OBJECTIVETo prepare the 4 candidate vaccine strains of H5N1 avian influenza virus isolated in China.

METHODSRecombinant viruses were rescued using reverse genetics. Neuraminidase (NA) and hemagglutinin (HA) segments of the A/Xinjiang/1/2006, A/Guangxi/1/2009, A/Hubei/1/2010, and A/Guangdong/1/2011 viruses were amplified by RT-PCR. Multibasic amino acid cleavage site of HA was removed and ligated into the pCIpolI vector for virus rescue. The recombinant viruses were evaluated by trypsin dependent assays. Their embryonate survival and antigenicity were compared with those of the respective wild-type viruses.

RESULTSThe 4 recombinant viruses showed similar antigenicity compared with wild-type viruses, chicken embryo survival and trypsin-dependent characteristics.

CONCLUSIONThe 4 recombinant viruses rescued using reverse genetics meet the criteria for classification of low pathogenic avian influenza strains, thus supporting the use of them for the development of seeds and production of pre-pandemic vaccines.

Animals ; Chick Embryo ; Chickens ; China ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; metabolism ; Influenza A Virus, H5N1 Subtype ; immunology ; Influenza Vaccines ; immunology ; Influenza in Birds ; prevention & control ; virology ; Neuraminidase ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Vaccines, Synthetic ; immunology

Infectious bronchitis virus (IBV) poses a severe threat to the poultry industry and causes heavy economic losses worldwide. Vaccination is the most effective method of preventing infection and controlling the spread of IBV, but currently available inactivated and attenuated virus vaccines have some disadvantages. We developed a chimeric virus-like particle (VLP)-based candidate vaccine for IBV protection. The chimeric VLP was composed of matrix 1 protein from avian influenza H5N1 virus and a fusion protein neuraminidase (NA)/spike 1 (S1) that was generated by fusing IBV S1 protein to the cytoplasmic and transmembrane domains of NA protein of avian influenza H5N1 virus. The chimeric VLPs elicited significantly higher S1-specific antibody responses in intramuscularly immunized mice and chickens than inactivated IBV viruses. Furthermore, the chimeric VLPs induced significantly higher neutralization antibody levels than inactivated H120 virus in SPF chickens. Finally, the chimeric VLPs induced significantly higher IL-4 production in mice. These results demonstrate that chimeric VLPs have the potential for use in vaccines against IBV infection.
Animals ; Antibodies, Viral/blood ; *Chickens ; Chimera/genetics/immunology ; Coronavirus Infections/prevention & control/*veterinary/virology ; Female ; *Immunity, Innate ; Infectious bronchitis virus/genetics/*immunology ; Influenza A Virus, H5N1 Subtype/genetics/immunology ; Injections, Intramuscular/veterinary ; Mice ; Mice, Inbred BALB C ; Neuraminidase/genetics ; Poultry Diseases/*prevention & control/virology ; Recombinant Fusion Proteins/genetics/immunology ; Spike Glycoprotein, Coronavirus/genetics/*immunology ; Vaccines, Synthetic/administration & dosage/genetics/immunology ; Vaccines, Virus-Like Particle/administration & dosage/genetics/*immunology ; Viral Proteins/genetics

