83 results on '"Chinglai Yang"'
Search Results
2. Intradermal Immunization of EBOV VLPs in Guinea Pigs Induces Broader Antibody Responses Against GP Than Intramuscular Injection
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Ying Liu, Zhiyuan Wen, Ricardo Carrion, Jerritt Nunneley, Hilary Staples, Anysha Ticer, Jean L. Patterson, Richard W. Compans, Ling Ye, and Chinglai Yang
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ebola ,vaccine ,intradermal immunization ,antibody response ,VLP ,Microbiology ,QR1-502 - Abstract
Ebolavirus (EBOV) infection in humans causes severe hemorrhagic fevers with high mortality rates that range from 30 to 80% as shown in different outbreaks. Thus the development of safe and efficacious EBOV vaccines remains an important goal for biomedical research. We have shown in early studies that immunization with insect cell-produced EBOV virus-like particles (VLPs) is able to induce protect vaccinated mice against lethal EBOV challenge. In the present study, we investigated immune responses induced by Ebola VLPs via two different routes, intramuscular and intradermal immunizations, in guinea pigs. Analyses of antibody responses revealed that similar levels of total IgG antibodies against the EBOV glycoprotein (GP) were induced by the two different immunization methods. However, further characterization showed that the EBOV GP-specific antibodies induced by intramuscular immunization were mainly of the IgG2 subtype whereas both IgG1 and IgG2 antibodies against EBOV GP were induced by intradermal immunization. In contrast, antibody responses against the EBOV matrix protein VP40 induced by intramuscular or intradermal immunizations exhibited similar IgG1 and IgG2 profiles. More interestingly, we found that the sites that the IgG1 antibodies induced by intradermal immunizations bind to in GP are different from those that bind to the IgG2 antibodies induced by intramuscular immunization. Further analyses revealed that sera from all vaccinated guinea pigs exhibited neutralizing activity against Ebola GP-mediated HIV pseudovirion infection at high levels. Moreover, all EBOV VLP-vaccinated guinea pigs survived the challenge by a high dose (1000 pfu) of guinea pig-adapted EBOV, while all control guinea pigs immunized with irrelevant VLPs succumbed to the challenge. The induction of both IgG1 and IgG2 antibody responses that recognized broader sites in GP by intradermal immunization of EBOV VLPs indicates that this approach may represent a more advantageous route of vaccination against virus infection.
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- 2020
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3. An immunogen containing four tandem 10E8 epitope repeats with exposed key residues induces antibodies that neutralize HIV-1 and activates an ADCC reporter gene
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Zhiwu Sun, Yun Zhu, Qian Wang, Ling Ye, Yanyan Dai, Shan Su, Fei Yu, Tianlei Ying, Chinglai Yang, Shibo Jiang, and Lu Lu
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ADCC ,HIV-1 ,neutralizing antibodies ,10E8 epitopes ,Infectious and parasitic diseases ,RC109-216 ,Microbiology ,QR1-502 - Abstract
After three decades of intensive research efforts, an effective vaccine against HIV-1 remains to be developed. Several broadly neutralizing antibodies to HIV-1, such as 10E8, recognize the membrane proximal external region (MPER) of the HIV-1 gp41 protein. Thus, the MPER is considered to be a very important target for vaccine design. However, the MPER segment has very weak immunogenicity and tends to insert its epitope residues into the cell membrane, thereby avoiding antibody binding. To address this complication in vaccine development, we herein designed a peptide, designated 10E8-4P, containing four copies of the 10E8 epitope as an immunogen. As predicted by structural simulation, 10E8-4P exhibits a well-arranged tandem helical conformation, with the key residues in the 10E8 epitope oriented at different angles, thus suggesting that some of these key residues may be exposed outside of the lipid membrane. Compared with a peptide containing a single 10E8 epitope (10E8-1P), 10E8-4P not only exhibited better antigenicity but also elicited neutralizing antibody response against HIV-1 pseudoviruses, whereas 10E8-1P could not induce detectable neutralizing antibody response. Importantly, antibodies elicited by 10E8-4P also possessed a strong ability to activate an antibody-dependent cell-mediated cytotoxicity (ADCC) reporter gene, thus suggesting that they may have ADCC activity. Therefore, this strategy shows promise for further optimization and application in future HIV-1 vaccine design.Emerging Microbes and Infections (2016) 5, e65; doi:10.1038/emi.2016.86; published online 22 June 2016
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- 2016
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4. Phenotypic and Genetic Characterization of Avian Influenza H5N2 Viruses with Intra- and Inter-Duck Variations in Taiwan.
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Yao-Tsun Li, Hui-Ying Ko, Chang-Chun David Lee, Ching-Yu Lai, Chuan-Liang Kao, Chinglai Yang, Won-Bo Wang, and Chwan-Chuen King
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Medicine ,Science - Abstract
BackgroundHuman infections with avian influenza viruses (AIVs) have frequently raised global concerns of emerging, interspecies-transmissible viruses with pandemic potential. Waterfowl, the predominant reservoir of influenza viruses in nature, harbor precursors of different genetic lineages that have contributed to novel pandemic influenza viruses in the past.MethodsTwo duck influenza H5N2 viruses, DV518 and DV413, isolated through virological surveillance at a live-poultry market in Taiwan, showed phylogenetic relatedness but exhibited different replication capabilities in mammalian Madin-Darby Canine Kidney (MDCK) cells. This study characterizes the replication properties of the two duck H5N2 viruses and the determinants involved.ResultsThe DV518 virus replicated more efficiently than DV413 in both MDCK and chicken DF1 cells. Interestingly, the infection of MDCK cells by DV518 formed heterogeneous plaques with great differences in size [large (L) and small (S)], and the two viral strains (p518-L and p518-S) obtained from plaque purification exhibited distinguishable replication kinetics in MDCK cells. Nonetheless, both plaque-purified DV518 strains still maintained their growth advantages over the plaque-purified p413 strain. Moreover, three amino acid substitutions in PA (P224S), PB2 (E72D), and M1 (A128T) were identified in intra-duck variations (p518-L vs p518-S), whereas other changes in HA (N170D), NA (I56T), and NP (Y289H) were present in inter-duck variations (DV518 vs DV413). Both p518-L and p518-S strains had the N170D substitution in HA, which might be related to their greater binding to MDCK cells. Additionally, polymerase activity assays on 293T cells demonstrated the role of vRNP in modulating the replication capability of the duck p518-L viruses in mammalian cells.ConclusionThese results demonstrate that intra-host phenotypic variation occurs even within an individual duck. In view of recent human infections by low pathogenic AIVs, this study suggests possible determinants involved in the stepwise selection of virus variants from the duck influenza virus population which may facilitate inter-species transmission.
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- 2015
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5. Emerged HA and NA mutants of the pandemic influenza H1N1 viruses with increasing epidemiological significance in Taipei and Kaohsiung, Taiwan, 2009-10.
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Chuan-Liang Kao, Ta-Chien Chan, Chu-Han Tsai, Kuan-Ying Chu, Shu-Fang Chuang, Chang-Chun Lee, Zheng-Rong Tiger Li, Ko-Wen Wu, Luan-Yin Chang, Yea-Huei Shen, Li-Min Huang, Ping-Ing Lee, Chinglai Yang, Richard Compans, Barry T Rouse, and Chwan-Chuen King
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Medicine ,Science - Abstract
The 2009 influenza pandemic provided an opportunity to observe dynamic changes of the hemagglutinin (HA) and neuraminidase (NA) of pH1N1 strains that spread in two metropolitan areas--Taipei and Kaohsiung. We observed cumulative increases of amino acid substitutions of both HA and NA that were higher in the post-peak than in the pre-peak period of the epidemic. About 14.94% and 3.44% of 174 isolates had one and two amino acids changes, respective, in the four antigenic sites. One unique adaptive mutation of HA2 (E374K) was first detected three weeks before the epidemic peak. This mutation evolved through the epidemic, and finally emerged as the major circulated strain, with significantly higher frequency in the post-peak period than in the pre-peak (64.65% vs 9.28%, p
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- 2012
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6. Characterization of immune responses induced by immunization with the HA DNA vaccines of two antigenically distinctive H5N1 HPAIV isolates.
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Yulong Gao, Zhiyuan Wen, Ke Dong, Gongxun Zhong, Xiaomei Wang, Zhigao Bu, Hualan Chen, Ling Ye, and Chinglai Yang
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Medicine ,Science - Abstract
The evolution of the H5N1 highly pathogenic avian influenza virus (HPAIV) has resulted in high sequence variations and diverse antigenic properties in circulating viral isolates. We investigated immune responses induced by HA DNA vaccines of two contemporary H5N1 HPAIV isolates, A/bar-headed goose/Qinghai/3/2005 (QH) and A/chicken/Shanxi/2/2006 (SX) respectively, against the homologous as well as the heterologous virus isolate for comparison. Characterization of antibody responses induced by immunization with QH-HA and SX-HA DNA vaccines showed that the two isolates are antigenically distinctive. Interestingly, after immunization with the QH-HA DNA vaccine, subsequent boosting with the SX-HA DNA vaccine significantly augmented antibody responses against the QH isolate but only induced low levels of antibody responses against the SX isolate. Conversely, after immunization with the SX-HA DNA vaccine, subsequent boosting with the QH-HA DNA vaccine significantly augmented antibody responses against the SX isolate but only induced low levels of antibody responses against the QH isolate. In contrast to the antibody responses, cross-reactive T cell responses are readily detected between these two isolates at similar levels. These results indicate the existence of original antigenic sin (OAS) between concurrently circulating H5N1 HPAIV strains, which may need to be taken into consideration in vaccine development against the potential H5N1 HPAIV pandemic.
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- 2012
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7. Antigenic subversion: a novel mechanism of host immune evasion by Ebola virus.
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Gopi S Mohan, Wenfang Li, Ling Ye, Richard W Compans, and Chinglai Yang
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Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
In addition to its surface glycoprotein (GP(1,2)), Ebola virus (EBOV) directs the production of large quantities of a truncated glycoprotein isoform (sGP) that is secreted into the extracellular space. The generation of secreted antigens has been studied in several viruses and suggested as a mechanism of host immune evasion through absorption of antibodies and interference with antibody-mediated clearance. However such a role has not been conclusively determined for the Ebola virus sGP. In this study, we immunized mice with DNA constructs expressing GP(1,2) and/or sGP, and demonstrate that sGP can efficiently compete for anti-GP(12) antibodies, but only from mice that have been immunized by sGP. We term this phenomenon "antigenic subversion", and propose a model whereby sGP redirects the host antibody response to focus on epitopes which it shares with membrane-bound GP(1,2), thereby allowing it to absorb anti-GP(1,2) antibodies. Unexpectedly, we found that sGP can also subvert a previously immunized host's anti-GP(1,2) response resulting in strong cross-reactivity with sGP. This finding is particularly relevant to EBOV vaccinology since it underscores the importance of eliciting robust immunity that is sufficient to rapidly clear an infection before antigenic subversion can occur. Antigenic subversion represents a novel virus escape strategy that likely helps EBOV evade host immunity, and may represent an important obstacle to EBOV vaccine design.
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- 2012
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8. Induction of HIV neutralizing antibodies against the MPER of the HIV envelope protein by HA/gp41 chimeric protein-based DNA and VLP vaccines.
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Ling Ye, Zhiyuan Wen, Ke Dong, Xi Wang, Zhigao Bu, Huizhong Zhang, Richard W Compans, and Chinglai Yang
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Medicine ,Science - Abstract
Several conserved neutralizing epitopes have been identified in the HIV Env protein and among these, the MPER of gp41 has received great attention and is widely recognized as a promising target. However, little success has been achieved in eliciting MPER-specific HIV neutralizing antibodies by a number of different vaccine strategies. We investigated the ability of HA/gp41 chimeric protein-based vaccines, which were designed to enhance the exposure of the MPER in its native conformation, to induce MPER-specific HIV neutralizing antibodies. In characterization of the HA/gp41 chimeric protein, we found that by mutating an unpaired Cys residue (Cys-14) in its HA1 subunit to a Ser residue, the modified chimeric protein HA-C14S/gp41 showed increased reactivity to a conformation-sensitive monoclonal antibody against HA and formed more stable trimers in VLPs. On the other hand, HA-C14S/gp41 and HA/gp41 chimeric proteins expressed on the cell surfaces exhibited similar reactivity to monoclonal antibodies 2F5 and 4E10. Immunization of guinea pigs using the HA-C14S/gp41 DNA or VLP vaccines induced antibodies against the HIV gp41 as well as to a peptide corresponding to a segment of MPER at higher levels than immunization by standard HIV VLPs. Further, sera from vaccinated guinea pigs were found to exhibit HIV neutralizing activities. Moreover, sera from guinea pigs vaccinated by HA-C14S/gp41 DNA and VLP vaccines but not the standard HIV VLPs, were found to neutralize HIV pseudovirions containing a SIV-4E10 chimeric Env protein. The virus neutralization could be blocked by a MPER-specific peptide, thus demonstrating induction of MPER-specific HIV neutralizing antibodies by this novel vaccine strategy. These results show that induction of MPER-specific HIV neutralizing antibodies can be achieved through a rationally designed vaccine strategy.
