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1.
Retrovirology ; 13(1): 82, 2016 Nov 28.
Article in English | MEDLINE | ID: mdl-27894306

ABSTRACT

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-1NL4-3) 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-1NL4-3 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-1NL4-3-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.


Subject(s)
AIDS Vaccines , Antibodies, Neutralizing/blood , HIV Antibodies/blood , HIV Infections/prevention & control , HIV-1/immunology , Immunogenicity, Vaccine , AIDS Vaccines/administration & dosage , AIDS Vaccines/adverse effects , AIDS Vaccines/immunology , Adult , Animals , Antibodies, Neutralizing/immunology , Bees/genetics , Female , Gene Products, nef/genetics , HIV Antibodies/immunology , HIV Infections/immunology , HIV-1/genetics , Human Immunodeficiency Virus Proteins/genetics , Humans , Male , Middle Aged , Protein Sorting Signals , Vaccines, Inactivated/administration & dosage , Vaccines, Inactivated/adverse effects , Vaccines, Inactivated/immunology , Viral Regulatory and Accessory Proteins/genetics , Young Adult
2.
Virus Res ; 171(1): 168-77, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23207069

ABSTRACT

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-ß 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.


Subject(s)
Gene Expression , Genetic Vectors/genetics , Vesicular stomatitis New Jersey virus/genetics , Animals , Carrier Proteins/metabolism , Cell Line , Cricetinae , Gene Order , Hepacivirus/genetics , Humans , Interferons/biosynthesis , Intracellular Signaling Peptides and Proteins , Mice , Mutation , Proteolysis , Vesicular stomatitis New Jersey virus/growth & development , Vesicular stomatitis New Jersey virus/ultrastructure , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virion/ultrastructure , Virus Replication
3.
Appl Microbiol Biotechnol ; 74(3): 609-16, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17139509

ABSTRACT

Recombinant vector pJLECit (8,232 bp) was constructed using citrate permease gene contained in the 3,919-bp fragment of plasmid pCM1 (8,280 bp) isolated from Lactococcus lactis subsp. lactis biovar diacetylactis NIAI N-7, repA and ori from pLU1, and pMB1 ori and the erythromycin resistance gene from pJIR418. Lactobacillus casei L-49-4 (plasmid-free mutant of strain L-49) harboring the constructed pJLECit converted citrate into diacetyl/acetoin. Citrate uptake rate of resting cells was the highest at pH 5.5 and 10 mM citrate concentration. Diacetyl formation activity by the cell-free extracts of Lb. casei L-49-4 (pJLECit) grown in de Man-Rogosa-Sharpe (MRS) broth was higher than that of cells grown in MRS broth without citrate. On the other hand, diacetyl reductase activity of cells grown in MRS broth was lower than that of cells grown in MRS broth without citrate.


Subject(s)
Bacterial Proteins/biosynthesis , Gene Expression , Lacticaseibacillus casei/genetics , Lactococcus lactis/enzymology , Organic Anion Transporters/biosynthesis , Acetoin/metabolism , Acetoin Dehydrogenase/analysis , Bacterial Proteins/genetics , Citric Acid/metabolism , Cloning, Molecular , Culture Media , DNA Helicases/genetics , Diacetyl/metabolism , Genetic Vectors , Hydrogen-Ion Concentration , Lacticaseibacillus casei/metabolism , Lactococcus lactis/genetics , Methyltransferases/genetics , Organic Anion Transporters/genetics , Plasmids/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics
4.
Plasmid ; 55(2): 128-34, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16330102

ABSTRACT

The complete nucleotide sequence of plasmid pLC494 isolated from Lactobacillus casei L-49 was determined. Plasmid pLC494 is an 8846-bp long circular molecule with a G+C content of 41.5%. Two putative open reading frames, ORF4 (282 amino acids) and ORF5 (169 amino acids), were identified as replication proteins A and B that revealed 100 and 99% similarity, respectively, with the replication proteins of plasmid pLA103 from Lactobacillus acidophilus TK8912. Upstream of ORF4 were the four repeat regions (three perfect 22-bp repeats and one imperfect motif), a putative ribosome binding site, a -10 region, and a -35 region. The shuttle vector pJLE4942 (5318 bp) was constructed using repA from pLC494, a multiple cloning site, ColE1 ori, the ori of gram-negative bacteria from vector pUC19, and the chloramphenicol resistance gene from pJIR418 as a selection marker. Transformation of several lactic acid bacteria with the vector pJLE4942 indicated that this vector might be useful as a genetic tool for the intestinal lactobacilli.


Subject(s)
Escherichia coli/genetics , Gastrointestinal Tract/microbiology , Genetic Vectors , Lacticaseibacillus casei/genetics , Plasmids/genetics , Sequence Analysis, DNA , Base Sequence , Cloning, Molecular , Humans , Molecular Sequence Data , Plasmids/chemistry , Transformation, Bacterial
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