Nucleoprotein (NP) of influenza virus is highly conserved and type-specific. NP can trigger strong cell-mediated immune responses in host and is involved in the protection against the challenges with different subtype influenza viruses. Here, NP of an avian H5N1 (A/Hubei/1/2010, HB) was expressed by baculovirus surface-display technology and its immunogenicity as well as protective mechanism was investigated in mice infection model. Western blot and immunolabeled electron microscopy assay showed NP was displayed on baculovirus surface. ELISA results showed NP could induce high level of anti-NP IgG in the sera from NP-Bac-inoculated mice. Two cellular immune peptides (NP57-74 IQNSITIERMVLSAFDER and NP441-458 RTEIIKMMESARPEDLSF) were identified by IFN-gamma ELISPOT assay. NP57-66 and NP441-450 and NP protein could be able to trigger the activation of CD4+ and CD8+ T cells, and the response of CD8+ T was more predominant. The challenge study of mice-adapted virus A/PR/8/34 (H1N1) showed that NP-Bac could reduce viral load and attenuate the damage to lung tissue. 50% protection ratio against the virus could be detected.
Animals ; Antibodies, Viral ; immunology ; Baculoviridae ; genetics ; metabolism ; Cross Protection ; Enzyme-Linked Immunospot Assay ; Female ; Humans ; Immunity, Cellular ; Influenza A Virus, H1N1 Subtype ; genetics ; immunology ; Influenza A Virus, H5N1 Subtype ; genetics ; immunology ; Influenza, Human ; immunology ; virology ; Mice ; Mice, Inbred BALB C ; RNA-Binding Proteins ; genetics ; immunology ; T-Lymphocytes ; immunology ; Viral Core Proteins ; genetics ; immunology

This study aimed to develop an effective experimental vaccine against highly pathogenic H5N1 Avian Influenza (HPAI) virus and to optimize their immunization programs. As reported previously, various DNA-based or recombinant vaccinia viral(Tiantan)-based H5N1 vaccine candidates, which containing a single cistronic construct (HAop, or NAop) or a bicistronic construct (HAop/M2 or NAop/M1) of H5N1 influenza virus (Anhui strain) were constructed and characterized in our lab. In this study, we further analysed the immunogenicity in mice of these vaccine candidates by various protocols (single or combined immunization). Our results showed that: comparing with immunization with DNA-based or rTTV-based H5N1 vaccine only, combined DNA-based with rTTV-based H5N1 vaccine immunization via prime-boost strategy enhanced immune response significantly against multi-H5N1 antigens detected by hemagglutination inhibition (HI) assay, NA- or M1- or M2-specific antibody detection, and micro-neutralizing antibody test and IFN-gamma ELISpot assay. Priming with DNA-based vaccine induced higher level of humoral response against HA or NA antigen than priming with rTTV-based vaccine; In contract, M1 and M2-specific antibody levels were higher among that of priming with rTTV -based vaccine. These findings provide a basis for further development of novel H5N1 vaccines and for the optimization of the immunization programs of combined multi-antigens vaccine candidates.
Animals ; Antigens, Viral ; genetics ; immunology ; Female ; Immunization ; methods ; Influenza A Virus, H5N1 Subtype ; genetics ; immunology ; Influenza Vaccines ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Vaccination ; methods ; Vaccines, DNA ; genetics ; immunology ; Vaccines, Synthetic ; genetics ; immunology ; Vaccinia ; genetics ; immunology

In order to evaluate the response to vector-expressed M1 and HA genes of influenza virus in mice, we prepared recombinant plasmid pStar-M1/HA and recombinant adenovirus Ad-M1/HA containing both the full-length matrix protein 1(M1) and hemagglutinin (HA) genes of human H5N1 influenza virus strain A/Anhui/1/2005. We then combined the DNA vaccine and adenoviral vaccine in immunization of BALB/c mice with a prime-boost regime. We immunized the mice with DNA vaccine at day 0 and 28 and with recombinant adenoviral vaccines at day 14 and 42. We took blood samples before each injection and 14 days after the final injection for detection of humoral immune responses. At day 56, we sacrificed the mice and collected splenocytes for detection of cellular immune responses. ELISA and hemagglutination inhibition (HI) assay showed that specific IgG Abs against H5N1 influenza virus was induced in serum of the immunized mice. ELISPOT results confirmed that the specific cellular immune responses were successfully induced against the M1 and HA proteins of H5N1 influenza virus. This study provides new strategy for development of novel influenza vaccines.
Adenoviridae ; genetics ; metabolism ; Animals ; Antibodies, Viral ; blood ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; immunology ; Immunization ; Influenza A Virus, H5N1 Subtype ; immunology ; Influenza Vaccines ; immunology ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; genetics ; immunology ; Vaccines, DNA ; immunology ; Viral Matrix Proteins ; genetics ; immunology