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- 2011
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9. Corrigendum to 'Newcastle disease virus vectored Nipah encephalitis vaccines induce B and T cell responses in mice and long-lasting neutralizing antibodies in pigs' [Virology 432 (2012) 327–335]
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Dongni Kong, Zhiyuan Wen, Hua Su, Jinying Ge, Weiye Chen, Xijun Wang, Chao Wu, Chinglai Yang, Hualan Chen, and Zhigao Bu
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Virology - Published
- 2022
10. An ultra-low-cost electroporator with microneedle electrodes (ePatch) for SARS-CoV-2 vaccination
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Mark R. Prausnitz, Dengning Xia, Gaurav Byagathvalli, Chao-Yi Lu, M. Saad Bhamla, Huan Yu, Rui Jin, Chinglai Yang, and Ling Ye
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COVID-19 Vaccines ,Coronavirus disease 2019 (COVID-19) ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,Transfection ,Proof of Concept Study ,DNA vaccination ,Mice ,Immunology and Inflammation ,Engineering ,Genes, Reporter ,Vaccines, DNA ,Electrode array ,Animals ,Humans ,Medicine ,Intradermal injection ,Rats, Wistar ,Pandemics ,Skin ,Mice, Inbred BALB C ,Multidisciplinary ,piezoelectricity ,SARS-CoV-2 ,business.industry ,Electroporation ,Vaccination ,microneedle array ,COVID-19 ,Equipment Design ,COVID-19 DNA vaccine ,Biological Sciences ,Rats ,Needles ,Physical Sciences ,Electrode ,Costs and Cost Analysis ,Female ,business ,Microelectrodes ,skin electroporation ,Biomedical engineering - Abstract
Significance Low-cost and rapidly distributable vaccines are urgently needed to combat COVID-19 and future pandemics, especially for developing countries and other low-resource settings. DNA vaccines are inexpensive, rapidly developed, and safe, but require bulky and expensive electroporation devices for effective vaccination, which presents challenges to affordable and mass vaccination. We developed an ultra-low-cost (, Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other pathogens with pandemic potential requires safe, protective, inexpensive, and easily accessible vaccines that can be developed and manufactured rapidly at a large scale. DNA vaccines can achieve these criteria, but induction of strong immune responses has often required bulky, expensive electroporation devices. Here, we report an ultra-low-cost (
- Published
- 2021
11. Intradermal Immunization of EBOV VLPs in Guinea Pigs Induces Broader Antibody Responses Against GP Than Intramuscular Injection
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Ricardo Carrion, Hilary M. Staples, Richard W. Compans, Anysha Ticer, Jerritt Nunneley, Jean L. Patterson, Zhiyuan Wen, Chinglai Yang, Ling Ye, and Ying Liu
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Microbiology (medical) ,lcsh:QR1-502 ,Microbiology ,Virus ,lcsh:Microbiology ,03 medical and health sciences ,Pseudovirion ,Immune system ,VP40 ,ebola ,vaccine ,VLP ,Medicine ,030304 developmental biology ,Original Research ,intradermal immunization ,0303 health sciences ,biology ,030306 microbiology ,business.industry ,antibody response ,biochemical phenomena, metabolism, and nutrition ,Virology ,Vaccination ,Immunization ,biology.protein ,Antibody ,business ,Intramuscular injection - Abstract
Ebolavirus (EBOV) infection in humans causes severe hemorrhagic fevers with high mortality rates that range from 30 to 80% as shown in different outbreaks. Thus the development of safe and efficacious EBOV vaccines remains an important goal for biomedical research. We have shown in early studies that immunization with insect cell-produced EBOV virus-like particles (VLPs) is able to induce protect vaccinated mice against lethal EBOV challenge. In the present study, we investigated immune responses induced by Ebola VLPs via two different routes, intramuscular and intradermal immunizations, in guinea pigs. Analyses of antibody responses revealed that similar levels of total IgG antibodies against the EBOV glycoprotein (GP) were induced by the two different immunization methods. However, further characterization showed that the EBOV GP-specific antibodies induced by intramuscular immunization were mainly of the IgG2 subtype whereas both IgG1 and IgG2 antibodies against EBOV GP were induced by intradermal immunization. In contrast, antibody responses against the EBOV matrix protein VP40 induced by intramuscular or intradermal immunizations exhibited similar IgG1 and IgG2 profiles. More interestingly, we found that the sites that the IgG1 antibodies induced by intradermal immunizations bind to in GP are different from those that bind to the IgG2 antibodies induced by intramuscular immunization. Further analyses revealed that sera from all vaccinated guinea pigs exhibited neutralizing activity against Ebola GP-mediated HIV pseudovirion infection at high levels. Moreover, all EBOV VLP-vaccinated guinea pigs survived the challenge by a high dose (1000 pfu) of guinea pig-adapted EBOV, while all control guinea pigs immunized with irrelevant VLPs succumbed to the challenge. The induction of both IgG1 and IgG2 antibody responses that recognized broader sites in GP by intradermal immunization of EBOV VLPs indicates that this approach may represent a more advantageous route of vaccination against virus infection.
- Published
- 2020
12. Blockage of regulatory T cells augments induction of protective immune responses by influenza virus-like particles in aged mice
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Chinglai Yang, Ling Ye, Zhigao Bu, Ke Dong, Zhiyuan Wen, Xi Wang, and Huizhong Zhang
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0301 basic medicine ,Aging ,Regulatory T cell ,Immunology ,Antibodies, Viral ,T-Lymphocytes, Regulatory ,Microbiology ,Article ,Virus ,Mice ,03 medical and health sciences ,Immunogenicity, Vaccine ,Influenza A Virus, H1N1 Subtype ,0302 clinical medicine ,Immune system ,Orthomyxoviridae Infections ,medicine ,Animals ,Vaccines, Virus-Like Particle ,IL-2 receptor ,Mice, Inbred BALB C ,biology ,Immunogenicity ,Vaccination ,Antibodies, Monoclonal ,Th1 Cells ,Virology ,030104 developmental biology ,Infectious Diseases ,medicine.anatomical_structure ,Immunization ,Influenza Vaccines ,Immunoglobulin G ,Antibody Formation ,biology.protein ,Female ,Antibody ,030215 immunology - Abstract
Elderly humans over 65 years old are at great risk to pathogenesis by influenza virus infection. However, although influenza vaccines provide effective protection in healthy young adults, protection of elderly adults is substantially lower even with a good match between the vaccine and the circulating influenza virus. To gain insight of the underlying mechanism for the reduced immunogenicity of influenza vaccines in the aged population, we investigated immunogenicity of influenza virus-like particle vaccines in aged mice, which represent a useful model for studying aging associated impairment in immune responses. Specifically, we investigated the effect of inhibiting regulatory T cells in aged mice on induction of protective immune responses by influenza vaccines. Our results showed that injecting anti-CD25 antibodies could down-regulate CD25 on the surface of regulatory T cells and significantly increase the levels of antibody responses induced by VLP immunization in aged mice. Further, the profiles of antibody responses were also changed towards Th1 type by regulatory T cell blockage in aged mice. Moreover, aged mice that were treated by anti-CD25 antibodies prior to vaccination were more effectively protected against lethal influenza virus challenge.
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- 2017
13. An ultra-low-cost electroporator with microneedle electrodes (ePatch) for SARS-CoV-2 vaccination.
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Dengning Xia, Rui Jin, Byagathvalli, Gaurav, Huan Yu, Ling Ye, Chao-Yi Lu, Saad Bhamla, M., Chinglai Yang, and Prausnitz, Mark R.
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SARS-CoV-2 ,DNA vaccines ,VACCINATION ,INTRADERMAL injections ,INTRAMUSCULAR injections - Abstract
Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other pathogens with pandemic potential requires safe, protective, inexpensive, and easily accessible vaccines that can be developed and manufactured rapidly at a large scale. DNA vaccines can achieve these criteria, but induction of strong immune responses has often required bulky, expensive electroporation devices. Here, we report an ultra-low-cost (<1 USD), handheld (<50 g) electroporation system utilizing a microneedle electrode array (“ePatch”) for DNA vaccination against SARS-CoV-2. The low cost and small size are achieved by combining a thumb-operated piezoelectric pulser derived from a common household stove lighter that emits microsecond, bipolar, oscillatory electric pulses and a microneedle electrode array that targets delivery of high electric field strength pulses to the skin’s epidermis. Antibody responses against SARS-CoV-2 induced by this electroporation system in mice were strong and enabled at least 10-fold dose sparing compared to conventional intramuscular or intradermal injection of the DNA vaccine. Vaccination was well tolerated with mild, transient effects on the skin. This ePatch system is easily portable, without any battery or other power source supply, offering an attractive, inexpensive approach for rapid and accessible DNA vaccination to combat COVID-19, as well as other epidemics. [ABSTRACT FROM AUTHOR]
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- 2021
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14. Enhanced Stability of Inactivated Influenza Vaccine Encapsulated in Dissolving Microneedle Patches
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Richard W. Compans, Mark R. Prausnitz, Leonard Y. Chu, Chinglai Yang, Ling Ye, and Ke Dong
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0301 basic medicine ,Hot Temperature ,Influenza vaccine ,Drug Storage ,Pharmacology toxicology ,Transdermal Patch ,Pharmaceutical Science ,02 engineering and technology ,Article ,Mice ,03 medical and health sciences ,Drug Delivery Systems ,Influenza A Virus, H1N1 Subtype ,Orthomyxoviridae Infections ,Influenza, Human ,Animals ,Humans ,Medicine ,Pharmacology (medical) ,Pharmacology ,business.industry ,Organic Chemistry ,Vaccine Stability ,021001 nanoscience & nanotechnology ,Virology ,030104 developmental biology ,Vaccines, Inactivated ,Influenza Vaccines ,Needles ,Molecular Medicine ,Immunization ,0210 nano-technology ,business ,Biotechnology - Abstract
This study tested the hypothesis that encapsulation of influenza vaccine in microneedle patches increases vaccine stability during storage at elevated temperature.Whole inactivated influenza virus vaccine (A/Puerto Rico/8/34) was formulated into dissolving microneedle patches and vaccine stability was evaluated by in vitro and in vivo assays of antigenicity and immunogenicity after storage for up to 3 months at 4, 25, 37 and 45°C.While liquid vaccine completely lost potency as determined by hemagglutination (HA) activity within 1-2 weeks outside of refrigeration, vaccine in microneedle patches lost 40-50% HA activity during or shortly after fabrication, but then had no significant additional loss of activity over 3 months of storage, independent of temperature. This level of stability required reduced humidity by packaging with desiccant, but was not affected by presence of oxygen. This finding was consistent with additional stability assays, including antigenicity of the vaccine measured by ELISA, virus particle morphological structure captured by transmission electron microscopy and protective immune responses by immunization of mice in vivo.These data show that inactivated influenza vaccine encapsulated in dissolving microneedle patches has enhanced stability during extended storage at elevated temperatures.
- Published
- 2015
15. 163. Enhancing the Immunogenicity of a Novel, Low-cost Ebola Vaccine
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Samuel D. Stampfer, Rui Jin, and Chinglai Yang
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Dilution technique ,Ebola virus ,Ebola vaccine ,business.industry ,Cost effectiveness ,Immunogenicity ,Tissue membrane ,medicine.disease_cause ,Virology ,AcademicSubjects/MED00290 ,Infectious Diseases ,Oncology ,Poster Abstracts ,Medicine ,business ,Dilute (action) - Abstract
Background Ebolaviruses cause viral hemorrhagic fever with high mortality rates. Nearly all Ebola vaccines in development use Ebola glycoprotein (GP) as the immunizing antigen. GP is present on the viral membrane and functions in cell entry by binding the cellular receptor and mediating membrane fusion; antibodies to GP induce protective immunity. Ebola also produces sGP: a smaller, secreted form of GP containing the receptor-binding domain; it is also able to induce protective immunity. sGP naturally refolds after thermal denaturation and thus may be more stable than GP, and may also be more cost effective as it is produced easily in high quantities. sGP is a homodimer that is covalently linked by a cysteine near its C-terminus. In this work, we explored how modifications to sGP that affect its ability to dimerize also alter its immunogenicity. Methods sGP mutants were generated in the pCAGGS mammalian expression plasmid, and injected into mice as a DNA vaccine. Mouse sera was tested by ELISA against sGP and GP proteins, and in a neutralization assay against GP-typed pseudovirions. Results We generated 4 different mutants of sGP that had altered abilities to form inter-protomer disulfide bonds. All had a mutated or deleted cysteine at position 306; two had disulfide-bonding restored by introduction of an engineered inter-protomer disulfide bond. Mice were immunized with a DNA vaccine encoding either an sGP mutant or wild-type sGP, and sera were collected. We found that sera from sGP mutants with reduced interprotomer disulfide bonds had significantly higher antibody titers to sGP and GP than sera from our wild-type sGP and engineered-disulfide sGP immunized mice. Antibody titers were similar between sGP and GP; these titers correlated with neutralization ability. Relative binding to sGP & GP (by ELISA OD) & relative % neutralization of pseudovirions at 1:10 dilution Conclusion Immunogenicity of Ebola sGP was enhanced significantly when mutations were introduced to reduce its ability to covalently dimerize. Immunogenicity correlated with induction of neutralizing antibodies, implying that our mutants may outperform wild-type sGP when used as a vaccine in vivo. This work helps paves the way for an alternative Ebola vaccine that has the potential to be more cost-effective and heat-stable than the currently-licensed vaccine. Disclosures Samuel D. Stampfer, MD/PhD, Gilead (Shareholder)
- Published
- 2020
16. Intradermal Vaccination With Adjuvanted Ebola Virus Soluble Glycoprotein Subunit Vaccine by Microneedle Patches Protects Mice Against Lethal Ebola Virus Challenge
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Richard W. Compans, Yasmine A. Gomaa, Chinglai Yang, Ling Ye, Mark R. Prausnitz, Jean L. Patterson, Gregory M. Glenn, Hua Wu, Fang Lin, Ricardo Carrion, David Flyer, Gale Smith, and Ying Liu
- Subjects
0301 basic medicine ,medicine.medical_treatment ,Supplement Articles ,medicine.disease_cause ,Antibodies, Viral ,Injections, Intramuscular ,03 medical and health sciences ,Mice ,Immune system ,Antigen ,Adjuvants, Immunologic ,medicine ,Immunology and Allergy ,Animals ,Humans ,Ebola Vaccines ,chemistry.chemical_classification ,Mice, Inbred BALB C ,Ebola virus ,biology ,business.industry ,Vaccination ,Hemorrhagic Fever, Ebola ,Virology ,030104 developmental biology ,Infectious Diseases ,HEK293 Cells ,Immunization ,chemistry ,Antibody Formation ,Vaccines, Subunit ,biology.protein ,Female ,Antibody ,business ,Glycoprotein ,Adjuvant ,HeLa Cells - Abstract
In this study, we investigated immune responses induced by purified Ebola virus (EBOV) soluble glycoprotein (sGP) subunit vaccines via intradermal immunization with microneedle (MN) patches in comparison with intramuscular (IM) injection in mice. Our results showed that MN delivery of EBOV sGP was superior to IM injection in eliciting higher levels and longer lasting antibody responses against EBOV sGP and GP antigens. Moreover, sGP-specific immune responses induced by MN or IM immunizations were effectively augmented by formulating sGP with a saponin-based adjuvant, and they were shown to confer complete protection of mice against lethal mouse-adapted EBOV (MA-EBOV) challenge. In comparison, mice that received sGP without adjuvant by MN or IM immunizations succumbed to lethal MA-EBOV challenge. These results show that immunization with EBOV sGP subunit vaccines with adjuvant by MN patches, which have been shown to provide improved safety and thermal stability, is a promising approach to protect against EBOV infection.
