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- Materials and methods
Influenza continues to pose a threat to human health on a seasonal basis, with an average of 25 470 influenza-associated respiratory and circulatory deaths each year. Pandemic outbreaks occur when an antigenically unique virus, such as the swine-origin strain in 2009, infects and causes disease in an immunologically naïve population. Immunity is largely attributed to antibodies specific for hemagglutinin (HA), the predominant glycoprotein on the virus surface that is responsible for binding to cellular sialic acid-containing receptors. HA is therefore the primary antigen contained in licensed inactivated influenza vaccines, with vaccine efficacy correlating with HA-inhibition (HI) titers.[2, 3]
Until the early 1980s, influenza vaccine potency was measured by its ability to agglutinate chicken cells as this functional property of HA is indicative of its native conformation. This assay, however, cannot be used to differentiate between influenza A and B viruses, or between H1N1 and H3N2 subtypes that are included in the trivalent vaccine, and this measure of agglutination did not always correlate with the vaccine's immunogenicity in man. This test was replaced by the single radial immunodiffusion (SRID) assay. This assay is specific for virus type and subtype, using sheep antiserum specific for native HA in agar to precipitate HA that is present in its native conformation. The assay is stability indicating, because denatured HA does not precipitate with this antiserum.
Denaturation of HA resulting in a decreased SRID measurement can be achieved by heat treatment, freeze-thawing, and acidification. It is well recognized that acidification is required for virus entry, with pH approximately 5·5 resulting in conformational change within the late endosome, exposing a fusogenic peptide that facilitates fusion of viral and endosomal membranes to permit entry of the viral genome into the cytoplasm of the infected cell. Gaspar et al., demonstrated a similar change in conformation when virus is treated with hydrostatic pressure at neutral pH, showing fusogenic properties of HA are increased after pressure treatment. Importantly, these authors demonstrate a large decrease in the infectivity of Sindbis, loss in hemagglutination of influenza, and suggest exposure to pressure may be a useful method to inactivate whole virus for vaccine production. While Sindbis and influenza viruses are enveloped, others have reported inactivation of a non-enveloped virus, foot-and-mouth disease virus, after treatment with pressure. In these reports, it is implied that immunogenic properties are not impacted by this treatment, but antibody responses to specific viral proteins after vaccination with live and inactivated virus preparations were not compared.
The extent of pressure-induced denaturation of HA to generate its fusogenic form is not known, and therefore, it is not known whether the antigenic structure of HA is retained following pressure treatment. In this report, we examine the impact of hydrostatic pressure on the antigenic form of HA from each of the viruses included in the 2011/2012 seasonal trivalent influenza vaccine, A/California/07/2009 (H1N1), A/Victoria/210/2009 (H3N2), and B/Brisbane/60/2008.
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- Materials and methods
Our results show that the antigenic structure of HA is changed by pressure treatment. The reduction in antigenic form and protection of loss in potency by allantoic fluid is dependent on virus strain. These observations are consistent with data showing HA is present in a metastable state, allowing conformational changes that are an essential element of viral entry. These changes include folding back of HA1 of the cleaved HA0 molecule, and exposure of the fusogenic peptide in acidic endosomes. Our results as well as those of Gaspar et al., suggest that acidification is not absolutely necessary for structural changes that result in loss of hemagglutination and increase in fusogenic activity – hydrostatic pressure at neutral pH results in a similar functional change.
