Avian influenza, subtype H5N1, causes occasional but serious infections in humans and efforts to produce vaccines against this strain continue. Current influenza vaccines are prophylactic and utilize the two major ant...Avian influenza, subtype H5N1, causes occasional but serious infections in humans and efforts to produce vaccines against this strain continue. Current influenza vaccines are prophylactic and utilize the two major antigens, hemagglutinin and neuraminidase. Nucleocapsid protein (NP) is an attractive alternative antigen because it is highly conserved across all influenza strains, has been shown to increase the rate of viral clearance, and potential therapeutic vaccines would elicit cytotoxic T lymphocyte responses in an infected person. The NP antigen from H5N1 was characterized using a variety of physico-chemical methods to gain insights into both the biological and physical properties of the antigen which are important from a regulatory viewpoint when considering therapeutic vaccines. Results obtained to date show that NP is relatively unstable and indicate that the conformation of the H5N1 NP antigen is highly dependent upon purification procedure, buffer conditions, pH and the presence or absence of RNA. These factors will need to be clearly defined and taken into consideration when manufacturing and regulating NP vaccine preparations.展开更多
In the course of isolating the attenuated Japanese encephalitis vaccine SA14-14-2,two attenuated strains SA14-9-7 and SA14-5-3 were also obtained that elicited low antibody responses in humans(o10%and 62%,respectively...In the course of isolating the attenuated Japanese encephalitis vaccine SA14-14-2,two attenuated strains SA14-9-7 and SA14-5-3 were also obtained that elicited low antibody responses in humans(o10%and 62%,respectively)and exerted much weaker immune protection in animal challenge experiments.However,the reason for these differences remains unknown.In order to understand why SA14-14-2 is superior to SA14-9-7 and SA14-5-3,we employed a reverse genetics method to identify the key mutations in the virus genome that determine the immunogenicity of live attenuated Japanese encephalitis viruses.We first sequenced the full genomic sequences of SA14-9-7 and SA14-5-3 and found mutations that changed four amino-acid base pairs when compared to the envelope gene of SA14-14-2.We mutated the genome of SA14-14-2 to generate these mutations both singly(E-177,E-264,E-279 and E-315)and in combination(E-177/264,E-279/315 and E-177/264/279/315)and tested these mutants along with parental strains SA14-14-2,SA14-9-7 and SA14-5-3 for their immunogenicity in vivo.When mice were immunized with SA14-9-7 and SA14-5-3,lower levels of neutralizing antibodies were generated compared with the immune response to SA14-14-2.Furthermore,SA14-5-3 was more immunogenic than SA14-9-7,which replicated the results previously seen in humans.Point mutations E-177,E-264,E-279 and E-315 diminished the immunogenicity of SA14-14-2 with E-264 and E-315,contributing the most to this phenotype.The mutant rJEV(E-177/E-264/E-279/E-315)containing all four point mutations exhibited the lowest immunogenicity with a seroconversion rate of 0 at an inoculation dose of 103 PFU(plaque-forming unit).We have identified the key amino acids in the envelope protein that account for the superior immunogenicity of SA14-14-2.展开更多
文摘Avian influenza, subtype H5N1, causes occasional but serious infections in humans and efforts to produce vaccines against this strain continue. Current influenza vaccines are prophylactic and utilize the two major antigens, hemagglutinin and neuraminidase. Nucleocapsid protein (NP) is an attractive alternative antigen because it is highly conserved across all influenza strains, has been shown to increase the rate of viral clearance, and potential therapeutic vaccines would elicit cytotoxic T lymphocyte responses in an infected person. The NP antigen from H5N1 was characterized using a variety of physico-chemical methods to gain insights into both the biological and physical properties of the antigen which are important from a regulatory viewpoint when considering therapeutic vaccines. Results obtained to date show that NP is relatively unstable and indicate that the conformation of the H5N1 NP antigen is highly dependent upon purification procedure, buffer conditions, pH and the presence or absence of RNA. These factors will need to be clearly defined and taken into consideration when manufacturing and regulating NP vaccine preparations.
基金This study was funded by the Chinese mega project of science research for major new drugs innovationdevelopment research for quality control of JE live attenuated vaccine and polio vaccine(Grant No.20142014ZX09304316-003).
文摘In the course of isolating the attenuated Japanese encephalitis vaccine SA14-14-2,two attenuated strains SA14-9-7 and SA14-5-3 were also obtained that elicited low antibody responses in humans(o10%and 62%,respectively)and exerted much weaker immune protection in animal challenge experiments.However,the reason for these differences remains unknown.In order to understand why SA14-14-2 is superior to SA14-9-7 and SA14-5-3,we employed a reverse genetics method to identify the key mutations in the virus genome that determine the immunogenicity of live attenuated Japanese encephalitis viruses.We first sequenced the full genomic sequences of SA14-9-7 and SA14-5-3 and found mutations that changed four amino-acid base pairs when compared to the envelope gene of SA14-14-2.We mutated the genome of SA14-14-2 to generate these mutations both singly(E-177,E-264,E-279 and E-315)and in combination(E-177/264,E-279/315 and E-177/264/279/315)and tested these mutants along with parental strains SA14-14-2,SA14-9-7 and SA14-5-3 for their immunogenicity in vivo.When mice were immunized with SA14-9-7 and SA14-5-3,lower levels of neutralizing antibodies were generated compared with the immune response to SA14-14-2.Furthermore,SA14-5-3 was more immunogenic than SA14-9-7,which replicated the results previously seen in humans.Point mutations E-177,E-264,E-279 and E-315 diminished the immunogenicity of SA14-14-2 with E-264 and E-315,contributing the most to this phenotype.The mutant rJEV(E-177/E-264/E-279/E-315)containing all four point mutations exhibited the lowest immunogenicity with a seroconversion rate of 0 at an inoculation dose of 103 PFU(plaque-forming unit).We have identified the key amino acids in the envelope protein that account for the superior immunogenicity of SA14-14-2.
