| Summary: | 博士 === 國立陽明大學 === 生化暨分子生物研究所 === 99 === H5N1 influenza viruses have spread extensively among wild birds and domestic poultry. Cross-species transmission of these viruses to humans has been documented in over 502 cases, with a mortality rate of ~60%. There is a great concern that a H5N1 virus would acquire the ability to spread efficiently between humans, thereby becoming a pandemic threat. A H5N1 influenza vaccine, therefore, become an integral part of any pandemic preparedness plan. However, traditional methods of making influenza vaccines have yet to produce the candidates that could induce potent neutralizing antibodies against divergent strains of H5N1 influenza viruses.
To address this need, we generated a consensus H5N1 hemagglutinin (HA) sequence based on data available in early 2006. This sequence was then optimized for protein expression before being inserted into a DNA plasmid (pCHA5). Immunizing mice with pCHA5 by electroporation (EP), elicited antibodies that neutralized a panel of virions that have been pseudotyped with the HA from various H5N1 viruses (in-vitro). Moreover, immunization with pCHA5 in mice conferred complete (clades 1 and 2.2) or significant (clade 2.1) protection from H5N1 virus challenges (in-vivo). The consensus HA DNA vaccine combined with EP delivery gave a great protection against H5N1 influenza virus in mouse model. This is the first time that consensus DNA vaccine via IM/EP injection that can elicit the broadest cross-neutralization and cross-protection activity to against all the divergent influenza H5N1 viruses in different clades.
Furthermore, the establishment of correlation of in-vitro neutralization and in-vivo challenge studies also enabled us to conduct a comprehensive analysis on serotyping characteristics of the HA from representative H5N1 viruses. The results not only confirmed that cross-clade immunity of consensus DNA vaccine was indeed contributed by the DNA sequence, but also established a platform to efficiently evaluate the cross-protection profiles of a given HA sequence. With this platform, the correlation between HA genotype and serotype can be successfully investigated for the vaccine strain candidate to be decided precisely. The vaccine antigen designed based on consensus strategy showed the superior cross-neutralization activity than WHO-suggested vaccine strains. Particularly for the second-generation consensus HA sequence, pCHA5II, can induce antiserum with better cross-neutralization activity than pCHA5 does, especially to the most recent circulating clade 2.3 viruses. It is noteworthy that, our cross-neutralization results are highly correlated to in-vivo challenge results. Based on our research here, we conclude that HA DNA vaccine which can induce cross-immunity (e.g., pCHA5/pCHA5II), combined with EP delivery is a promising strategy to induce antibodies with better cross-reactivities against divergent H5N1 influenza viruses. Thus, we conclude that this consensus HA-based vaccine could induce broad protection against divergent H5N1 influenza viruses. This is the first time by using HA DNA vaccine-induced antiserum against a set of H5N1 pseudotyped viruses to establish the correlation between the H5N1 HA antigenicity within genotype and immunogenicity within serotype. The highly reliable results could be the one of references for vaccine strain decision.
Moreover, we were interested in examining the amino acid mutation or glycosylation modification in the HA globular region of pCHA5-immune escaping virus strains. Based on genetic alignment revealed that most of the amino acid differences between CHA5 and those insusceptible strains were at the receptor binding domain with the most significant one at the 157th residue. When we immunized mice with HA harboring 157P, the elicited antibodies showed increased neutralizing activity against the clade 2.3 viruses. Likewise, the viruses pseudotyped with hemagglutinin containing 157S become more susceptible to neutralization. The correlation between escape capability from CHA5-vaccinated immunity and sialosides binding activities were confirmed by sugar binding analysis of HA containing 157P or 157S. It was concluded that the S157P amino acid substitution of hemagglutinin can alter antigenicity, immunogenicity, and binding avidity of H5N1 viruses. Overall, in this comprehensive study, we established a promising universal vaccine against H5N1 influenza virus and provided a system for vaccine strain decision and development. We also revealed the possible mechanism for influenza virus evading from vaccinated-immunity which combined with glycan microarray results that could be the highly sensitive surveillance system for flu pandemic.
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