- Published
- 2018
17. Intradermal immunization by Ebola virus GP subunit vaccines using microneedle patches protects mice against lethal EBOV challenge
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Mark R. Prausnitz, Ricardo Carrion, David Flyer, Jean L. Patterson, Fang Lin, Gregory M. Glenn, Richard W. Compans, Chinglai Yang, Ling Ye, Ying Liu, Yasmine A. Gomaa, Gale Smith, and Hua Wu
- Subjects
0301 basic medicine ,Injections, Intradermal ,medicine.medical_treatment ,lcsh:Medicine ,medicine.disease_cause ,Antibodies, Viral ,Article ,03 medical and health sciences ,Mice ,Immune system ,Adjuvants, Immunologic ,medicine ,Animals ,Humans ,Subunit vaccines ,Ebola Vaccines ,lcsh:Science ,Glycoproteins ,chemistry.chemical_classification ,Multidisciplinary ,Ebola virus ,biology ,business.industry ,lcsh:R ,Vaccination ,Hemorrhagic Fever, Ebola ,Ebolavirus ,Virology ,3. Good health ,030104 developmental biology ,Immunization ,chemistry ,Antibody Formation ,Vaccines, Subunit ,biology.protein ,lcsh:Q ,Antibody ,business ,Glycoprotein ,Adjuvant - Abstract
Development of a safe and efficacious filovirus vaccine is of high importance to public health. In this study, we compared immune responses induced by Ebola virus (EBOV) glycoprotein (GP) subunit vaccines via intradermal immunization with microneedle (MN) patches and the conventional intramuscular (IM) injection in mice, which showed that MN delivery of GP induced higher levels and longer lasting antibody responses against GP than IM injection. Further, we found that EBOV GP in formulation with a saponin-based adjuvant, Matrix-M, can be efficiently loaded onto MN patches. Co-delivery of Matrix-M with GP significantly enhanced induction of antibody responses by MN delivery, as also observed for IM injection. Results from challenge studies showed that all mice that received the GP/adjuvant formulation by MN or IM immunizations were protected from lethal EBOV challenge. Further, 4 out of 5 mice vaccinated by MN delivery of unadjuvanted GP also survived the challenge, whereas only 1 out of 5 mice vaccinated by IM injection of unadjuvanted GP survived the challenge. These results demonstrate that MN patch delivery of EBOV GP subunit vaccines, which is expected to enable improved safety and thermal stability, can confer effective protection against EBOV infection that is superior to IM vaccination.
- Published
- 2018
18. Characterization of Immune Responses Induced by Ebola Virus Glycoprotein (GP) and Truncated GP Isoform DNA Vaccines and Protection Against Lethal Ebola Virus Challenge in Mice
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Jean L. Patterson, Hilary M. Staples, Richard W. Compans, Wenfang Li, Ricardo Carrion, Anysha Ticer, Gopi S. Mohan, Jerritt Nunneley, Chinglai Yang, and Ling Ye
- Subjects
viruses ,Immunization, Secondary ,Biology ,Antibodies, Viral ,medicine.disease_cause ,Virus ,Epitope ,DNA vaccination ,Mice ,Viral Proteins ,Immune system ,Antigen ,Vaccines, DNA ,medicine ,Animals ,Humans ,Protein Isoforms ,Ebola and Marburg Viruses-Research, Outbreak Management, Epidemiology and Ecology ,Immunology and Allergy ,Ebola Vaccines ,Glycoproteins ,Mice, Inbred BALB C ,Ebola virus ,Vaccination ,Hemorrhagic Fever, Ebola ,Ebolavirus ,Virology ,HEK293 Cells ,Infectious Diseases ,Immunization ,Antibody Formation ,biology.protein ,Female ,Antibody - Abstract
In addition to its surface glycoprotein (GP), Ebola virus directs the production of large quantities of a truncated glycoprotein isoform (sGP) that is secreted into the extracellular space. We recently reported that sGP actively diverts host antibody responses against the epitopes that it shares with GP and thereby allows itself to absorb anti-GP antibodies, a phenomenon we termed “antigenic subversion.” To investigate the effect of antigenic subversion by sGP on protection against virus infection, we compared immune responses induced by different prime-boost immunization regimens with GP and sGP DNA vaccines in mice and their efficacy against lethal Ebola virus challenge. Similar levels of anti-GP antibodies were induced by 2 immunizations with sGP and GP DNA vaccines. However, 2 immunizations with GP but not sGP DNA vaccine fully protected mice from lethal challenge. Boosting with sGP or GP DNA vaccine in mice that had been primed by GP or sGP DNA vaccine augmented the levels of anti-GP antibody responses and further improved protective efficacy against Ebola virus infection. These results show that both the quality and the levels of anti-GP antibody responses affect the efficacy of protection against Ebola virus infection.
- Published
- 2015
19. Less Is More: Ebola Virus Surface Glycoprotein Expression Levels Regulate Virus Production and Infectivity
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Xiaoqian Lin, Brian Pollack, Wenfang Li, Chinglai Yang, Ling Ye, Ana C. Monteiro, Richard W. Compans, Gopi S. Mohan, and Bishu Sapkota
- Subjects
Gene Expression Regulation, Viral ,viruses ,Immunology ,Filoviridae ,Biology ,Virus Replication ,medicine.disease_cause ,Microbiology ,Virus ,Cell Line ,VP40 ,Virology ,medicine ,Humans ,Virus Release ,Ebolavirus ,Infectivity ,Membrane Glycoproteins ,Ebola virus ,Virus Internalization ,biology.organism_classification ,Virus-Cell Interactions ,Viral replication ,Insect Science ,RNA Editing - Abstract
The Ebola virus (EBOV) surface glycoprotein (GP 1,2 ) mediates host cell attachment and fusion and is the primary target for host neutralizing antibodies. Expression of GP 1,2 at high levels disrupts normal cell physiology, and EBOV uses an RNA-editing mechanism to regulate expression of the GP gene. In this study, we demonstrate that high levels of GP 1,2 expression impair production and release of EBOV virus-like particles (VLPs) as well as infectivity of GP 1,2 -pseudotyped viruses. We further show that this effect is mediated through two mechanisms. First, high levels of GP 1,2 expression reduce synthesis of other proteins needed for virus assembly. Second, viruses containing high levels of GP 1,2 are intrinsically less infectious, possibly due to impaired receptor binding or endosomal processing. Importantly, proteolysis can rescue the infectivity of high-GP 1,2 -containing viruses. Taken together, our findings indicate that GP 1,2 expression levels have a profound effect on factors that contribute to virus fitness and that RNA editing may be an important mechanism employed by EBOV to regulate GP 1,2 expression in order to optimize virus production and infectivity. IMPORTANCE The Ebola virus (EBOV), as well as other members of the Filoviridae family, causes severe hemorrhagic fever that is highly lethal, with up to 90% mortality. The EBOV surface glycoprotein (GP 1,2 ) plays important roles in virus infection and pathogenesis, and its expression is tightly regulated by an RNA-editing mechanism during virus replication. Our study demonstrates that the level of GP 1,2 expression profoundly affects virus particle production and release and uncovers a new mechanism by which Ebola virus infectivity is regulated by the level of GP 1,2 expression. These findings extend our understanding of EBOV infection and replication in adaptation of host environments, which will aid the development of countermeasures against EBOV infection.
- Published
- 2015
20. Rlim, an E3 ubiquitin ligase, influences the stability of Stathmin protein in human osteosarcoma cells
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Chinglai Yang, Huizhong Zhang, Xingyu Li, Xi Chen, Longfei Yang, Fang Lin, Xi Wang, Ke Dong, Jian-Jun Shen, Min Long, and Junxia Wei
- Subjects
Proteasome Endopeptidase Complex ,Ubiquitin-Protein Ligases ,Regulator ,Bone Neoplasms ,Stathmin ,macromolecular substances ,Cell Line, Tumor ,Humans ,RNA, Small Interfering ,Cell Proliferation ,Zinc finger ,Osteosarcoma ,Oncogene ,biology ,Ubiquitin ,Cell growth ,Ubiquitination ,Cell Cycle Checkpoints ,Cell Biology ,Cell cycle ,Ubiquitin ligase ,HEK293 Cells ,Proteasome ,Proteolysis ,biology.protein ,Cancer research ,RNA Interference ,Protein Processing, Post-Translational ,Protein Binding - Abstract
Stathmin is an oncoprotein and is expressed at high levels in a wide variety of human malignancies, which plays important roles in maintenance of malignant phenotypes. The regulation of Stathmin gene overexpression has been wildly explored, but the exact mechanism still needs to be elucidated. It is believed that regulation of an oncogene protein abundance through post-translational modifications is essential for maintenance of malignant phenotypes. Here we identified the Rlim, a Ring H2 zinc finger protein with intrinsic ubiquitin ligase activity, as a Stathmin-interacting protein that could increase Stathmin turnover through binding with this targeted protein and then induce its degradation by proteasome in a ubiquitin-dependent manner. Inhibition of endogenous Rlim expression by siRNA could increase the level of Stathmin protein, which further led to cell proliferation and cell cycle changes in human osteosarcoma cell lines. On the other hand, forced overexpression of Rlim could decrease the level of Stathmin protein. These results demonstrate that Rlim is involved in the negative regulation of Stathmin protein level through physical interaction and ubiquitin-mediated proteolysis. Hence, Rlim is a novel regulator of Stathmin protein in a ubiquitin-dependent manner, and represents a new pathway for malignant phenotype turnover by modulating the level of Stathmin protein in human osteosarcomas.
- Published
- 2014
21. Ebola Vaccination Using a DNA Vaccine Coated on PLGA-PLL/γPGA Nanoparticles Administered Using a Microneedle Patch
- Author
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Mark R. Prausnitz, Chinglai Yang, Ling Ye, Richard W. Compans, Hung-Wei Yang, and Xin Dong Guo
- Subjects
0301 basic medicine ,Biomedical Engineering ,Pharmaceutical Science ,Nanoparticle ,02 engineering and technology ,DNA vaccination ,Biomaterials ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,Immune system ,Immunogenicity, Vaccine ,Polylactic Acid-Polyglycolic Acid Copolymer ,Vaccines, DNA ,Animals ,Humans ,Polylysine ,Lactic Acid ,Ebola Vaccines ,Thermostability ,Immunogenicity ,021001 nanoscience & nanotechnology ,Virology ,Vaccination ,PLGA ,030104 developmental biology ,chemistry ,Polyglutamic Acid ,Needles ,Nanoparticles ,Delivery system ,0210 nano-technology ,Polyglycolic Acid ,HeLa Cells - Abstract
Ebola DNA vaccine is incorporated into PLGA-PLL/γPGA nanoparticles and administered to skin using a microneedle (MN) patch. The nanoparticle delivery system increases vaccine thermostability and immunogenicity compared to free vaccine. Vaccination by MN patch produces stronger immune responses than intramuscular administration.