Hydrostatic pressure has been a useful tool to investigate protein structure, because tertiary and quaternary structures are highly dependent on specific areas of hydration on the molecule surface and also hydrophobic, water-excluded cavities within the molecule. Studies of hydrostatic pressure-induced changes in purified proteins and viruses have facilitated an understanding of protein–DNA recognition and virus assembly, contributed to an understanding of the formation of protein aggregates that play a role in Parkinson's disease and transmissible spongiform encephalopathies. Importantly, these studies allowed identification of compounds that inhibit protein aggregation, providing drug candidates to prevent or treat these diseases. Our results demonstrating that addition of allantoic fluid protects hemagglutinins of influenza A but not B viruses from pressure-induced loss in potency suggest that specific interactions between molecules contained in allantoic fluid and HA preserve its antigenic structure. Given that aggregates can form when molecules are exposed to pressure, one possibility is that addition of allantoic fluid prevents the formation of large complexes that limit migration of HA through the agarose used in the potency assay. If this were the explanation, we would expect that a pressure-treated influenza sample would retain immunogenicity in mice – this was not the case. Our opinion is therefore that in the presence of allantoic fluid, pressure induces the fusion-active form of HA, resulting in loss of hemagglutination, but that the native structure of HA is preserved, perhaps through binding of specific glycoconjugates within the allantoic fluid of chicken eggs that bind to the HA of influenza A, but not influenza B viruses. Birds are not a natural host of influenza B viruses, and therefore, this may reflect differences in receptor binding that support replication of influenza A viruses in avian species. Further studies are needed to determine whether the difference in the protective capacity of allantoic fluid is observed more generally for larger numbers of influenza A and B viruses, and to identify components of allantoic fluid that contribute to the protection of HA's antigenic structure.
Our results show discordance between hemagglutination and potency assay results, and while pressure impacts both, the changes we measured were often independent of one another. This is not surprising considering the interactions between HA and receptors on red blood cells and between HA and specific antibodies are fundamentally different. Alternate potency assays are currently being considered for influenza vaccines because of the lengthy time needed to prepare reagents for SRID analysis. Careful thought should be given to avoid the use of assays that depend on HA's receptor-binding activity to capture or detect antigen because, as our results demonstrate, this is not always indicative of the immunogenic form of HA.
There are several industrial and research applications for hydrostatic pressure technology. It is used in the food industry to inactivate adventitious agents, and has been studied as a method to inactivate norovirus, hepatitis A virus, simian immunodeficiency virus, human immunodeficiency virus and highly pathogenic avian influenza virus. Pressure-inactivated vesicular stomatitis virus, rotavirus, foot-and-mouth disease virus, chicken infectious bursal disease virus, and yellow fever virus, have been tested as potential vaccines. While multiple pressure-induced changes could contribute to virus inactivation, our data suggest that disassembly of oligomers or changes in conformation of receptor-binding domains explain the lack of infectivity. For example, inactivation of influenza is likely due to induction of the fusion-active state, resulting in a loss in binding to receptors. In the case of chicken infectious bursal disease virus, the antigenic structure of the pressure-treated virus was reported as intact. This may not be the case for all antigens; in fact, the impact of pressure on the potency has not been reported for many viral vaccine candidates, including simian immunodeficiency virus and HIV-1, and a detrimental effect of pressure on antigenic structure may explain the low levels of neutralizing antibodies generated in response to pressure-inactivated yellow fever virus. Our data show that the antigenic form of HA and subsequent antibody response to HA is significantly reduced by pressure, suggesting that inactivation of influenza virus with pressure is unlikely to provide a suitable influenza vaccine candidate. Hydrostatic pressure technology does, however, provide a tool to identify buffer conditions or molecules that stabilize HA. Our data suggest that components of allantoic fluid protect the antigenic structure of HA without improving hemagglutination; further studies are needed to understand these results fully and to discover the molecular interactions that contribute to this observation.
In summary, we show that hydrostatic pressure changes the conformation of HA, resulting in loss of reactivity with antibodies generated against the native molecule. Not only is there a change in antigenic form, HA can no longer bind to receptors, shown as a loss of hemagglutination, although these measures are often independent of one another. Inclusion of allantoic fluid protected hemagglutinins of the influenza A (A/CA/09 and A/VI/09), but not B (B/FL/06 and B/BR/08), viruses from pressure-induced changes in structure, preserving antigenic structure in the absence of HA's ability to agglutinate red blood cells. Further studies are needed to determine whether this difference is observed more generally for larger numbers of influenza A and B viruses. Our data support the use of hydrostatic pressure technology as a tool to examine HA stability. As shown by inclusion of allantoic fluid, subjecting antigens to pressure may also provide the means to identify buffer conditions that improve stability of the antigen, and may therefore be helpful in generating formulations that extend vaccine shelf-life.