of N197D mutation of gp120, which should provide valuable information for a better understanding of the interplay between HIV-1 and VRC01/03.
    JAIDS Journal of Acquired Immune Deficiency Syndromes 02/2015; 69(3). DOI:10.1097/QAI.0000000000000595 · 4.39 Impact Factor
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    ABSTRACT: Increasing evidence shows that grains may play a role in disease prevention beyond the simple provision of energy and nutrients. It has been reported that some components contained in grains exert their functional effects on viral and bacterial infections and protect against various cancers. However, until now, hardly any intervention studies have investigated the effects of grains or grain based extracts on the inhibition of HIV-1 infection. In this study, the antiviral function of a zymolytic grain based extract (ZGE) was detected in vitro and in rats, and the antiviral mechanism was investigated. Results showed that ZGE had an inhibition effect on HIV-1 infection in vitro with low cytotoxic effects. The study of the mechanism demonstrated that this functional food possibly acted on the viral surface structure protein gp120 which is responsible for cell binding, as well as on the postattachment stage of the virus. The sera of model rats administrated with this food by gavage presented anti-infection abilities against HIV-1 in vitro during a serum concentration associated period of time. These findings provide valuable insights into the application of ZGE on the control of viral load, which may contribute to future anti-HIV treatment with less adverse effects.
    Evidence-based Complementary and Alternative Medicine 01/2015; 2015:642327. DOI:10.1155/2015/642327 · 1.88 Impact Factor
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    ABSTRACT: Background Fitness costs and slower disease progression are associated with a cytolytic T lymphocyte (CTL) escape mutation T242N in Gag in HIV-1-infected individuals carrying HLA-B*57/5801 alleles. However, the impact of different context in diverse HIV-1 strains on the fitness costs due to the T242N mutation has not been well characterized. To better understand the extent of fitness costs of the T242N mutation and the repair of fitness loss through compensatory amino acids, we investigated its fitness impact in different transmitted/founder (T/F) viruses.ResultsThe T242N mutation resulted in various levels of fitness loss in four different T/F viruses. However, the fitness costs were significantly compromised by preexisting compensatory amino acids in (Isoleucine at position 247) or outside (glutamine at position 219) the CTL epitope. Moreover, the transmitted T242N escape mutant in subject CH131 was as fit as the revertant N242T mutant and the elimination of the compensatory amino acid I247 in the T/F viral genome resulted in significant fitness cost, suggesting the fitness loss caused by the T242N mutation had been fully repaired in the donor at transmission. Analysis of the global circulating HIV-1 sequences in the Los Alamos HIV Sequence Database showed a high prevalence of compensatory amino acids for the T242N mutation and other T cell escape mutations.Conclusions Our results show that the preexisting compensatory amino acids in the majority of circulating HIV-1 strains could significantly compromise the fitness loss due to CTL escape mutations and thus increase challenges for T cell based vaccines.
    Retrovirology 11/2014; 11(1):101. DOI:10.1186/s12977-014-0101-0 · 4.77 Impact Factor

Publication Stats

2k Citations
360.84 Total Impact Points

Institutions

  • 2003–2015
    • Jilin University
      • College of Life Sciences
      Yung-chi, Jilin Sheng, China
  • 2013
    • Changchun University of Technology
      Huinan, Jilin Sheng, China
  • 2012
    • Peking University
      • School of Pharmaceutical Sciences
      Peping, Beijing, China
  • 2008
    • 302 Military Hospital of China
      Peping, Beijing, China
  • 2007
    • Changchun University of Science and Technology
      Changchun, Fujian, China
  • 2001–2003
    • Johns Hopkins University
      • Department of Molecular Microbiology and Immunology
      Baltimore, Maryland, United States
  • 2002
    • Johns Hopkins Bloomberg School of Public Health
      • Department of Epidemiology
      Baltimore, Maryland, United States