- Published
- 2016
22. An immunogen containing four tandem 10E8 epitope repeats with exposed key residues induces antibodies that neutralize HIV-1 and activates an ADCC reporter gene
- Author
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Tianlei Ying, Chinglai Yang, Yun Zhu, Zhiwu Sun, Ling Ye, Qian Wang, Yanyan Dai, Lu Lu, Shan Su, Shibo Jiang, and Fei Yu
- Subjects
0301 basic medicine ,Antigenicity ,Immunogen ,Epidemiology ,Immunology ,Enzyme-Linked Immunosorbent Assay ,Biology ,HIV Antibodies ,Microbiology ,Epitope ,03 medical and health sciences ,Epitopes ,10E8 epitopes ,Genes, Reporter ,Neutralization Tests ,Virology ,Drug Discovery ,Animals ,Humans ,neutralizing antibodies ,Neutralizing antibody ,Antibody-dependent cell-mediated cytotoxicity ,AIDS Vaccines ,Linear epitope ,Immunogenicity ,Circular Dichroism ,Antibody-Dependent Cell Cytotoxicity ,General Medicine ,Antibodies, Neutralizing ,3. Good health ,030104 developmental biology ,Infectious Diseases ,biology.protein ,HIV-1 ,Parasitology ,Original Article ,Rabbits ,Antibody ,ADCC ,Protein Binding - Abstract
After three decades of intensive research efforts, an effective vaccine against HIV-1 remains to be developed. Several broadly neutralizing antibodies to HIV-1, such as 10E8, recognize the membrane proximal external region (MPER) of the HIV-1 gp41 protein. Thus, the MPER is considered to be a very important target for vaccine design. However, the MPER segment has very weak immunogenicity and tends to insert its epitope residues into the cell membrane, thereby avoiding antibody binding. To address this complication in vaccine development, we herein designed a peptide, designated 10E8-4P, containing four copies of the 10E8 epitope as an immunogen. As predicted by structural simulation, 10E8-4P exhibits a well-arranged tandem helical conformation, with the key residues in the 10E8 epitope oriented at different angles, thus suggesting that some of these key residues may be exposed outside of the lipid membrane. Compared with a peptide containing a single 10E8 epitope (10E8-1P), 10E8-4P not only exhibited better antigenicity but also elicited neutralizing antibody response against HIV-1 pseudoviruses, whereas 10E8-1P could not induce detectable neutralizing antibody response. Importantly, antibodies elicited by 10E8-4P also possessed a strong ability to activate an antibody-dependent cell-mediated cytotoxicity (ADCC) reporter gene, thus suggesting that they may have ADCC activity. Therefore, this strategy shows promise for further optimization and application in future HIV-1 vaccine design.
- Published
- 2016
23. Production of Potent Fully Human Polyclonal Antibodies against Ebola Zaire Virus in Transchromosomal Cattle
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Steven A. Kwilas, Jin-an Jiao, Andrew S. Herbert, Thomas C. Luke, Chinglai Yang, Hana Golding, Ana I. Kuehne, Eddie Sullivan, Russell A. Bakken, Surender Khurana, Jay W. Hooper, Sandra Fuentes, Ling Ye, Ralph A. Tripp, Gregory M. Glenn, John M. Dye, Wenfang Li, Jennifer M. Brannan, Hua Wu, Gale Smith, Elizabeth M. Coyle, Richard W. Compans, and Ramon A. Ortiz
- Subjects
0301 basic medicine ,Enzyme-Linked Immunosorbent Assay ,medicine.disease_cause ,Antibodies, Viral ,Immunoglobulin G ,Article ,Hyperimmunization ,Animals, Genetically Modified ,03 medical and health sciences ,0302 clinical medicine ,Neutralization Tests ,medicine ,Animals ,Humans ,030212 general & internal medicine ,Ebolavirus ,Mice, Inbred BALB C ,Multidisciplinary ,Ebola virus ,biology ,business.industry ,Immunization, Passive ,Hemorrhagic Fever, Ebola ,Surface Plasmon Resonance ,Virology ,Titer ,Disease Models, Animal ,030104 developmental biology ,Treatment Outcome ,Immunization ,Polyclonal antibodies ,biology.protein ,Cattle ,Antibody ,business - Abstract
Polyclonal antibodies, derived from humans or hyperimmunized animals, have been used prophylactically or therapeutically as countermeasures for a variety of infectious diseases. SAB Biotherapeutics has successfully developed a transchromosomic (Tc) bovine platform technology that can produce fully human immunoglobulins rapidly, and in substantial quantities, against a variety of disease targets. In this study, two Tc bovines expressing high levels of fully human IgG were hyperimmunized with a recombinant glycoprotein (GP) vaccine consisting of the 2014 Ebola virus (EBOV) Makona isolate. Serum collected from these hyperimmunized Tc bovines contained high titers of human IgG against EBOV GP as determined by GP specific ELISA, surface plasmon resonance (SPR), and virus neutralization assays. Fully human polyclonal antibodies against EBOV were purified and evaluated in a mouse challenge model using mouse adapted Ebola virus (maEBOV). Intraperitoneal administration of the purified anti-EBOV IgG (100 mg/kg) to BALB/c mice one day after lethal challenge with maEBOV resulted in 90% protection; whereas 100% of the control animals succumbed. The results show that hyperimmunization of Tc bovines with EBOV GP can elicit protective and potent neutralizing fully human IgG antibodies rapidly and in commercially viable quantities.
- Published
- 2016
24. Molecular Basis of Neurovirulence of Flury Rabies Virus Vaccine Strains: Importance of the Polymerase and the Glycoprotein R333Q Mutation
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Hongyue Zhai, Xijun Wang, Chinglai Yang, Dongni Kong, Zhigao Bu, Lihong Tao, Hualan Chen, Bolin Zhao, Tao Hua, and Jinying Ge
- Subjects
Rabies ,Molecular Sequence Data ,Immunology ,Mutation, Missense ,Virus Replication ,medicine.disease_cause ,Microbiology ,Virus ,Cell Line ,Mice ,Viral Proteins ,Dogs ,Rabies vaccine ,Cricetinae ,Virology ,medicine ,Animals ,Dog Diseases ,Mononegavirales ,Lyssavirus ,Glycoproteins ,Genetics ,Mice, Inbred BALB C ,Mutation ,Base Sequence ,Virulence ,biology ,Rabies virus ,Brain ,DNA-Directed RNA Polymerases ,Rhabdoviridae ,biology.organism_classification ,Rabies Vaccines ,Viral replication ,Insect Science ,Pathogenesis and Immunity ,Female ,medicine.drug - Abstract
The molecular mechanisms associated with rabies virus (RV) virulence are not fully understood. In this study, the RV Flury low-egg-passage (LEP) and high-egg-passage (HEP) strains were used as models to explore the attenuation mechanism of RV. The results of our studies confirmed that the R333Q mutation in the glycoprotein (GR333Q) is crucial for the attenuation of Flury RV in mice. The R333Q mutation is stably maintained in the HEP genome background but not in the LEP genome background during replication in mouse brain tissue or cell culture. Further investigation using chimeric viruses revealed that the polymerase L gene determines the genetic stability of the GR333Qmutation during replication. Moreover, a recombinant RV containing the LEP G protein with the R333Q mutation and the HEP L gene showed significant attenuation, genetic stability, enhancement of apoptosis, and immunogenicity. These results indicate that attenuation of the RV Flury strain results from the coevolution of G and L elements and provide important information for the generation of safer and more effective modified live rabies vaccine.
- Published
- 2010
25. Immunization by influenza virus-like particles protects aged mice against lethal influenza virus challenge
- Author
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Chinglai Yang, Lei Pan, Ling Ye, Zhiyuan Wen, Yulong Gao, Richard W. Compans, Zhigao Bu, and Ke Dong
- Subjects
viruses ,Orthomyxoviridae ,Neuraminidase ,Hemagglutinin (influenza) ,Hemagglutinin Glycoproteins, Influenza Virus ,Antibodies, Viral ,complex mixtures ,Article ,Virus ,Cell Line ,Microbiology ,Mice ,Viral Proteins ,Influenza A Virus, H1N1 Subtype ,Virus-like particle ,Virology ,Influenza, Human ,Animals ,Humans ,Avidity ,Cells, Cultured ,Pharmacology ,Mice, Inbred BALB C ,biology ,Immunogenicity ,Age Factors ,Virion ,virus diseases ,Dendritic Cells ,biochemical phenomena, metabolism, and nutrition ,biology.organism_classification ,Immunization ,Influenza Vaccines ,biology.protein ,Cytokines ,Female - Abstract
Influenza virus-like particles (VLPs) were produced in Sf9 insect cells by co-expressing the matrix protein M1 and the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) using the recombinant baculovirus expression system. The VLPs were morphologically similar to influenza virions. Both HA and NA proteins were incorporated into VLPs and these proteins retained their functional activities. Further, influenza VLPs but not inactivated influenza viruses (IIV) stimulated secretion of inflammatory cytokines from mouse bone marrow-derived dendritic cells (BMDC). Immunogenicity of influenza VLPs and their protective efficacies against lethal influenza virus challenge were evaluated in young and aged mice. Immunization with influenza VLPs induced strong antibody responses against HA that inhibited hemagglutination by influenza virus, similar to IIV vaccines. Compared to young mice, antibody responses in aged mice immunized with a low dose of either influenza VLPs or IIV vaccines exhibited markedly reduced avidity for HA. However, immunization of aged mice with a high dose of influenza VLPs induced antibody responses with high avidity similar to those in young mice. Furthermore, all vaccinated animals survived a lethal challenge by a mouse-adapted influenza virus (A/PR/8/34), indicating that influenza VLPs are highly efficacious for protection against influenza virus infection in both young and aged mice.
- Published
- 2009
26. Immunization by vaccine-coated microneedle arrays protects against lethal influenza virus challenge
- Author
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Chinglai Yang, Ling Ye, Ioanna Skountzou, Mark R. Prausnitz, Harvinder S. Gill, Qiyun Zhu, Lei Pan, Zhiyuan Wen, Vladimir Zarnitsyn, Yulong Gao, and Richard W. Compans
- Subjects
Hemagglutination ,Influenza vaccine ,Orthomyxoviridae ,Enzyme-Linked Immunosorbent Assay ,Hemagglutinin Glycoproteins, Influenza Virus ,Biology ,Administration, Cutaneous ,medicine.disease_cause ,Antibodies ,Virus ,Mice ,Immunity ,Influenza A virus ,medicine ,Animals ,Mice, Inbred BALB C ,Vaccines ,Multidisciplinary ,Hemagglutination assay ,Biological Sciences ,biology.organism_classification ,Virology ,Immunization ,Influenza Vaccines ,Immunology ,Cytokines ,Female - Abstract
Influenza prophylaxis would benefit from a simple method to administer influenza vaccine into skin without the need for hypodermic needles. In this study, solid metal microneedle arrays (MNs) were investigated as a system for cutaneous vaccine delivery using influenza virus antigen. The MNs with 5 monument-shaped microneedles per array were produced and coated with inactivated influenza virus A/PR/8/34 (IIV). As much as 10 μg of viral proteins could be coated onto an array of 5 microneedles, and the coated IIV was delivered into skin at high efficiency within minutes. The coated MNs were used to immunize mice in comparison with conventional intramuscular injection at the same dose. Analysis of immune responses showed that a single immunization with IIV-coated MNs induced strong antibody responses against influenza virus, with significant levels of hemagglutination inhibition activities (>1:40), which were comparable to those induced by conventional intramuscular immunization. Moreover, mice immunized by a single dose of IIV coated on MNs were effectively protected against lethal challenge by a high dose of mouse-adapted influenza virus A/PR/8/34. These results show that MNs are highly effective as a simple method of vaccine delivery to elicit protective immune responses against virus infection.
- Published
- 2009
27. Protection against filovirus infection: virus-like particle vaccines
- Author
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Richard W. Compans, Chinglai Yang, and Ling Ye
- Subjects
Pharmacology ,viruses ,Viral Vaccine ,Therapeutic treatment ,Immunology ,Viral Vaccines ,Filoviridae ,Biology ,biology.organism_classification ,Virology ,Virus-Like Particle Vaccines ,Hemorrhagic Fevers ,Immune system ,Virus-like particle ,Drug Discovery ,Filoviridae Infections ,Animals ,Humans ,Molecular Medicine ,Effective treatment - Abstract
Significant progress has been made in vaccine development against infection by Ebola and Marburg viruses, members of the Filoviridae, which cause severe hemorrhagic fevers in humans with no effective treatment and a mortality rate of up to 90%. Several vaccine strategies have been shown to effectively protect immunized animals against filovirus infection. Among these candidate vaccine strategies, virus-like particles represent a promising approach and have been shown to protect small laboratory animals as well as nonhuman primates against lethal challenge by Ebola and/or Marburg viruses. This review briefly summarizes filovirus epidemiology and pathogenesis, and focuses on the discussion of recent advances in filovirus vaccine development and the current understanding of protective immune responses against filovirus infection with an emphasis on the progress and challenge of filovirus virus-like particle vaccine development.
