Barbara A. Pokorny

The total influenza vaccine failure of 1947 revisited: Major intrasubtypic antigenic change can explain failure of vaccine in a post-World War II epidemic

Although vaccine-induced immunity to influenza A virus is continually challenged by progressively selected mutations in the viruss major antigens (antigenic drift), virus strains within a subtype (e.g., H1N1) are antigenically cross-reactive. Although cross-immunity diminishes as further mutations accumulate, necessitating frequent changes in vaccine strains, older vaccines are usually partially protective. The post-World War II epidemic of 1947 is notable for the total failure of a vaccine previously effective in the 1943–44 and 1944–45 seasons. We have combined extensive antigenic characterization of the hemagglutinin and neuraminidase antigens of the 1943 and 1947 viruses with analysis of their nucleotide and amino acid sequences and have found marked antigenic and amino acid differences in viruses of the two years. Furthermore, in a mouse model, vaccination with the 1943 vaccine had no effect on infection with the 1947 strain. These findings are important, because complete lack of cross-immunogenicity has been found previously only with antigenic shift, in which antigenically novel antigens have been captured by reassortment of human and animal strains, sometimes leading to pandemics. Although the 1947 epidemic lacked the usual hallmarks of pandemic disease, including an extensive increase in mortality, it warns of the possibility that extreme intrasubtypic antigenic variation (if coupled with an increase in disease severity) could produce pandemic disease without the introduction of animal virus antigens.

Purified influenza A virus N2 neuraminidase vaccine is immunogenic and non-toxic in humans

The immunogenicity and toxicity of a purified influenza virus (N2) neuraminidase vaccine (NAV) were investigated in 88 human subjects aged 18-40, and compared to response to a conventional trivalent influenza vaccine, Fluogen (Parke-Davis). NAV doses ranged from 2.6 to 69.9 micrograms and were given intramuscularly. Serologic neuraminidase-inhibiting (NI) and neuraminidase-specific ELISA responses in this N2-primed population were roughly proportional to the dose administered. Maximal response was seen in 14-21 days and NI antibody titers persisted unabated for the 6-month post-vaccination follow-up period. All doses were well tolerated with respect to local and systemic reactions. NI tests performed with the putative (1975) priming N2 antigen demonstrated anamnestic response but did not reveal responses not already shown with the homologous (1992) antigen. Response to this purified, non-adjuvanted preparation encourages continuing investigation of the induction of infection-permissive immunity with influenza virus neuraminidase.

Receptor Specificity of Influenza A H3N2 Viruses Isolated in Mammalian Cells and Embryonated Chicken Eggs

ABSTRACT Isolation of human subtype H3N2 influenza viruses in embryonated chicken eggs yields viruses with amino acid substitutions in the hemagglutinin (HA) that often affect binding to sialic acid receptors. We used a glycan array approach to analyze the repertoire of sialylated glycans recognized by viruses from the same clinical specimen isolated in eggs or cell cultures. The binding profiles of whole virions to 85 sialoglycans on the microarray allowed the categorization of cell isolates into two groups. Group 1 cell isolates displayed binding to a restricted set of α2-6 and α2-3 sialoglycans, whereas group 2 cell isolates revealed receptor specificity broader than that of their egg counterparts. Egg isolates from group 1 showed binding specificities similar to those of cell isolates, whereas group 2 egg isolates showed a significantly reduced binding to α2-6- and α2-3-type receptors but retained substantial binding to specific O- and N-linked α2-3 glycans, including α2-3GalNAc and fucosylated α2-3 glycans (including sialyl Lewis x), both of which may be important receptors for H3N2 virus replication in eggs. These results revealed an unexpected diversity in receptor binding specificities among recent H3N2 viruses, with distinct patterns of amino acid substitution in the HA occurring upon isolation and/or propagation in eggs. These findings also suggest that clinical specimens containing viruses with group 1-like receptor binding profiles would be less prone to undergoing receptor binding or antigenic changes upon isolation in eggs. Screening cell isolates for appropriate receptor binding properties might help focus efforts to isolate the most suitable viruses in eggs for production of antigenically well-matched influenza vaccines.

