Free haemagglutinin in inactivated whole virus influenza vaccines

While studying the haemagglutinin content of whole virus inactivated influenza vaccines by the single radial diffusion test and quantitative electron microscopy, it was found that not all haemagglutinin measured by single radial diffusion was bound to virions, a part of it being in a free state. The influence of unbound haemagglutinin on the immunogenicity of whole virus inactivated influenza vaccine is discussed. In addition, the use of single radial diffusion for the assessment of unbound haemagglutinin is suggested.     

Analysis of genome integrity of influenza virus in formaldehyde-inactivated split vaccines

Although inactivated split-virus influenza vaccines are widely used to render protection against the viral infection, information regarding the effects of the vaccine preparation on the integrity of the viral genomes is currently very limited. In the present study, to gain insight into the physical and genetic alterations of RNA viral genomes in the course of the vaccine preparation, we analyzed the influenza virus A H1N1 hemagglutinin (HA) genome segment in a conventional split-virus influenza vaccine inactivated by formaldehyde, which encodes the major surface protein of the virus. The vaccine solutions were treated with proteinase K prior to RNA purification, and viral genomic RNA was successfully retrieved up to almost eightfold higher than samples without the treatment. Despite the high yield, no full-length bands of the influenza viral genomes were identifiable upon electrophoresis due to severe degradation. Interestingly, however, we were able to amplify a variety of genomic regions including a fragment covering the full-length HA gene using cDNA. Furthermore, sequencing of a series of the fragments for confirmation revealed that the rate of base alteration of the gene in the vaccine was approximately 0.1 %, which is comparable to the spontaneous error rate occurring during PCR. We next constructed a GST expression vector carrying the full-length HA fragment cloned from the vaccine and confirmed that the correct size of the expected GST-fused HA protein was expressed. Taken together, these results demonstrate that a full-length HA RNA genome fragment with comparatively intact sequence information may exist in the inactivated split-virus vaccine. Therefore, these experimental findings on the properties of the HA RNA in the influenza vaccine, may contribute to cautious use of the vaccine in a variety of research areas and protocols.     

Immune recognition of influenza virus haemagglutinin

Haemagglutinin glycoproteins are the components of influenza virus membranes against which infectivity-neutralizing antibodies are directed. Sequence analysis of natural and laboratory-selected variant haemagglutinins indicates the regions of the molecule recognized by antibodies and by helper T cells; the identity of these regions and the relations between them are discussed.     

The use of new polymethylmethacrylate adjuvants for split influenza vaccines.

Split influenza virus vaccines with varying antigen content were adjuvated with polymethylmethacrylate particles, produced by polymerizing monomeric methylmethacrylate in the presence of the subunits, or by addition of the subunits to previously polymerized methacrylate particles. Both adjuvants yielded higher antibody titers than aluminum hydroxide or fluid vaccines. The character of the antigen-adjuvant conjugate of both types of polymer adjuvants is discussed.     

The structure of a membrane fusion mutant of the influenza virus haemagglutinin.

The haemagglutinin glycoprotein (HA) of influenza virus specifically mediates fusion of the viral and host cell endosomal membranes at the acidic pH of endosomes. The HAs from mutant viruses with raised fusion pH optima contain amino acid substitutions in regions of the HA structure thought to be involved in the fusion process [Daniels et al. (1985b) Cell, 40, 431-439]. We have determined the neutral pH crystal structure of one such mutant, HA2 112 Asp----Gly. A water molecule appears to partially replace the aspartate side chain, and no changes are observed in the surrounding structure. It appears that four intra-chain hydrogen bonds that stabilize the location of the N-terminus of HA2 are lost in the mutant, resulting in a local destabilization that facilitates the extrusion of the N-terminus at higher pH.     

四价流感病毒裂解疫苗的稳定性

目的评价四价流感病毒裂解疫苗的稳定性,为疫苗的有效期提供依据。方法分别将A1(H1N1)、A3(H3N2)、B/Victoria和B/Yamagata等4株毒株制备单价原液,生产6批疫苗(不含硫柳汞),评价疫苗分别在(37±2)℃、(25±2)℃和(6±2)℃条件下的稳定性。结果四价流感病毒裂解疫苗在(37±2)℃保存10 d、在(25±2)℃保存3个月、在(6±2)℃保存15个月,疫苗各项指标均符合企业注册标准和《中华人民共和国药典》2015版(三部)的要求。结论四价流感病毒裂解疫苗具有良好的稳定性,在(6±2)℃可稳定保存15个月。     

Electron microscopy of the low pH structure of influenza virus haemagglutinin.

Influenza virus haemagglutinin mediates infection of cells by fusion of viral and endosomal membranes, triggered by low pH which induces a conformational change in the protein. We report studies of this change by electron microscopy, neutron scattering, sedimentation and photon correlation on X-31 (H3N2) haemagglutinin, both intact and bromelain cleaved, in various assemblies. HAs in all preparations showed a thinning at low pH, and a marked elongation which was removed on tryptic digestion, revealing altered features in the remaining stem portion of the molecule. A tentative model of the change is proposed, with reference to the known X-ray structure at neutral pH, in which major changes occur in the stem tertiary structure, while the top portion is only affected in its quaternary structure.