- Published
- 2008
28. Phenotypic and Genetic Characterization of Avian Influenza H5N2 Viruses with Intra- and Inter-Duck Variations in Taiwan
- Author
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Hui-Ying Ko, Chuan-Liang Kao, Won-Bo Wang, Chang-Chun D Lee, Yao-Tsun Li, Chinglai Yang, Ching-Yu Lai, and Chwan-Chuen King
- Subjects
Viral Plaque Assay ,Genotype ,viruses ,Science ,Population ,Taiwan ,Chick Embryo ,Genome, Viral ,Biology ,Virus Replication ,medicine.disease_cause ,H5N1 genetic structure ,Virus ,Cell Line ,Viral Proteins ,Genetic variation ,medicine ,Influenza A virus ,Animals ,Humans ,Amino Acid Sequence ,education ,Cells, Cultured ,Genetics ,education.field_of_study ,Multidisciplinary ,Genetic Variation ,virus diseases ,Biological Evolution ,Virology ,Influenza A virus subtype H5N1 ,Ducks ,Phenotype ,Amino Acid Substitution ,Viral replication ,Influenza in Birds ,Medicine ,Influenza A Virus, H5N2 Subtype ,Research Article - Abstract
BackgroundHuman infections with avian influenza viruses (AIVs) have frequently raised global concerns of emerging, interspecies-transmissible viruses with pandemic potential. Waterfowl, the predominant reservoir of influenza viruses in nature, harbor precursors of different genetic lineages that have contributed to novel pandemic influenza viruses in the past.MethodsTwo duck influenza H5N2 viruses, DV518 and DV413, isolated through virological surveillance at a live-poultry market in Taiwan, showed phylogenetic relatedness but exhibited different replication capabilities in mammalian Madin-Darby Canine Kidney (MDCK) cells. This study characterizes the replication properties of the two duck H5N2 viruses and the determinants involved.ResultsThe DV518 virus replicated more efficiently than DV413 in both MDCK and chicken DF1 cells. Interestingly, the infection of MDCK cells by DV518 formed heterogeneous plaques with great differences in size [large (L) and small (S)], and the two viral strains (p518-L and p518-S) obtained from plaque purification exhibited distinguishable replication kinetics in MDCK cells. Nonetheless, both plaque-purified DV518 strains still maintained their growth advantages over the plaque-purified p413 strain. Moreover, three amino acid substitutions in PA (P224S), PB2 (E72D), and M1 (A128T) were identified in intra-duck variations (p518-L vs p518-S), whereas other changes in HA (N170D), NA (I56T), and NP (Y289H) were present in inter-duck variations (DV518 vs DV413). Both p518-L and p518-S strains had the N170D substitution in HA, which might be related to their greater binding to MDCK cells. Additionally, polymerase activity assays on 293T cells demonstrated the role of vRNP in modulating the replication capability of the duck p518-L viruses in mammalian cells.ConclusionThese results demonstrate that intra-host phenotypic variation occurs even within an individual duck. In view of recent human infections by low pathogenic AIVs, this study suggests possible determinants involved in the stepwise selection of virus variants from the duck influenza virus population which may facilitate inter-species transmission.
- Published
- 2015
29. Antigenic properties of a transport-competent influenza HA/HIV Env chimeric protein
- Author
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Richard W. Compans, Yuliang Sun, Qingyang Wu, Shibo Jiang, Zhigao Bu, Chinglai Yang, David A. Steinhauer, Ling Ye, and Jianguo Lin
- Subjects
medicine.drug_class ,Recombinant Fusion Proteins ,Protein subunit ,viruses ,Hemagglutinins, Viral ,HIV Infections ,HIV Antibodies ,Biology ,Monoclonal antibody ,medicine.disease_cause ,Gp41 ,Genes, env ,Epitope ,Mice ,03 medical and health sciences ,Virology ,medicine ,Influenza A virus ,Animals ,030304 developmental biology ,AIDS Vaccines ,chemistry.chemical_classification ,Mice, Inbred BALB C ,0303 health sciences ,Influenza A Virus, H3N2 Subtype ,030302 biochemistry & molecular biology ,virus diseases ,HIV ,Fusion protein ,Molecular biology ,gp41 ,HIV Envelope Protein gp41 ,Transmembrane protein ,3. Good health ,chemistry ,HIV-1 ,Female ,Immunization ,Chimeric protein ,Glycoprotein ,Vaccine - Abstract
The transmembrane subunit (gp41) of the HIV Env glycoprotein contains conserved neutralizing epitopes which are not well-exposed in wild-type HIV Env proteins. To enhance the exposure of these epitopes, a chimeric protein, HA/gp41, in which the gp41 of HIV-1 89.6 envelope protein was fused to the C-terminus of the HA1 subunit of the influenza HA protein, was constructed. Characterization of protein expression showed that the HA/gp41 chimeric proteins were expressed on cell surfaces and formed trimeric oligomers, as found in the HIV Env as well as influenza HA proteins. In addition, the HA/gp41 chimeric protein expressed on the cell surface can also be cleaved into 2 subunits by trypsin treatment, similar to the influenza HA. Moreover, the HA/gp41 chimeric protein was found to maintain a pre-fusion conformation. Interestingly, the HA/gp41 chimeric proteins on cell surfaces exhibited increased reactivity to monoclonal antibodies against the HIV Env gp41 subunit compared with the HIV-1 envelope protein, including the two broadly neutralizing monoclonal antibodies 2F5 and 4E10. Immunization of mice with a DNA vaccine expressing the HA/gp41 chimeric protein induced antibodies against the HIV gp41 protein and these antibodies exhibit neutralizing activity against infection by an HIV SF162 pseudovirus. These results demonstrate that the construction of such chimeric proteins can provide enhanced exposure of conserved epitopes in the HIV Env gp41 and may represent a novel vaccine design strategy for inducing broadly neutralizing antibodies against HIV.
- Published
- 2006
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30. Development of vaccines for prevention of Ebola virus infection
- Author
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Chinglai Yang and Ling Ye
- Subjects
medicine.medical_specialty ,Ebola vaccine ,Immune protection ,business.industry ,viruses ,Public health ,Immunology ,virus diseases ,Outbreak ,Hemorrhagic Fever, Ebola ,Ebolavirus ,Microbiology ,Virology ,West africa ,Hemorrhagic Fevers ,Disease Models, Animal ,Infectious Diseases ,Communicable Disease Control ,Medicine ,Effective treatment ,Animals ,Humans ,Ebola Vaccines ,business ,Ebola virus infection - Abstract
Ebola virus infection causes severe hemorrhagic fevers with high fatality rates up to 90% in humans, for which no effective treatment is currently available. The ongoing Ebola outbreak in West Africa that has caused over 14,000 human infections and over 5000 deaths underscores its serious threat to the public health. While licensed vaccines against Ebola virus infection are still not available, a number of vaccine approaches have been developed and shown to protect against lethal Ebola virus infection in animal models. This review aims to summarize the advancement of different strategies for Ebola vaccine development with a focus on the discussion of their protective efficacies and possible limitations. In addition, the development of animal models for efficacy evaluation of Ebola vaccines and the mechanism of immune protection against Ebola virus infection are also discussed.
- Published
- 2014
31. Enhancement of immunogenicity of an HIV Env DNA vaccine by mutation of the Tyr-based endocytosis motif in the cytoplasmic domain
- Author
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Richard W. Compans, Chinglai Yang, Ling Ye, Andrei N. Vzorov, Dahnide Taylor, and Zhigao Bu
- Subjects
Cytoplasm ,T-Lymphocytes ,viruses ,Amino Acid Motifs ,Molecular Sequence Data ,Mutant ,Cell Count ,HIV Infections ,HIV Antibodies ,Biology ,Virus ,DNA vaccination ,Mice ,Virology ,Serine ,Vaccines, DNA ,Animals ,Amino Acid Sequence ,Tyrosine ,Gene ,Cells, Cultured ,AIDS Vaccines ,Immunogenicity ,Wild type ,HIV ,virus diseases ,Molecular biology ,Endocytosis ,HIV Envelope Protein gp41 ,Protein Structure, Tertiary ,Amino Acid Substitution ,Mutation ,Female ,Immunization ,DNA construct ,Spleen - Abstract
We investigated the effect of the conserved tyrosine-based endocytosis motif (YXXΦ) in the cytoplasmic domain of the human immunodeficiency viruses (HIV) envelope protein (Env) on its immunogenicity. Genes with codons optimized for mammalian expression were synthesized for the HIV 89.6 Env with a truncated cytoplasmic domain and a mutant Env in which the tyrosine residue in the YXXΦ motif was changed into a serine. Mutation of the Tyr residue enhanced surface expression of the Env protein. Analysis of immune responses induced by DNA immunization of mice showed that the DNA construct for the Tyr mutant Env induced moderately higher levels of T cell responses. More interestingly, the DNA construct for the mutant Env induced significantly higher levels of antibody responses against the Env protein in comparison to the construct for the wild type Env. Our results suggest that the YXXΦ motif in the HIV Env cytoplasmic domain may play a role in virus evasion of host immune responses through affecting its immunogenicity.
- Published
- 2004
32. Palmitoylation of the Murine Leukemia Virus Envelope Protein Is Critical for Lipid Raft Association and Surface Expression
- Author
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Min Li, Richard W. Compans, Armin Weidmann, Chinglai Yang, and Suxiang Tong
- Subjects
Octoxynol ,viruses ,Immunology ,Mutant ,Palmitic Acid ,Replication ,Biology ,Giant Cells ,Membrane Fusion ,Microbiology ,Cell Line ,Cell membrane ,Mice ,Membrane Microdomains ,Viral Envelope Proteins ,Palmitoylation ,Virology ,Murine leukemia virus ,medicine ,Animals ,Lipid raft ,Syncytium ,Cell Membrane ,virus diseases ,Lipid bilayer fusion ,3T3 Cells ,Raft ,biology.organism_classification ,Molecular biology ,Leukemia Virus, Murine ,medicine.anatomical_structure ,Solubility ,Biochemistry ,Insect Science ,Mutation ,lipids (amino acids, peptides, and proteins) - Abstract
To investigate the association of the murine leukemia virus (MuLV) Env protein with lipid rafts, we compared wild-type and palmitoylation-deficient mutant Env proteins by using extraction with the mild detergent Triton X-100 (TX-100) followed by a sucrose gradient flotation assay. We found that the wild-type MuLV Env protein was resistant to ice-cold TX-100 treatment and floated to the top of the gradients. In contrast, we observed that the palmitoylation-deficient mutant Env protein was mostly soluble when extracted by ice-cold TX-100 and stayed at the bottom of the gradients. Both the wild-type and mutant Env proteins were found to be soluble when treated with methyl-β-cyclodextrin before extraction with ice-cold TX-100 or when treated with ice-cold octyl-β-glucoside instead of TX-100. These results indicate that the MuLV Env protein is associated with lipid rafts and that palmitoylation of the Env protein is critical for lipid raft association. Although the palmitoylation-deficient Env mutant was synthesized at a level similar to that of the wild-type Env, it was found to be expressed at reduced levels on the cell surface. We observed syncytium formation activity with both the wild-type and mutant Env proteins, indicating that palmitoylation or raft association is not required for MuLV viral fusion activity.
- Published
- 2002
33. The Emergence and Evolution of Avian H5N2 Influenza viruses in Chickens in Taiwan
- Author
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Hongbo Zhu, CL Cheung, David K. Smith, Tommy Tsan-Yuk Lam, Yao-Tsun Li, Chinglai Yang, Liuxia Peng, Richard W. Compans, Pei-Yu Huang, Chwan-Chuen King, Chun-Hung Yip, Yun-Cheng Chang, Chang-Chun D Lee, and Yi Guan
- Subjects
animal structures ,viruses ,Immunology ,Reassortment ,Molecular Sequence Data ,Taiwan ,Hemagglutinin (influenza) ,Neuraminidase ,Hemagglutinin Glycoproteins, Influenza Virus ,Biology ,medicine.disease_cause ,Microbiology ,H5N1 genetic structure ,Virus ,Evolution, Molecular ,Viral Proteins ,Virology ,Reassortant Viruses ,medicine ,Animals ,Cluster Analysis ,Phylogeny ,Attenuated vaccine ,Sequence Analysis, DNA ,Influenza A virus subtype H5N1 ,Genetic Diversity and Evolution ,Insect Science ,Influenza in Birds ,biology.protein ,Enzootic ,Influenza A Virus, H5N2 Subtype ,Chickens - Abstract
Sporadic activity by H5N2 influenza viruses has been observed in chickens in Taiwan from 2003 to 2012. The available information suggests that these viruses were generated by reassortment between a Mexican-like H5N2 virus and a local enzootic H6N1 virus. Yet the origin, prevalence, and pathogenicity of these H5N2 viruses have not been fully defined. Following the 2012 highly pathogenic avian influenza (HPAI) outbreaks, surveillance was conducted from December 2012 to July 2013 at a live-poultry wholesale market in Taipei. Our findings showed that H5N2 and H6N1 viruses cocirculated at low levels in chickens in Taiwan. Phylogenetic analyses revealed that all H5N2 viruses had hemagglutinin (HA) and neuraminidase (NA) genes derived from a 1994 Mexican-like virus, while their internal gene complexes were incorporated from the enzootic H6N1 virus lineage by multiple reassortment events. Pathogenicity studies demonstrated heterogeneous results even though all tested viruses had motifs (R-X-K/R-R) supportive of high pathogenicity. Serological surveys for common subtypes of avian viruses confirmed the prevalence of the H5N2 and H6N1 viruses in chickens and revealed an extraordinarily high seroconversion rate to an H9N2 virus, a subtype that is not found in Taiwan but is prevalent in mainland China. These findings suggest that reassortant H5N2 viruses, together with H6N1 viruses, have become established and enzootic in chickens throughout Taiwan and that a large-scale vaccination program might have been conducted locally that likely led to the introduction of the 1994 Mexican-like virus to Taiwan in 2003. IMPORTANCE H5N2 avian influenza viruses first appeared in chickens in Taiwan in 2003 and caused a series of outbreaks afterwards. Phylogenetic analyses show that the chicken H5N2 viruses have H5 and N2 genes that are closely related to those of a vaccine strain originating from Mexico in 1994, while the contemporary duck H5N2 viruses in Taiwan belong to the Eurasian gene pool. The unusually high similarity of the chicken H5N2 viruses to the Mexican vaccine strain suggests that these viruses might have been introduced to Taiwan by using inadequately inactivated or attenuated vaccines. These chicken H5N2 viruses are developing varying levels of pathogenicity that could lead to significant consequences for the local poultry industry. These findings emphasize the need for strict quality control and competent oversight in the manufacture and usage of avian influenza virus vaccines and indicate that alternatives to widespread vaccination may be desirable.