Protection of Mice with Recombinant Influenza Virus Neuraminidase

Contemporary influenza vaccines are standardized with respect to their content of hemagglutinin, the major virus antigen. Although the immunizing effect of viral neuraminidase--the less abundant of the 2 major surface glycoproteins--has been well documented in experimental animals, the importance of the purified recombinant protein has not yet been adequately assessed in animals or humans. We demonstrate that different lots of a baculovirus-derived recombinant N2 protein, in the absence of other influenza virus proteins, can induce neuraminidase-specific antibodies, reduce the replication of both homologous and heterovariant virus in mice, and suppress disease, as it is manifested by total body weight loss.

Gene Constellation of Influenza A Virus Reassortants with High Growth Phenotype Prepared as Seed Candidates for Vaccine Production

Background Influenza A virus vaccines undergo yearly reformulations due to the antigenic variability of the virus caused by antigenic drift and shift. It is critical to the vaccine manufacturing process to obtain influenza A seed virus that is antigenically identical to circulating wild type (wt) virus and grows to high titers in embryonated chicken eggs. Inactivated influenza A seasonal vaccines are generated by classical reassortment. The classical method takes advantage of the ability of the influenza virus to reassort based on the segmented nature of its genome. In ovo co-inoculation of a high growth or yield (hy) donor virus and a low yield wt virus with antibody selection against the donor surface antigens results in progeny viruses that grow to high titers in ovo with wt origin hemagglutinin (HA) and neuraminidase (NA) glycoproteins. In this report we determined the parental origin of the remaining six genes encoding the internal proteins that contribute to the hy phenotype in ovo. Methodology The genetic analysis was conducted using reverse transcription-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP). The characterization was conducted to determine the parental origin of the gene segments (hy donor virus or wt virus), gene segment ratios and constellations. Fold increase in growth of reassortant viruses compared to respective parent wt viruses was determined by hemagglutination assay titers. Significance In this study fifty-seven influenza A vaccine candidate reassortants were analyzed for the presence or absence of correlations between specific gene segment ratios, gene constellations and hy reassortant phenotype. We found two gene ratios, 6∶2 and 5∶3, to be the most prevalent among the hy reassortants analyzed, although other gene ratios also conferred hy in certain reassortants.

Supplementation of conventional trivalent influenza vaccine with purified viral N1 and N2 neuraminidases induces a balanced immune response without antigenic competition

Influenza viruses neuraminidase (NA) were chromatographically extracted from influenza viruses A/Nanchang/933/95 H3(NC)N2(NC) [R] and A/Johannesburg/82/96 H1(JH)N1(JH) [R] and used to supplement conventional inactivated trivalent influenza vaccine. Immunization of mice with this preparation resulted in high titers of antibodies to both hemagglutinins (HA) and neuraminidases (NA); there were no significant differences in the anti-HA antibody titers between the conventional and the supplemented vaccine preparation. Likewise, there were no significant differences in anti-NA antibody titers between the supplemented vaccine and titers from mice immunized with a neuraminidase vaccine containing a mixture of N1-NA and N2-NA. There was no evidence of a diminution of the immune response to the HA components of the vaccine despite the presence of antigenically equivalent amounts of both N1-NA and N2-NAs. Homotypic and distantly related heterotypic infections for both H1, N1 and H3N2 subtypes were suppressed and greater reduction in pulmonary virus titers (PVT) were observed in the trivalent vaccine supplemented with purified neuraminidase from each subtype, N1 and N2. Effects on the influenza B viral components were not studied. Previous studies on supplementation of conventional influenza vaccine focused only on monovalent H3N2 vaccine preparations; this study demonstrates in a mouse model system that supplementation of trivalent influenza vaccine with both influenza A subtype neuraminidases, N1 and N2 is highly immunogenic for HA and NA of each subtype and efficacious in protecting against influenza from homotypic and heterotypic infectious challenges of either subtype.

Preparation and characterization of a purified influenza virus neuraminidase vaccine.

Influenza virus neuraminidase (NA) has been shown to induce protective but infection-permissive immunity in experimental animals. Challenge infection following such immunization is attended by decreased viral replication and disease manifestations but is sufficient to provide antigenic stimulation and definitive immunity to the virus. The present report describes the preparation and characterization of a purified NA vaccine (NAV) used in Phase 1 (immunogenicity and toxicity) trials in humans. In essence, virion NA was isolated from detergent-disrupted virus by affinity chromatography on oxamic acid-agarose, treated with formalin and tested for its enzymatic activity and for its immunogenicity in Balb/c mice and New Zealand rabbits. The preparation was essentially free of viral hemagglutinin but contained residual NP and M1 proteins. Both dispersed and aggregated NA tetrameric heads were seen in electron micrographs. Enzymatic activity was preserved, and minimal immunogenic doses in mice and rabbits, respectively, were 3.7 and 0.027 micrograms per kg.