Secretory IgA (SIgA) antibodies in external secretions play an important role in mucosal immune response. Polymeric SIgA was advantageous over monomeric IgA (mIgA) and IgG in several aspects. To express secretory IgA antibody against H5N1 virus, we constructed the secretory component and immunoglobulin J expressing plasmids and co-transfected the plasmids into the Chinese hamster ovary cells (CHO) stably expressing immunoglobulin A. Then we used Zeocin to select the positive clone cells, monoclonal cells stably secreting SIgA was screened through fold dilution method at last. The SIgA antibody secreted from the CHO cells was confirmed by Western blotting, which demonstrated that we had got the complete SIgA molecular. The successful expression of this polymeric anti-H5N1 SIgA in CHO cells will contribute to the production of recombinant SIgA as a preventive agent for infectious disease control.
Animals ; Antibodies, Viral ; biosynthesis ; genetics ; CHO Cells ; Cloning, Molecular ; Cricetinae ; Cricetulus ; Genetic Vectors ; Immunoglobulin A ; immunology ; Immunoglobulin A, Secretory ; biosynthesis ; genetics ; immunology ; Influenza A Virus, H5N1 Subtype ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

In our previous study, a panel of 52 broadly cross-reactive H5-specific monoclonal antibodies (MAbs) were generated and characterized. The 13D4, one of these MAbs, has been demonstrated to protect mice against lethal challenge by 4 strains of H5N1 avian influenza virus representing the currently prevailing genetic populations, clades 1, 2.1, 2.2, and 2.3. Here, we further cloned the gene of the 13D4 MAb and constructed a single-chain variable fragment. Then, the 13D4 single-chain antibody (scFv) was expressed in secretory maner in Pichia pastoris. The supernatant of the culture was concentrated and subjected to ammonium sulfate precipitation. The purity of the 13D4 scFv was around 90% in SDS-PAGE following ion-exchange chromatography. We further investigated its binding property using hemagglutination inhibition (HI) test and blocking ELISA. The results indicated that the 13D4 scFv shared the same binding sites and comparable HI titer with the prototype murine 13D4 Mab. In conclusion, an anti-H5 single-chain wide-spectrum neutralizing antibody is prepared successfully in yeast system.
Antibodies, Viral ; genetics ; Hemagglutination Inhibition Tests ; Immunoglobulin Fragments ; genetics ; immunology ; Influenza A Virus, H5N1 Subtype ; immunology ; Pichia ; genetics ; Single-Chain Antibodies ; genetics ; immunology

We developed vectors expressing two antigen of H5N1 influenza virus. Based on the human H5N1 avian influenza virus strain A/Anhui/1/2005 isolated in China, we amplified the matrix protein 2 (M2) and Hemagglutinin (HA) genes by PCR and subcloned them into pStar vector to construct two genes co-expressing recombinant DNA vaccine pStar-M2/HA. After transfection of 293 cells with the plasmid, we confirmed with indirect immunofluorescence assay (IFA) that M2 and HA genes cloned on plasmid pStar co-expressed successfully. Using Ad-Easy adenovirus vector system, by homologous recombination in bacteria and packaging in 293 cells, we constructed two recombinant adenoviruses, namely Ad-M2 and Ad-HA. After infection of 293 cells with the recombinant adenoviruses, we confirmed with IFA that M2 and HA genes cloned into adenoviruses expressed successfully. We then combined the recombinant DNA vaccine and adenoviral vector vaccines in immunization of BALB/c mice with a prime-boost regime. On day 0 and day 28, we immunized the mice with DNA vaccine and on day 14 and day 42, with recombinant adenovirus vaccines. We took blood samples before each injection and 14 days after the final injection. On day 56, we collected splenocytes from the mice. ELISA and hemagglutination inhibition (HI) assay showed that the vaccines successfully induced specific IgG antibodies against HA protein in serum of the immunized mice. ELISPOT confirmed that the vaccines successfully induced the special cellular immune response to M2 and HA protein of H5N1 influenza virus. The study on combined immunization with M2 and HA genes provided basis for development of novel influenza vaccine.
Adenoviridae ; genetics ; metabolism ; Animals ; Female ; Genetic Vectors ; genetics ; Hemagglutinin Glycoproteins, Influenza Virus ; biosynthesis ; genetics ; Influenza A Virus, H5N1 Subtype ; genetics ; immunology ; Influenza Vaccines ; immunology ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Vaccination ; Vaccines, DNA ; immunology ; Viral Matrix Proteins ; biosynthesis ; genetics