- Published
- 2014
34. Mutations in the Cytoplasmic Tail of Murine Leukemia Virus Envelope Protein Suppress Fusion Inhibition by R Peptide
- Author
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Chinglai Yang, Richard W. Compans, and Min Li
- Subjects
Immunoprecipitation ,viruses ,Molecular Sequence Data ,Immunology ,Mutant ,Peptide ,Biology ,Membrane Fusion ,Microbiology ,Cell Line ,Mice ,Viral Envelope Proteins ,Virology ,Murine leukemia virus ,Animals ,Humans ,Amino Acid Sequence ,Peptide sequence ,chemistry.chemical_classification ,Syncytium ,Lipid bilayer fusion ,biology.organism_classification ,Molecular biology ,Peptide Fragments ,Virus-Cell Interactions ,Amino acid ,Leukemia Virus, Murine ,chemistry ,Insect Science ,Mutagenesis, Site-Directed ,HeLa Cells - Abstract
During viral maturation, the cytoplasmic tail of the murine leukemia virus (MuLV) envelope (Env) protein undergoes proteolytic cleavage by the viral protease to release the 16-amino-acid R peptide, and this cleavage event activates the Env protein's fusion activity. We introduced Gly and/or Ser residues at different positions upstream of the R peptide in the cytoplasmic tail of the Friend MuLV Env protein and investigated their effects on fusion activity. Expression in HeLa T 4 cells of a mutant Env protein with a single Gly insertion after I619, five amino acids upstream from the R peptide, induced syncytium formation with overlaid XC cells. Env proteins containing single or double Gly-Ser insertions after F614, 10 amino acids upstream from the R peptide, induced syncytium formation, and mutant proteins with multiple Gly insertions induced various levels of syncytium formation between HeLa T 4 and XC cells. Immunoprecipitation and surface biotinylation assays showed that most of the mutants had surface expression levels comparable to those of the wild-type or R peptide-truncated Env proteins. Fluorescence dye redistribution assays also showed no hemifusion in the Env proteins which did not induce fusion. Our results indicate that insertion mutations in the cytoplasmic tail of the MuLV Env protein can suppress the inhibitory effect of the R peptide on membrane fusion and that there are differences in the effects of insertions in two regions in the cytoplasmic tail upstream of the R peptide.
- Published
- 2001
35. Analysis of the murine leukemia virus R peptide: delineation of the molecular determinants which are important for its fusion inhibition activity
- Author
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Richard W. Compans and Chinglai Yang
- Subjects
Cytoplasm ,viruses ,Molecular Sequence Data ,Immunology ,Mutant ,Peptide ,Membrane Fusion ,Microbiology ,Structure-Activity Relationship ,Virology ,Murine leukemia virus ,Humans ,Amino Acid Sequence ,Peptide sequence ,Sequence Deletion ,chemistry.chemical_classification ,Cell fusion ,biology ,C-terminus ,Gene Products, env ,Lipid bilayer fusion ,biology.organism_classification ,Molecular biology ,Peptide Fragments ,Amino acid ,Leukemia Virus, Murine ,chemistry ,Insect Science ,Sequence Alignment ,HeLa Cells ,Research Article - Abstract
In previous studies, the C-terminal R peptide of the murine leukemia virus (MuLV) Env protein was shown to be a potent inhibitor of viral fusion activity. In the present study, we investigated the molecular determinants in the MuLV Env protein cytoplasmic tail which are important for the fusion inhibition activity of the R peptide. We constructed a series of mutant MuLV env genes which express Env proteins with serial truncations, internal deletions, or amino acid substitutions in the cytoplasmic tail. To analyze their cell fusion activity, we employed a quantitative fusion assay. We found that truncations of up to 7 amino acids from the C terminus of the cytoplasmic tail had no detectable effect on the lack of fusion activity of the full-length Env protein; however, further truncations resulted in a progressive increase in cell fusion activity. Studies of mutant proteins with amino acid substitutions in the cytoplasmic tail showed that Leu-627 plays an important role in fusion inhibition by the R peptide, while most of the other amino acids in the R peptide were not essential for fusion inhibition. Studies of mutant proteins with internal deletions upstream of the cleavage site in the cytoplasmic tail showed that this region is also involved in fusion inhibition by the R peptide, although only to a limited extent. The results are consistent with a model in which the MuLV R peptide exhibits its fusion inhibition activity through interaction with a cellular factor(s).
- Published
- 1997
36. Palmitoylation of the Murine Leukemia Virus Envelope Glycoprotein Transmembrane Subunits
- Author
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Richard W. Compans and Chinglai Yang
- Subjects
Protein subunit ,Molecular Sequence Data ,Palmitic Acid ,Palmitic Acids ,Biology ,Mice ,Viral Envelope Proteins ,Viral envelope ,Palmitoylation ,Virology ,Animals ,Humans ,chemistry.chemical_classification ,Binding Sites ,Base Sequence ,Virus Assembly ,Virion ,Biological Transport ,3T3 Cells ,Transmembrane protein ,Friend murine leukemia virus ,Amino acid ,Kinetics ,Transmembrane domain ,Biochemistry ,chemistry ,DNA, Viral ,Fatty acylation ,HeLa Cells ,Cysteine - Abstract
The envelope protein of Friend murine leukemia virus is modified by fatty acylation of the transmembrane (TM) protein subunit. The labeling by [3H]palmitic acid was found to be sensitive to treatment with the reducing reagents 2-mercaptoethanol and hydroxylamine, indicating the presence of a thioester linkage. Pulse-chase experiments showed that the precursor protein can be labeled by [3H]palmitic acid prior to its cleavage into the surface and TM subunits. By using site-directed mutagenesis, we determined that palmitoylation occurs on a cysteine residue, Cys 606, located in the transmembrane domain. A thin-layer chromatography assay after acid hydrolysis showed that incorporated label comigrated with palmitic acid. When another cysteine residue was introduced into the cytoplasmic tail 22 amino acids from the transmembrane domain, no palmitoylation was observed to occur on this cysteine residue, demonstrating the importance of the position of the cysteine residue for palmitoylation. Sequence comparison revealed that most retrovirus envelope proteins have one or two conserved cysteine residues in their transmembrane domain. Mutations that change the palmitoylation state of the murine leukemia virus envelope protein did not affect its transport, processing, surface expression, or cell fusion activity. The palmitate-deficient viral envelope proteins were incorporated into virus particles, and replication of the virusin vitrowas not affected significantly by the mutation of the palmitoylation site.
- Published
- 1996
37. The human and simian immunodeficiency virus envelope glycoprotein transmembrane subunits are palmitoylated
- Author
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Chinglai Yang, Claus Peter Spies, and Richard W. Compans
- Subjects
Protein Conformation ,viruses ,Protein subunit ,Molecular Sequence Data ,Palmitic Acid ,Palmitic Acids ,HIV Envelope Protein gp120 ,Biology ,Gp41 ,Membrane Fusion ,HIV Envelope Protein gp160 ,Palmitoylation ,Humans ,Amino Acid Sequence ,Protein Precursors ,chemistry.chemical_classification ,Multidisciplinary ,Base Sequence ,Sequence Homology, Amino Acid ,Gene Products, env ,virus diseases ,HIV envelope protein ,Molecular biology ,HIV Envelope Protein gp41 ,Recombinant Proteins ,Transmembrane protein ,Amino acid ,Biochemistry ,chemistry ,HIV-1 ,lipids (amino acids, peptides, and proteins) ,Simian Immunodeficiency Virus ,Fatty acylation ,Glycoprotein ,Protein Processing, Post-Translational ,HeLa Cells ,Research Article - Abstract
The envelope proteins of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) were found to be modified by fatty acylation of the transmembrane protein subunit gp41. The precursor gp160 was also palmitoylated prior to its cleavage into the gp120 and gp41 subunits. The palmitic acid label was sensitive to treatment with hydroxylamine or 2-mercaptoethanol, indicating that the linkage is through a thioester bond. Treatment with cycloheximide did not prevent the incorporation of [3H]palmitic acid into the HIV envelope protein, indicating that palmitoylation is a posttranslation modification. In contrast to other glycoproteins, which are palmitoylated at cysteine residues within or close to the membrane-spanning hydrophobic domain, the palmitoylation of the HIV-1 envelope proteins occurs on two cysteine residues, Cys-764 and Cys-837, which are 59 and 132 amino acids, respectively, from the proposed membrane-spanning domain of gp41. Sequence comparison revealed that one of these residues (Cys-764) is conserved in the cytoplasmic domains of almost all HIV-1 isolates and is located very close to an amphipathic region which has been postulated to bind to the plasma membrane.
- Published
- 1995
38. Newcastle disease virus-vectored Nipah encephalitis vaccines induce B and T cell responses in mice and long-lasting neutralizing antibodies in pigs
- Author
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Jinying Ge, Zhiyuan Wen, Hualan Chen, Chao Wu, Hua Su, Zhigao Bu, Chinglai Yang, Xijun Wang, Dongni Kong, and Weiye Chen
- Subjects
Time Factors ,Paramyxoviridae ,Swine ,T-Lymphocytes ,Genetic Vectors ,Newcastle disease virus ,Antibodies, Viral ,Newcastle disease ,Virus ,Microbiology ,Cell Line ,Mice ,Viral Envelope Proteins ,Virology ,medicine ,Animals ,Humans ,Recombinant Newcastle disease virus ,Encephalitis, Viral ,Neutralizing antibody ,Henipavirus Infections ,B-Lymphocytes ,Mice, Inbred BALB C ,Vaccines, Synthetic ,biology ,Viral Vaccine ,Immunogenicity ,Nipah Virus ,Viral Vaccines ,biology.organism_classification ,medicine.disease ,Antibodies, Neutralizing ,Nipah encephalitis virus ,biology.protein ,Female ,Antibody ,Glycoprotein ,Vaccine ,Encephalitis - Abstract
Nipah virus (NiV), a member of the Paramyxoviridae family, causes deadly encephalitis in humans and huge economic losses to the pig industry. Here, we generated recombinant avirulent Newcastle disease virus (NDV) LaSota strains expressing the NiV G and F proteins respectively (designated as rLa-NiVG and rLa-NiVF), and evaluated their immunogenicity in mice and pigs. Both rLa-NiVG and rLa-NiVF displayed growth properties similar to those of LaSota virus in chicken eggs. Co-infection of rLa-NiVG and rLa-NiVF caused marked syncytia formation, while intracerebral co-inoculation of these viruses in mice showed they were safe in at least one mammalian species. Animal immunization studies showed rLa-NiVG and rLa-NiVF induced NiV neutralizing antibody responses in mice and pigs, and F protein-specific CD8+ T cell responses in mice. Most importantly, rLa-NiVG and rLa-NiVF administered alone or together, induced a long-lasting neutralizing antibody response in pigs. Recombinant rLa-NiVG/F thus appear to be promising NiV vaccine candidates for pigs and potentially humans.
