Publications by authors named "Kunyu Wu"

5 Publications

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First Phase I human clinical trial of a killed whole-HIV-1 vaccine: demonstration of its safety and enhancement of anti-HIV antibody responses.

Retrovirology 2016 Nov 28;13(1):82. Epub 2016 Nov 28.

Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, The University of Western Ontario, 1400 Western Road, London, ON, N6G 2V4, Canada.

Background: Vaccination with inactivated (killed) whole-virus particles has been used to prevent a wide range of viral diseases. However, for an HIV vaccine this approach has been largely negated due to inherent safety concerns, despite the ability of killed whole-virus vaccines to generate a strong, predominantly antibody-mediated immune response in vivo. HIV-1 Clade B NL4-3 was genetically modified by deleting the nef and vpu genes and substituting the coding sequence for the Env signal peptide with that of honeybee melittin signal peptide to produce a less virulent and more replication efficient virus. This genetically modified virus (gmHIV-1) was inactivated and formulated as a killed whole-HIV vaccine, and then used for a Phase I human clinical trial (Trial Registration: Clinical Trials NCT01546818). The gmHIV-1 was propagated in the A3.01 human T cell line followed by virus purification and inactivation with aldrithiol-2 and γ-irradiation. Thirty-three HIV-1 positive volunteers receiving cART were recruited for this observer-blinded, placebo-controlled Phase I human clinical trial to assess the safety and immunogenicity.

Results: Genetically modified and killed whole-HIV-1 vaccine, SAV001, was well tolerated with no serious adverse events. HIV-1-specific PCR showed neither evidence of vaccine virus replication in the vaccine virus-infected human T lymphocytes in vitro nor in the participating volunteers receiving SAV001 vaccine. Furthermore, SAV001 with adjuvant significantly increased the pre-existing antibody response to HIV-1 proteins. Antibodies in the plasma of vaccinees were also found to recognize HIV-1 envelope protein on the surface of infected cells as well as showing an enhancement of broadly neutralizing antibodies inhibiting tier I and II of HIV-1 B, D, and A subtypes.

Conclusion: The killed whole-HIV vaccine, SAV001, is safe and triggers anti-HIV immune responses. It remains to be determined through an appropriate trial whether this immune response prevents HIV infection.
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http://dx.doi.org/10.1186/s12977-016-0317-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5126836PMC
November 2016

Creation of matrix protein gene variants of two serotypes of vesicular stomatitis virus as prime-boost vaccine vectors.

J Virol 2015 Jun 8;89(12):6338-51. Epub 2015 Apr 8.

Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada

Unlabelled: To take advantage of live recombinant vesicular stomatitis viruses (rVSVs) as vaccine vectors for their high yield and for their induction of strong and long-lasting immune responses, it is necessary to make live vaccine vectors safe for use without losing their immunogenicity. We have generated safer and highly efficient recombinant VSV vaccine vectors by combining the M51R mutation in the M gene of serotype VSV-Indiana (VSVInd) with a temperature-sensitive mutation (tsO23) of the VSVInd Orsay strain. In addition, we have generated two new serotype VSV-New Jersey (VSVNJ) vaccine vectors by combining M48R and M51R mutations with G22E and L110F mutations in the M gene, rVSVNJ(G22E M48R M51R) [rVSVNJ(GMM)] and VSVNJ(G22E M48R M51R L110F) [rVSVNJ(GMML)]. The combined mutations G21E, M51R, and L111F in the M protein of VSVInd significantly reduced the burst size of the virus by up to 10,000-fold at 37°C without affecting the level of protein expression. BHK21 cells and SH-SY5Y human neuroblastoma cells infected with rVSVInd(GML), rVSVNJ(GMM), and rVSVNJ(GMML) showed significantly reduced cytopathic effects in vitro at 37°C, and mice injected with 1 million infectious virus particles of these mutants into the brain showed no neurological dysfunctions or any other adverse effects. In order to increase the stability of the temperature-sensitive mutant, we have replaced the phenylalanine with alanine. This will change all three nucleotides from UUG (leucine) to GCA (alanine). The resulting L111A mutant showed the temperature-sensitive phenotype of rVSVInd(GML) and increased stability. Twenty consecutive passages of rVSVInd(GML) with an L111A mutation did not convert back to leucine (UUG) at position 111 in the M protein gene.

Importance: Recombinant vesicular stomatitis viruses as live vaccine vectors are very effective in expressing foreign genes and inducing adaptive T cell and B cell immune responses. As with any other live viruses in humans or animals, the use of live rVSVs as vaccine vectors demands the utmost safety. Our strategy to attenuate rVSVInd by utilizing a temperature-sensitive assembly-defective mutation of L111A and combining it with an M51R mutation in the M protein of rVSVInd significantly reduced the pathogenicity of the virus while maintaining highly effective virus production. We believe our new temperature-sensitive M gene mutant of rVSVInd(GML) and M gene mutants of rVSVNJ(GMM) and rVSVNJ(GMML) add excellent vaccine vectors to the pool of live viral vectors.
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http://dx.doi.org/10.1128/JVI.00222-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4474318PMC
June 2015

Genetically modified VSV(NJ) vector is capable of accommodating a large foreign gene insert and allows high level gene expression.

Virus Res 2013 Jan 30;171(1):168-77. Epub 2012 Nov 30.

Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, Siebens-Drake Research Institute, Western University, London, ON N6G 2V4, Canada.

