RESUMO
The overwhelming majority of influenza vaccines are prepared with the use of chicken embryo allantoic fluid. The presence of ovalbumin (this protein constitutes >60% total protein in the allantoic fluid) in the vaccine can lead to severe allergy. Hence, effective reduction of ovalbumin content is of crucial importance for vaccine production. We compared two methods of purification and concentration of influenza virus: zonal gradient ultracentrifugation and combined ultrafiltration/diafiltration and exclusion chromatography protocol, used for fabrication of seasonal vaccines. Combined chromatography is comparable with zonal centrifugation protocol by the results of ovalbumin removal (to meet standard requirements).
Assuntos
Cromatografia em Agarose/métodos , Vacinas contra Influenza/isolamento & purificação , Ovalbumina/isolamento & purificação , Ultracentrifugação/métodos , Ultrafiltração/métodos , Líquido Amniótico/química , Líquido Amniótico/virologia , Animais , Embrião de Galinha , Humanos , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/biossíntese , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Influenza Humana/virologiaRESUMO
The recombinant strain Flu-NS1-124-Omp16 (H5N1) of the influenza virus expressing the brucellar Omp16 gene was constructed on the basis of the technology of reverse genetics for the purpose of developing vector anti-brucellosis vaccine. The obtained recombinant strain is a genetically stable construction. This stability is confirmed by the comparative analysis of the nucleotide sequences of the HA, NA, and NS genes of the recombinant vector virus Flu-NS1-124-Omp16 (H5N1) expressing the Omp16 gene of the Brucella abortus (GenBank: AAA59360.1). The comparative analysis showed that the nucleotide sequence of the NS gene of the first and the fifth passage level of the Flu-NS1-124-Omp16 (H5N1) virus corresponded for 100% to the initial part of 12AAS2TC_124 Omp16g containing the chimera NS1-124-Omp16 in the composition of DNA (deoxyribonucleic acid) plasmids pHW2000. Total identity with HA and NA genes of the strain A/AstanaRG/6:2/2009 (H5N1) was shown by the comparative analysis of the nucleotide sequences of HA and NA genes of the first and the fifth passage level of the recombinant strain Flu-NS1-124-Omp16 (H5N1). The recombinant vector virus Flu-NS1-124-Omp16 (H5N1) expressing the brucella Omp16 gene maintains the genetic stability during 5 passages in 10-day developing chicken embryos.
Assuntos
Proteínas da Membrana Bacteriana Externa , Brucella abortus/genética , Genes Bacterianos , Vetores Genéticos , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Virus da Influenza A Subtipo H5N1 , Neuraminidase , Proteínas não Estruturais Virais , Proteínas Virais , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Embrião de Galinha , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/metabolismo , Neuraminidase/genética , Neuraminidase/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
AIM: Development of technological stages of preparation of experimental influenza whole-virion inactivated adsorbed vaccine based on recombinant influenza virus strains NIBRG-14 and A/Astana/RG/6:2/2009. MATERIALS AND METHODS: 2 recombinant vaccines influenza strains were used in the study--NIBRG-14 and A/Astana/RG/6:2/2009. Purification of native virus-containing allantoic fluid was performed by ion-exchange chromatography. The virus was inactivated by formaldehyde. Merthiolate at concentration of 0.1 mg/ml was added to the vaccine as a preserving substance. Aluminium hydroxide was used as an adjuvant. Harmlessness and immunogenicity (HI) of the constructed preparation are determining. RESULTS: Virus-containing materials from recombinant strains with biological activity of 8.5 - 9.0 lg EID50/cm3 and hemagglutination activity of 1:256 - 1:1024 in chicken embryos were obtained. Optimal inactivation regimen of non-purified suspensions by formaldehyde was established and combined scheme of purification and concentration of influenza virus was selected that provide harmlessness and immunogenicity of experimental samples of inactivated vaccines against highly pathogenic influenza A/H5N1 in experiments in mice. CONCLUSION: The data obtained on quality parameters of intermediate products and final vaccine give evidence on their compliance with normative parameters for whole-virion influenza purified vaccine.
Assuntos
Anticorpos Antivirais/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Infecções por Orthomyxoviridae/prevenção & controle , Adjuvantes Imunológicos , Hidróxido de Alumínio/imunologia , Animais , Anticorpos Antivirais/sangue , Embrião de Galinha , Formaldeído/química , Hemaglutinação , Humanos , Imunização , Vacinas contra Influenza/biossíntese , Influenza Humana/imunologia , Influenza Humana/virologia , Cazaquistão , Camundongos , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Vacinas de Produtos Inativados , Vacinas Sintéticas , Vírion/imunologia , Vírion/isolamento & purificaçãoRESUMO
This report describes the first isolation and characterization of porcine circovirus 2 (PCV2) in the Republic of Kazakhstan. The virus was isolated from a dead piglet that did not exhibit any typical clinical symptoms of porcine circovirus disease at a pig factory in North Kazakhstan oblast (region). The isolated virus belongs to genotype 2 (PCV2) and shares 96.6% sequence homology with one isolate and two strains from China and two French strains in group 1, cluster 1ab and 96.4% homology with two strains isolated in China and one strain from Hungary. Electron microscopy revealed the isolated virus had the typical morphological structure of PCV. This is the evidence of occurrence of porcine circovirus 2 isolation and characterization in Kazakhstan.