THE INFLUENCE OF CORTISONE ON EXPERIMENTAL VIRAL INFECTION III. EFFECTS ON CERTAIN DYNAMICS OF INFLUENZA VIRUS INCREASE

The administration, of 5.0 mg of cortisone 6 to 24 hr prior to the intravenous injection of mice with NDV was associated with a marked reduction in the interferon response as measured in serum and spleen. Reduced concentrations of interferon following cortisone pretreatment were demonstrable from 6 to 16 hr following injection of virus.

Molecular Signature of High Yield (Growth) Influenza A Virus Reassortants Prepared as Candidate Vaccine Seeds

Background Human influenza virus isolates generally grow poorly in embryonated chicken eggs. Hence, gene reassortment of influenza A wild type (wt) viruses is performed with a highly egg adapted donor virus, A/Puerto Rico/8/1934 (PR8), to provide the high yield reassortant (HYR) viral ‘seeds’ for vaccine production. HYR must contain the hemagglutinin (HA) and neuraminidase (NA) genes of wt virus and one to six ‘internal’ genes from PR8. Most studies of influenza wt and HYRs have focused on the HA gene. The main objective of this study is the identification of the molecular signature in all eight gene segments of influenza A HYR candidate vaccine seeds associated with high growth in ovo. Methodology The genomes of 14 wt parental viruses, 23 HYRs (5 H1N1; 2, 1976 H1N1-SOIV; 2, 2009 H1N1pdm; 2 H2N2 and 12 H3N2) and PR8 were sequenced using the high-throughput sequencing pipeline with big dye terminator chemistry. Results Silent and coding mutations were found in all internal genes derived from PR8 with the exception of the M gene. The M gene derived from PR8 was invariant in all 23 HYRs underlining the critical role of PR8 M in high yield phenotype. None of the wt virus derived internal genes had any silent change(s) except the PB1 gene in X-157. The highest number of recurrent silent and coding mutations was found in NS. With respect to the surface antigens, the majority of HYRs had coding mutations in HA; only 2 HYRs had coding mutations in NA. Significance In the era of application of reverse genetics to alter influenza A virus genomes, the mutations identified in the HYR gene segments associated with high growth in ovo may be of great practical benefit to modify PR8 and/or wt virus gene sequences for improved growth of vaccine ‘seed’ viruses.

Development of high yield reassortants for influenza type B viruses and analysis of their gene compositions.

A critical step in producing the annual inactivated influenza vaccine is the development of high yield (hy) seed viruses by reassortment for improved growth in ovo. Although hy reassortants for type A influenza viruses have been developed for many years, hy B influenza reassortant virus development for vaccine production has proven difficult. In this study, we have developed fourteen hy influenza type B reassortants as vaccine candidate strains with B/Lee/40 as the donor virus. Upon characterization by the Influenza Division at the Centers for Disease Control and Prevention (CDC) and the verification of HA by sequencing, all B reassortants were found to be antigenically indistinguishable from the wild type (wt) parents and suitable for vaccine production. However, only one hy reassortant seed virus from this group was used by a manufacturer for vaccine production. In general, hy reassortants showed an increase in hemagglutination (HA) titers over their wt parents by approximately 8 fold (range 1-32 fold). Gene compositions of the hy B reassortants were analyzed by restriction fragment length polymorphism (RFLP) and the wt origin of the HA and neuraminidase (NA) were confirmed. However, in contrast to hy A reassortants which require the M gene (hy donor A/PR/8/34) for high yield, all fourteen hy B reassortants obtained the NP gene from the hy donor strain (B/Lee/40). The parental source for the remaining genes varied among the hy B reassortants. The results indicate that the B/Lee/40 NP and PB1 gene segments are important contributors to high yield growth in influenza B reassortant viruses for both Yamagata and Victoria lineages. The B/Lee/40 PB2 gene along with wt NS gene also contributed to the improved growth for hy reassortants of Yamagata lineage.