It is desirable to develop a RNA virus vector capable of accommodating large foreign genes for high level gene expression. Vesicular stomatitis virus (VSV) has been used as a gene expression vector, especially Indiana serotype (VSV(Ind)), but less with New Jersey serotype (VSV(NJ)). Here, we report constructions of genetically modified rVSV(NJ) vector carrying various lengths of human hepatitis C virus (HCV) non-structural (NS) protein genes, level of inserted gene expression and characterization of rVSV(NJ). We modified the M gene of VSV(NJ) by changing methionine to arginine at positions 48 and 51 (rVSV(NJ)-M) (Kim and Kang, 2007) for construction of rVSV(NJ) with various lengths of HCV non-structural genes. The NS polyprotein genes of HCV were inserted between the G and L genes of the rVSV(NJ)-M vector, and recombinant VSV(NJ)-M viruses with HCV gene inserts were recovered by the reverse genetics. The recombinant VSV(NJ)-M vector with the HCV NS genes express high levels of all different forms of the NS proteins. The electron microscopic examination showed that lengths of recombinant VSV(NJ)-M without gene of interests, VSV(NJ)-M with a gene of HCV NS3 and NS4A (VSV(NJ)-M-NS3/4A), VSV(NJ)-M with a gene of HCV NS4AB plus NS5AB (VSV(NJ)-M-NS4AB/5AB), and VSV(NJ)-M carrying a gene of HCV NS3, NS4AB, and NS5AB (VSV(NJ)-M-NS3/4AB/5AB) were 172±10.5 nm, 201±12.5 nm, 226±12.9 nm, and 247±18.2 nm, respectively. The lengths of recombinant VSVs increased approximately 10nm by insertion of 1kb of foreign genes. The diameter of these recombinant viruses also increased slightly by longer HCV gene inserts. Our results showed that the recombinant VSV(NJ)-M vector can accommodate as much as 6000 bases of the foreign gene. We compared the magnitude of the IFN induction in mouse fibroblast L(Y) cells infected with rVSV(NJ) wild type and rVSV(NJ) M mutant viruses and show that the rVSV(NJ) M mutant virus infection induced a higher level of the IFN-β compare to the wild type virus. In addition, we showed that the NS protein expression level in IFN-incompetent cells (Mouse-L) infected with rVSV(NJ)-M viruses was higher than in IFN-competent L(Y) cells. In addition, we confirmed that HCV NS protein genes were expressed and properly processed. We also confirmed that NS3 protein expressed from the rVSV(NJ)-M cleaves NS polyprotein at junctions and that NS4A plays an important role as a co-factor for NS3 protease to cleave at the NS4B/5A site and at the NS5A/5B site.
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http://dx.doi.org/10.1016/j.virusres.2012.11.007DOI Listing
January 2013

Expression and processing of human immunodeficiency virus type 1 gp160 using the vesicular stomatitis virus New Jersey serotype vector system.

J Gen Virol 2009 May 4;90(Pt 5):1135-1140. Epub 2009 Mar 4.

Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, Siebens-Drake Research Institute, The University of Western Ontario, London, ON N6G 2V4, Canada.

The Indiana serotype of vesicular stomatitis virus (VSV(IND)), but not the New Jersey serotype (VSV(NJ)), has been widely used as a gene expression vector. In terms of prime-boost-based vaccine strategies, it would be desirable to use two different VSV serotypes to avoid immunity against the priming viral vector. Here, we report that we have applied the VSV(NJ) vector system for expression of the env gene of human immunodeficiency virus type 1 (HIV-1). The HIV-1 env gene was inserted into the VSV(NJ) vector system at two different sites: between the P and M genes (NP-gp160-MGL) and between the G and L genes (NPMG-gp160-L). The HIV-1 env gene product, gp160, was efficiently expressed and processed in cells infected with either of these two recombinant VSV-HIV-1(gp160) viruses. In this study, we have investigated the applicability of the VSV(NJ) vector system for foreign gene expression.
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http://dx.doi.org/10.1099/vir.0.009019-0DOI Listing
May 2009

[Characterization of genome of A/Guangzhou/333/99(H9N2) virus].

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi 2002 Jun;16(2):142-5

Institute of Virology, Chinese Academy of Preventive Medicine, Beijing 100052, China.

Background: To understand the characterization of genome of a strain of avian influenza A H9N2 virus repeatedly isolated from a child with influenza illness. Thereafter to reveal the origin of this H9N2 virus.

Methods: Viruses were passed in embryonated hen eggs and virion RNA was extracted from allantoic fluid and reverse transcribed to synthesize cDNA. cDNA was amplified by PCR and the PCR product was purified with a purification kit. Afterwards RNA sequence analysis was performed by dideoxynucleotide chain termination and a cloning method. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (Version 1.03) and Editseg (Version 3.69) softwares.

Results: Genome of A/Guangzhou/333/99 (H9N2) virus was closely related to avian influenza A H9N2 virus, but obvious difference from that of A/Duck/Hong Kong/Y439/97(H9N2) virus, as well as its genome did not include any RNA segment derived from human influenza A virus. However, the genes encoding the HA,NA,NP and NS proteins of A/Guangzhou/333/99 virus were derived from those of G9 lineage virus, the rest genes encoding the M and three polymerase (PB2,PB1 and PA) proteins were derived from G1 lineage strain.

Conclusions: A/Guangzhou/333/99 virus was a reassortant derived from reassortment betweenG9 and G1 lineages of avian influenzaA(H9N2) viruses. Therefore, the most possibility is that it is derived from avian influenza A virus directly. The results do not only demonstrate that avian influenza A (H9N2) virus could infect men, but also firstly prove that the genetic reassortment could be occurred between different genetic lineages of avian influenza A (H9N2) viruses in the nature.
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June 2002