This study aims to develop inexpensive and effective experimental vaccines against highly pathogenic H5N1 Avian Influenza (HPAI) virus and to optimize their immunization programs. To this end, we first synthesized the codon-optimized hemagglutinin gene (HAop) and neuraminidase gene (NAop), both of which were derived from a H5N1 virus (Anhui strain), and constructed successfully the DNA vaccines containing a single cistronic construct (HAwt, HAop, or NAop) or a bicistronic construct (HAop/M2 or NAop/M1) of H5N1 influenza virus origin. Their expression was confirmed by indirect immunofluorescent assay (IFA) and Western blotting. Then twice vaccination of mice with the DNA vaccines by injection intramuscularly or in vivo electroporation (EP) via two different routes was evaluated and analyzed by hemagglutination inhibition (HI) assay, NA-specific antibody detection, micro-neutralizing antibody test and IFN-gamma ELISpot assay. Our results showed that the DNA vaccines with coden-optimized HAop and NAop constructs could quickly elicit a strong immune response by in vivo EP, especially the cellular immune response against HA and NA; the in vivo EP via intradermal route induced stronger humoral immune responses than those via intramuscular route. Our findings will pave a way for further development of novel DNA-based H5N1 vaccine and for the optimization of the immunization programs of DNA vaccine.
Animals ; Antigens, Viral ; immunology ; Codon ; genetics ; Electroporation ; Female ; Humans ; Influenza A Virus, H5N1 Subtype ; immunology ; Mice ; Mice, Inbred BALB C ; Vaccination ; methods ; Vaccines, DNA ; genetics ; immunology ; metabolism ; Viral Structural Proteins ; immunology

Influenza vaccine strains have been traditionally developed by annual reassortment between vaccine donor strain and the epidemic virulent strains. The classical method requires screening and genotyping of the vaccine strain among various reassortant viruses, which are usually laborious and time-consuming. Here we developed an efficient reverse genetic system to generate the 6:2 reassortant vaccine virus from cDNAs derived from the influenza RNAs. Thus, cDNAs of the two RNAs coding for surface antigens, haemagglutinin and neuraminidase from the epidemic virus and the 6 internal genes from the donor strain were transfected into cells and the infectious viruses of 6:2 defined RNA ratio were rescued. X-31 virus (a high-growth virus in embryonated eggs) and its cold-adapted strain X-31 ca were judiciously chosen as donor strains for the generation of inactivated vaccine and live-attenuated vaccine, respectively. The growth properties of these recombinant viruses in embryonated chicken eggs and MDCK cell were indistinguishable as compared to those generated by classical reassortment process. Based on the reverse genetic system, we generated 6 + 2 reassortant avian influenza vaccine strains corresponding to the A/Chicken/Korea/MS96 (H9N2) and A/Indonesia/5/2005 (H5N1). The results would serve as technical platform for the generation of both injectable inactivated vaccine and the nasal spray live attenuated vaccine for the prevention of influenza epidemics and pandemics.
Animals ; Chick Embryo ; Chickens ; Genetic Engineering ; Hemagglutinins, Viral/genetics/metabolism ; Humans ; Influenza A Virus, H5N1 Subtype/*genetics/immunology ; Influenza A Virus, H9N2 Subtype/*genetics/immunology ; Influenza Vaccines/*genetics/metabolism ; Influenza in Birds/immunology/virology ; Influenza, Human/immunology/*prevention & control/virology ; Neuraminidase/genetics/metabolism ; Transgenes ; Vaccines, Attenuated/*genetics/metabolism ; Viral Proteins/genetics/metabolism