- Published
- 2012
39. Emerged HA and NA mutants of the pandemic influenza H1N1 viruses with increasing epidemiological significance in Taipei and Kaohsiung, Taiwan, 2009-10
- Author
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Chinglai Yang, Richard W. Compans, Li-Min Huang, Chu-Han Tsai, Yea-Huei Shen, Chwan-Chuen King, Barry T. Rouse, Chuan-Liang Kao, Zheng-Rong Tiger Li, Shu-Fang Chuang, Luan-Ying Chang, Ko-Wen Wu, Chang-Chun Lee, Kuan-Ying Chu, Ta-Chien Chan, and Ping-Ing Lee
- Subjects
Oseltamivir ,Viral Diseases ,Epidemiology ,Science ,Population ,Taiwan ,Hemagglutinin (influenza) ,Neuraminidase ,medicine.disease_cause ,Real-Time Polymerase Chain Reaction ,Microbiology ,Herd immunity ,chemistry.chemical_compound ,Influenza A Virus, H1N1 Subtype ,Virology ,Pandemic ,Influenza, Human ,Influenza A virus ,medicine ,Genetics ,Humans ,education ,Biology ,education.field_of_study ,Multidisciplinary ,biology ,business.industry ,Vaccination ,Infectious Diseases ,Hemagglutinins ,chemistry ,Mutation ,biology.protein ,Medicine ,business ,Research Article - Abstract
The 2009 influenza pandemic provided an opportunity to observe dynamic changes of the hemagglutinin (HA) and neuraminidase (NA) of pH1N1 strains that spread in two metropolitan areas--Taipei and Kaohsiung. We observed cumulative increases of amino acid substitutions of both HA and NA that were higher in the post-peak than in the pre-peak period of the epidemic. About 14.94% and 3.44% of 174 isolates had one and two amino acids changes, respective, in the four antigenic sites. One unique adaptive mutation of HA2 (E374K) was first detected three weeks before the epidemic peak. This mutation evolved through the epidemic, and finally emerged as the major circulated strain, with significantly higher frequency in the post-peak period than in the pre-peak (64.65% vs 9.28%, p
- Published
- 2012
40. Induction of HIV neutralizing antibodies against the MPER of the HIV envelope protein by HA/gp41 chimeric protein-based DNA and VLP vaccines
- Author
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Zhiyuan Wen, Huizhong Zhang, Zhigao Bu, Richard W. Compans, Chinglai Yang, Ling Ye, Xi Wang, and Ke Dong
- Subjects
medicine.drug_class ,Protein subunit ,Recombinant Fusion Proteins ,viruses ,Guinea Pigs ,Molecular Sequence Data ,lcsh:Medicine ,Enzyme-Linked Immunosorbent Assay ,HIV Infections ,HIV Antibodies ,Gp41 ,Monoclonal antibody ,03 medical and health sciences ,Antigen ,medicine ,Animals ,Humans ,Amino Acid Sequence ,lcsh:Science ,Antigens, Viral ,Virology/Vaccines ,030304 developmental biology ,AIDS Vaccines ,0303 health sciences ,Multidisciplinary ,biology ,030306 microbiology ,lcsh:R ,Antibodies, Monoclonal ,HIV ,virus diseases ,HIV envelope protein ,DNA ,Infectious Diseases/HIV Infection and AIDS ,Fusion protein ,Virology ,Antibodies, Neutralizing ,HIV Envelope Protein gp41 ,3. Good health ,Hemagglutinins ,Immunology/Immune Response ,biology.protein ,Immunization ,lcsh:Q ,Antibody ,Research Article - Abstract
Several conserved neutralizing epitopes have been identified in the HIV Env protein and among these, the MPER of gp41 has received great attention and is widely recognized as a promising target. However, little success has been achieved in eliciting MPER-specific HIV neutralizing antibodies by a number of different vaccine strategies. We investigated the ability of HA/gp41 chimeric protein-based vaccines, which were designed to enhance the exposure of the MPER in its native conformation, to induce MPER-specific HIV neutralizing antibodies. In characterization of the HA/gp41 chimeric protein, we found that by mutating an unpaired Cys residue (Cys-14) in its HA1 subunit to a Ser residue, the modified chimeric protein HA-C14S/gp41 showed increased reactivity to a conformation-sensitive monoclonal antibody against HA and formed more stable trimers in VLPs. On the other hand, HA-C14S/gp41 and HA/gp41 chimeric proteins expressed on the cell surfaces exhibited similar reactivity to monoclonal antibodies 2F5 and 4E10. Immunization of guinea pigs using the HA-C14S/gp41 DNA or VLP vaccines induced antibodies against the HIV gp41 as well as to a peptide corresponding to a segment of MPER at higher levels than immunization by standard HIV VLPs. Further, sera from vaccinated guinea pigs were found to exhibit HIV neutralizing activities. Moreover, sera from guinea pigs vaccinated by HA-C14S/gp41 DNA and VLP vaccines but not the standard HIV VLPs, were found to neutralize HIV pseudovirions containing a SIV-4E10 chimeric Env protein. The virus neutralization could be blocked by a MPER-specific peptide, thus demonstrating induction of MPER-specific HIV neutralizing antibodies by this novel vaccine strategy. These results show that induction of MPER-specific HIV neutralizing antibodies can be achieved through a rationally designed vaccine strategy.
- Published
- 2011
41. Immunization with a Mixture of HIV Env DNA and VLP Vaccines Augments Induction of CD8 T Cell Responses
- Author
-
Richard W. Compans, Lei Pan, Zhiyuan Wen, Zhigao Bu, Ke Dong, Huizhong Zhang, Chinglai Yang, and Ling Ye
- Subjects
Article Subject ,Health, Toxicology and Mutagenesis ,viruses ,lcsh:Biotechnology ,Human immunodeficiency virus (HIV) ,lcsh:Medicine ,lcsh:Chemical technology ,medicine.disease_cause ,lcsh:Technology ,complex mixtures ,DNA vaccination ,chemistry.chemical_compound ,Immune system ,lcsh:TP248.13-248.65 ,Genetics ,Medicine ,Cytotoxic T cell ,lcsh:TP1-1185 ,Molecular Biology ,biology ,lcsh:T ,business.industry ,lcsh:R ,virus diseases ,General Medicine ,biochemical phenomena, metabolism, and nutrition ,Virology ,Antibody response ,chemistry ,Immunization ,Immunology ,biology.protein ,Molecular Medicine ,bacteria ,Antibody ,business ,DNA ,Research Article ,Biotechnology - Abstract
The immune response induced by immunization with HIV Env DNA and virus-like particle (VLP) vaccines was investigated. Immunization with the HIV Env DNA vaccine induced a strong CD8 T cell response but relatively weak antibody response against the HIV Env whereas immunization with VLPs induced higher levels of antibody responses but little CD8 T cell response. Interestingly, immunization with a mixture the HIV Env DNA and VLP vaccines induced enhanced CD8 T cell and antibody responses. Further, it was observed that the mixing of DNA and VLP vaccines during immunization is necessary for augmenting induction of CD8 T cell responses and such augmentation of CD8 T cell responses was also observed by mixing the HIV Env DNA vaccine with control VLPs. These results show that immunization with a mixture of DNA and VLP vaccines combines advantages of both vaccine platforms for eliciting high levels of both antibody and CD8 T cell responses.
- Published
- 2010
42. Protection against lethal challenge by Ebola virus-like particles produced in insect cells
- Author
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Richard W. Compans, Jean L. Patterson, Kathleen M. Brasky, E. Ellen Schwegler, Chinglai Yang, Zhiyuan Wen, Ling Ye, Young Tae Ro, Anysha Ticer, Yuliang Sun, and Ricardo Carrion
- Subjects
viruses ,Lethal challenge ,Immunization, Secondary ,Viremia ,Spodoptera ,Antibodies, Viral ,medicine.disease_cause ,Virus ,Article ,Cell Line ,Microbiology ,Mice ,03 medical and health sciences ,Ebola virus ,Immune system ,Virus-like particle ,Neutralization Tests ,Virology ,medicine ,Animals ,Ebola Vaccines ,Antibody ,030304 developmental biology ,Mice, Inbred BALB C ,0303 health sciences ,Insect cell ,biology ,030306 microbiology ,virus diseases ,Virus like particle ,Hemorrhagic Fever, Ebola ,biochemical phenomena, metabolism, and nutrition ,Ebolavirus ,medicine.disease ,Survival Analysis ,3. Good health ,Vaccines, Virosome ,Immunization ,biology.protein ,Baculoviridae ,Vaccine - Abstract
Ebola virus-like particles (VLPs) were produced in insect cells using a recombinant baculovirus expression system and their efficacy for protection against Ebola virus infection was investigated. Two immunizations with 50 microg Ebola VLPs (high dose) induced a high level of antibodies against Ebola GP that exhibited strong neutralizing activity against GP-mediated virus infection and conferred complete protection of vaccinated mice against lethal challenge by a high dose of mouse-adapted Ebola virus. In contrast, two immunizations with 10 microg Ebola VLPs (low dose) induced 5-fold lower levels of antibodies against GP and these mice were not protected against lethal Ebola virus challenge, similar to control mice that were immunized with 50 microg SIV Gag VLPs. However, the antibody responses against GP were boosted significantly after a third immunization with 10 microg Ebola VLPs to similar levels as those induced by two immunizations with 50 microg Ebola VLPs, and vaccinated mice were also effectively protected against lethal Ebola virus challenge. Furthermore, serum viremia levels in protected mice were either below the level of detection or significantly lower compared to the viremia levels in control mice. These results show that effective protection can be achieved by immunization with Ebola VLPs produced in insect cells, which give high production yields, and lend further support to their development as an effective vaccine strategy against Ebola virus.
- Published
- 2008
43. Delivery of DNA HIV-1 Vaccine to the Liver Induces High and Long-lasting Humoral Immune Responses
- Author
-
Zdenek Hel, Chinglai Yang, Zina Moldoveanu, Richard W. Compans, Jiri Mestecky, Lea Novak, Milan Raska, Jan Novak, and Jadranka Bozja
- Subjects
Blotting, Western ,Immunization, Secondary ,Enzyme-Linked Immunosorbent Assay ,Biology ,CD8-Positive T-Lymphocytes ,HIV Envelope Protein gp120 ,Article ,DNA vaccination ,law.invention ,chemistry.chemical_compound ,Interferon-gamma ,Mice ,Immune system ,Antigen ,law ,Neutralization Tests ,Vaccines, DNA ,Animals ,Immunity, Mucosal ,Administration, Intranasal ,AIDS Vaccines ,Mice, Inbred BALB C ,Vaccines, Synthetic ,General Veterinary ,General Immunology and Microbiology ,Public Health, Environmental and Occupational Health ,Virology ,Vaccination ,Infectious Diseases ,chemistry ,Liver ,Humoral immunity ,Immunology ,Antibody Formation ,Injections, Intravenous ,Vagina ,biology.protein ,Recombinant DNA ,HIV-1 ,Molecular Medicine ,Electrophoresis, Polyacrylamide Gel ,Female ,Antibody ,DNA ,Plasmids - Abstract
The quality of immune responses induced by DNA vaccination depends on the site of DNA administration, the expression, and the properties of the encoded antigen. In the present study, we demonstrate that intravenous hydrodynamic HIV-1 envelope DNA injection resulted in high levels of expression of HIV-1 envelope antigen in the liver. When compared to the administration of DNA by i.n., i.d., i.m., and i.splenic routes, hydrodynamic vaccination induced, upon DNA boosting, levels of HIV-1 envelope-specific antibodies 40-fold higher than those elicited by the other routes tested. Hydrodynamic vaccination with 1 microg DNA induced higher humoral responses than 100 microg DNA given intramuscularly in the prime-boost regimen. High levels of envelope-specific IgG and IgA antibodies were induced in genital tract secretions after two doses of DNA followed by intranasal boosting with recombinant HIV-1 gp120 protein. Furthermore, two doses of 100 microg DNA generated interferon-gamma production in approximately 4.3+/-1.7% of CD8(+) splenocytes after in vitro stimulation with HIV-1 envelope peptides. These results demonstrate that DNA vaccines targeted to tissues with high proteosynthetic activity, such as the liver, results in enhanced immune responses.
- Published
- 2008
44. A naturally occurring deletion in its NS gene contributes to the attenuation of an H5N1 swine influenza virus in chickens
- Author
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Peirong Jiao, Huanliang Yang, Guohua Deng, Weiye Chen, Hualan Chen, Yoshihiro Kawaoka, Gongxun Zhong, Wenyan Cao, Zhigao Bu, Qiyun Zhu, Chinglai Yang, and Kangzhen Yu
- Subjects
viruses ,Immunology ,Mutant ,Orthomyxoviridae ,Virulence ,Chick Embryo ,Viral Nonstructural Proteins ,Recombinant virus ,medicine.disease_cause ,Kidney ,Microbiology ,Virus ,Lethal Dose 50 ,Mice ,Interferon ,Virology ,medicine ,Influenza A virus ,Animals ,Lung ,Sequence Deletion ,Mice, Inbred BALB C ,biology ,Influenza A Virus, H5N1 Subtype ,Brain ,biology.organism_classification ,Molecular biology ,Reverse genetics ,Insect Science ,Influenza in Birds ,Pathogenesis and Immunity ,Female ,Interferons ,Chickens ,Spleen ,medicine.drug - Abstract
In 2001 and 2003, we isolated two H5N1 viruses, A/swine/Fujian/1/01 (SW/FJ/01) and A/swine/Fujian/1/03 (SW/FJ/03), from pigs in Fujian Province, southern China. Genetically, these two viruses are similar, although the NS gene of the SW/FJ/03 virus has a 15-nucleotide deletion at coding positions 612 to 626. The SW/FJ/01 virus is highly lethal for chickens, whereas the SW/FJ/03 virus is nonpathogenic for chickens when administrated intravenously or intranasally. To understand the molecular basis for the difference in virulence, we used reverse genetics to create a series of single-gene recombinants of both viruses. We found that a recombinant virus containing the mutated NS gene from the SW/FJ/03 virus in the SW/FJ/01 virus background was completely attenuated in chickens. We also found that viruses expressing the mutant NS1 protein of SW/FJ/03 did not antagonize the induction of interferon (IFN) protein. Conversely, only the recombinant virus containing the wild-type SW/FJ/01 NS gene in the SW/FJ/03 background was lethal in chickens and antagonized IFN protein levels. Further, we proved that the NS1 genes of the two viruses differ in their stabilities in the host cells and in their abilities to interact with the chicken cleavage and polyadenylation specificity factor. These results indicate that the deletion of amino acids 191 to 195 of the NS1 protein is critical for the attenuation of the SW/FJ/03 virus in chickens and that this deletion affects the ability of the virus to antagonize IFN induction in host cells.
- Published
- 2007
45. Newcastle Disease Virus-Based Live Attenuated Vaccine Completely Protects Chickens and Mice from Lethal Challenge of Homologous and Heterologous H5N1 Avian Influenza Viruses▿
- Author
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Sen Hu, Chinglai Yang, Yong Wang, Kangzhen Yu, Jinying Ge, Yongping Jiang, Zhigao Bu, Guobing Tian, Jianzhong Shi, Yanbing Li, Hualan Chen, Xijun Wang, Zhiyuan Wen, and Guohua Deng
- Subjects
Attenuated vaccine ,animal structures ,biology ,Influenza vaccine ,Viral Vaccine ,viruses ,animal diseases ,Immunology ,Orthomyxoviridae ,virus diseases ,biology.organism_classification ,medicine.disease_cause ,Microbiology ,Virology ,Newcastle disease ,Influenza A virus subtype H5N1 ,Insect Science ,embryonic structures ,medicine ,Influenza A virus ,Pathogenesis and Immunity ,Mononegavirales - Abstract
H5N1 highly pathogenic avian influenza virus (HPAIV) has continued to spread and poses a significant threat to both animal and human health. Current influenza vaccine strategies have limitations that prevent their effective use for widespread inoculation of animals in the field. Vaccine strains of Newcastle disease virus (NDV), however, have been used successfully to easily vaccinate large numbers of animals. In this study, we used reverse genetics to construct a NDV that expressed an H5 subtype avian influenza virus (AIV) hemagglutinin (HA). Both a wild-type and a mutated HA open reading frame (ORF) from the HPAIV wild bird isolate, A/Bar-headed goose/Qinghai/3/2005 (H5N1), were inserted into the intergenic region between the P and M genes of the LaSota NDV vaccine strain. The recombinant viruses stably expressing the wild-type and mutant HA genes were found to be innocuous after intracerebral inoculation of 1-day-old chickens. A single dose of the recombinant viruses in chickens induced both NDV- and AIV H5-specific antibodies and completely protected chickens from challenge with a lethal dose of both velogenic NDV and homologous and heterologous H5N1 HPAIV. In addition, BALB/c mice immunized with the recombinant NDV-based vaccine produced H5 AIV-specific antibodies and were completely protected from homologous and heterologous lethal virus challenge. Our results indicate that recombinant NDV is suitable as a bivalent live attenuated vaccine against both NDV and AIV infection in poultry. The recombinant NDV vaccine may also have potential use in high-risk human individuals to control the pandemic spread of lethal avian influenza.
- Published
- 2006
46. Murine leukemia virus R Peptide inhibits influenza virus hemagglutinin-induced membrane fusion
- Author
-
David A. Steinhauer, Chinglai Yang, Zhu Nan Li, Min Li, Richard W. Compans, and Qizhi Yao
- Subjects
Protein Conformation ,viruses ,Recombinant Fusion Proteins ,Immunology ,Orthomyxoviridae ,Hemagglutinins, Viral ,Peptide ,Hemagglutinin Glycoproteins, Influenza Virus ,Microbiology ,Membrane Fusion ,Virus ,Mice ,Protein structure ,Virology ,Murine leukemia virus ,Animals ,Trypsin ,Gammaretrovirus ,chemistry.chemical_classification ,biology ,Structure and Assembly ,Lipid bilayer fusion ,Gene Products, env ,Hydrogen-Ion Concentration ,biology.organism_classification ,Fusion protein ,Molecular biology ,Leukemia Virus, Murine ,chemistry ,Insect Science ,Oligopeptides - Abstract
The cytoplasmic tail of the murine leukemia virus (MuLV) envelope (Env) protein is known to play an important role in regulating viral fusion activity. Upon removal of the C-terminal 16 amino acids, designated as the R peptide, the fusion activity of the Env protein is activated. To extend our understanding of the inhibitory effect of the R peptide and investigate the specificity of inhibition, we constructed chimeric influenza virus-MuLV hemagglutinin (HA) genes. The influenza virus HA protein is the best-studied membrane fusion model, and we investigated the fusion activities of the chimeric HA proteins. We compared constructs in which the coding sequence for the cytoplasmic tail of the influenza virus HA protein was replaced by that of the wild-type or mutant MuLV Env protein or in which the cytoplasmic tail sequence of the MuLV Env protein was added to the HA cytoplasmic domain. Enzyme-linked immunosorbent assays and Western blot analysis showed that all chimeric HA proteins were effectively expressed on the cell surface and cleaved by trypsin. In BHK21 cells, the wild-type HA protein had a significant ability after trypsin cleavage to induce syncytium formation at pH 5.1; however, neither the chimeric HA protein with the full-length cytoplasmic tail of MuLV Env nor the full-length HA protein followed by the R peptide showed any syncytium formation. When the R peptide was truncated or mutated, the fusion activity was partially recovered in the chimeric HA proteins. A low-pH conformational-change assay showed that similar conformational changes occurred for the wild-type and chimeric HA proteins. All chimeric HA proteins were capable of promoting hemifusion and small fusion pore formation, as shown by a dye redistribution assay. These results indicate that the R peptide of the MuLV Env protein has a sequence-dependent inhibitory effect on influenza virus HA protein-induced membrane fusion and that the inhibitory effect occurs at a late stage in fusion pore enlargement.
- Published
- 2006
47. Chimeric influenza virus hemagglutinin proteins containing large domains of the Bacillus anthracis protective antigen: protein characterization, incorporation into infectious influenza viruses, and antigenicity
- Author
-
Zhu-Nan Li, Scott N. Mueller, Chinglai Yang, Zhigao Bu, Ling Ye, Rafi Ahmed, and David A. Steinhauer
- Subjects
Signal peptide ,Models, Molecular ,Anthrax toxin ,Immunology ,Orthomyxoviridae ,Bacterial Toxins ,Genetic Vectors ,Hemagglutinin (influenza) ,Hemagglutinin Glycoproteins, Influenza Virus ,Vaccinia virus ,Chick Embryo ,Biology ,medicine.disease_cause ,Antibodies, Viral ,Microbiology ,Virus ,law.invention ,Mice ,law ,Virology ,Vaccines and Antiviral Agents ,Influenza A virus ,medicine ,Animals ,Humans ,Administration, Intranasal ,Recombination, Genetic ,Antigens, Bacterial ,Vaccines, Synthetic ,Vaccination ,biology.organism_classification ,Fusion protein ,Antibodies, Bacterial ,Mice, Inbred C57BL ,Influenza Vaccines ,Insect Science ,Bacillus anthracis ,biology.protein ,Recombinant DNA ,Female ,Plasmids - Abstract
Large polypeptides of the Bacillus anthracis protective antigen (PA) were inserted into an influenza A virus hemagglutinin glycoprotein (HA), and the chimeric proteins were functionally characterized and incorporated into infectious influenza viruses. PA domain 1′, the region responsible for binding to the other toxin components, the lethal factor and edema factor, and domain 4, the receptor binding domain (RBD), were inserted at the C-terminal flank of the HA signal peptide and incorporated into the HA1 subunit of HA. The chimeric proteins, designated as LEF/HA (90 amino acid insertion) and RBD/HA (140 amino acid insertion), were initially analyzed following expression using recombinant vaccinia viruses. Both chimeric proteins were shown to display functional phenotypes similar to that of the wild-type HA. They transport to the cell surface, can be cleaved into the HA1 and HA2 subunits by trypsin to activate membrane fusion potential, are able to undergo the low-pH-induced conformational changes required for fusion, and are capable of inducing the fusion process. We were also able to generate recombinant influenza viruses containing the chimeric RBD/HA and LEF/HA genes, and the inserted PA domains were maintained in the HA gene segments following several passages in MDCK cells or embryonated chicken eggs. Furthermore, DNA immunization of mice with plasmids that express the chimeric RBD/HA and LEF/HA proteins, and the recombinant viruses containing them, induced antibody responses against both the HA and PA components of the protein. These approaches may provide useful tools for vaccines against anthrax and other diseases.
- Published
- 2005
48. Ebola virus-like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutralizing antibodies
- Author
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Richard W. Compans, Chinglai Yang, Yuliang Sun, Ling Ye, Michael K. Lo, Zhigao Bu, Soumaya Bennouna, Jianguo Lin, Qingyang Wu, and Bali Pulendran
- Subjects
Gene Expression Regulation, Viral ,Insecta ,viruses ,Sf9 ,medicine.disease_cause ,Antibodies, Viral ,complex mixtures ,Virus ,Cell Line ,Viral Matrix Proteins ,Mice ,VP40 ,Virus-like particle ,Neutralization Tests ,Virology ,medicine ,Animals ,Humans ,Neutralizing antibody ,Ebola virus ,biology ,virus diseases ,Dendritic cell ,Dendritic Cells ,biochemical phenomena, metabolism, and nutrition ,Ebolavirus ,Immunoglobulin G ,Ebola ,biology.protein ,Cytokine secretion ,Female ,Vaccine ,Baculoviridae - Abstract
Recombinant baculoviruses (rBV) expressing Ebola virus VP40 (rBV-VP40) or GP (rBV-GP) proteins were generated. Infection of Sf9 insect cells by rBV-VP40 led to assembly and budding of filamentous particles from the cell surface as shown by electron microscopy. Ebola virus-like particles (VLPs) were produced by coinfection of Sf9 cells with rBV-VP40 and rBV-GP, and incorporation of Ebola GP into VLPs was demonstrated by SDS-PAGE and Western blot analysis. Recombinant baculovirus infection of insect cells yielded high levels of VLPs, which were shown to stimulate cytokine secretion from human dendritic cells similar to VLPs produced in mammalian cells. The immunogenicity of Ebola VLPs produced in insect cells was evaluated by immunization of mice. Analysis of antibody responses showed that most of the GP-specific antibodies were of the IgG2a subtype, while no significant level of IgG1 subtype antibodies specific for GP was induced, indicating the induction of a Th1-biased immune response. Furthermore, sera from Ebola VLP immunized mice were able to block infection by Ebola GP pseudotyped HIV virus in a single round infection assay, indicating that a neutralizing antibody against the Ebola GP protein was induced. These results show that production of Ebola VLPs in insect cells using recombinant baculoviruses represents a promising approach for vaccine development against Ebola virus infection.
- Published
- 2005
49. Modified HIV envelope proteins with enhanced binding to neutralizing monoclonal antibodies
- Author
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Chinglai Yang, Richard W. Compans, Ling Ye, Lizheng Guo, Fu-Shi Quan, Sang-Moo Kang, and Chunzi Huang
- Subjects
Models, Molecular ,Glycosylation ,medicine.drug_class ,Protein Conformation ,Mutant ,Biology ,Monoclonal antibody ,Giant Cells ,Epitope ,Cell Line ,Antigen-Antibody Reactions ,03 medical and health sciences ,chemistry.chemical_compound ,Epitopes ,0302 clinical medicine ,Protein structure ,Virology ,medicine ,Animals ,Humans ,Binding site ,030304 developmental biology ,Modified envelope proteins ,0303 health sciences ,Wild type ,Antibodies, Monoclonal ,Gene Products, env ,Human immunodeficiency virus (HIV) ,Molecular biology ,Enhanced antigenicity ,Recombinant Proteins ,3. Good health ,chemistry ,CD4 Antigens ,biology.protein ,HIV-1 ,Mutagenesis, Site-Directed ,Antibody ,030215 immunology - Abstract
The target for neutralizing antibodies against human immunodeficiency virus (HIV) is the trimeric Env protein on the native virion. Conserved neutralizing epitopes of receptor binding sites are located in the recessed core of the Env protein, partially masked by glycosylations and variable loops. In this study, we have investigated the effects of modifications of the HIV Env protein by glycosylation site mutations, deletions of variable loops, or combinations of both types of mutations on their protein functions and reactivities with neutralizing antibodies. Modified Env proteins were expressed in insect or mammalian cells, and their reactivity with epitope-specific broadly neutralizing monoclonal antibodies (Mabs) was determined by flow cytometry. A unique mutant designated 3G with mutations in three glycosylation motifs within the V3/C3 domains surrounding the CD4 binding site showed higher levels of binding to most broadly neutralizing Mabs (b12 and 2F5) in both insect and mammalian expression systems. Mutants with a deletion of both V1 and V2 loop domains or with a unique combination of both types of mutations also bound to most neutralizing Mabs at higher levels compared to the wild-type control. Most mutants maintained the ability to bind CD4 and to induce syncytium formation at similar or higher levels as compared to that of the wild-type Env protein, except for a mutant with a combination of variable loop deletions and deglycosylation mutations. Our study suggests that modified HIV Env proteins with reduced glycosylation in domains surrounding the CD4 binding site or variable loop-deleted mutants expose important neutralizing epitopes at higher levels than wild type and may provide novel vaccine immunogens.
- Published
- 2004
50. Surface Stability and Immunogenicity of the Human Immunodeficiency Virus Envelope Glycoprotein: Role of the Cytoplasmic Domain
- Author
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Chinglai Yang, Ling Ye, Zhigao Bu, Andrei N. Vzorov, Dahnide Taylor, and Richard W. Compans
- Subjects
Gene Expression Regulation, Viral ,Cytoplasm ,Immunoprecipitation ,viruses ,Immunology ,Amino Acid Motifs ,Biology ,CD8-Positive T-Lymphocytes ,HIV Antibodies ,Microbiology ,Virus ,Mice ,Viral envelope ,Virology ,Vaccines and Antiviral Agents ,Animals ,Humans ,Tyrosine ,Gene ,chemistry.chemical_classification ,Mice, Inbred BALB C ,Immunogenicity ,virus diseases ,Gene Products, env ,Endocytosis ,chemistry ,Insect Science ,DNA, Viral ,Mutation ,HIV-1 ,DNA construct ,Female ,Immunization ,Glycoprotein - Abstract
The effects of two functional domains, the membrane-proximal YXXΦ motif and the membrane-distal inhibitory sequence in the long cytoplasmic tail of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env), on immunogenicity of the envelope protein were investigated. Genes with codons optimized for mammalian expression were synthesized for the HIV 89.6 Env and a truncated Env with 50 amino acids in the cytoplasmic domain to delete the membrane distal inhibitory sequence for surface expression. Additional genes were generated in which the tyrosine residue in the YXXΦ motif was changed into a serine. Pulse-chase radioactive labeling and immunoprecipitation studies indicated that both domains can mediate endocytosis of the HIV Env, and removal of both domains is required to enhance HIV Env protein surface stability. Analysis of immune responses induced by DNA immunization of mice showed that the DNA construct for the mutant Env exhibiting enhanced surface stability induced significantly higher levels of antibody responses against the HIV Env protein. Our results suggest that the HIV Env cytoplasmic domain may play important roles in virus infection and pathogenesis by modulating its immunogenicity.
- Published
- 2004
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