HA1 (Hemagglutinin) quantitation for influenza A H1N1 and H3N2 high yield reassortant vaccine candidate seed viruses by RP-UPLC.

The only effective measure to decrease morbidity and mortality caused by the influenza virus in the human population is worldwide vaccination. Vaccination produces neutralizing antibodies that target the HA1 subunit of the HA (hemagglutinin) protein and are strain specific. The effectiveness of new influenza vaccines are linked to two factors, the correct prediction of the circulating strains in the population in a particular season and the concentration of the HA1 protein in the vaccine formulation. With the advent of the licensing of quadrivalent vaccines, pharmaceutical manufacturers are under considerable pressure due to time constraints and dedicated resources to deliver 194-198 million doses (2020-2021 U.S. market) of vaccine. Considering the valuable resources needed to produce the influenza vaccine in a timely manner, the efficient quantitation of the HA1 protein (the main component in the influenza vaccine) is required. Currently the only method approved by regulatory agencies for quantitation of the HA antigen in vaccines is the single radial immunodiffusion assay (SRID), an antibody dependent assay that is not time efficient. Time efficient methods that are antibody independent e.g. reverse phase-high performance liquid chromatography (RP-HPLC) or size exclusion-HPLC (SE-HPLC) are available. An improved method implementing reverse phase-ultra performance liquid chromatography (RP-UPLC) has been developed to quantitate the HA1 protein antigen present in the high yield reassortant vaccine seed viruses from influenza A H1N1 and H3N2 subtypes harvested from inoculated embryonated chicken eggs. This method differentiates between high yield and lower yielding reassortants in order to select the best vaccine candidate seed virus with the highest growth 'in ovo'. This direct capability to monitor the HA1 concentration of potential reassortant seed viruses and to choose the best yielding HA influenza reassortant when faced with multiple viral seed candidates provides a major advantage on the industrial scale to the influenza vaccine process.

Synthesis of 5-O-oligoglucosyl extended α-(2→4)-Kdo disaccharides corresponding to inner core fragments of Moraxellaceae lipopolysaccharides.

The heptose-deficient inner core of the lipopolysaccharide of several pathogenic strains of the Moraxellaceae family (Moraxella, Acinetobacter) and of Bartonella henselae, respectively, comprises an α-D-glucopyranose attached to position 5 of Kdo. In continuation of the synthesis of fragments of Acinetobacter haemolyticus LPS, the branched α-Glcp-(1 → 5)[α-Kdo-(2 → 4)]-α-Kdo trisaccharide motif was elaborated. The glycosylation of a suitably protected, α-(2 → 4)-interlinked Kdo-disaccharide was achieved in high yield and fair anomeric selectivity using a 4,6-O-benzylidene N-phenyltrifluoroacetimidate glucosyl donor. Subsequent regioselective reductive benzylidene opening afforded a trisaccharide acceptor, which was extended with ß-D-glucopyranosyl and isomaltosyl residues. Global deprotection provided tri- to pentasaccharide structures corresponding to the inner core region of A. haemolyticus lipopolysaccharide.

Synthesis of chlamydia lipopolysaccharide haptens through the use of α-specific 3-iodo-Kdo fluoride glycosyl donors.

A scalable approach towards high-yielding and (stereo)selective glycosyl donors of the 2-ulosonic acid Kdo (3-deoxy-D-manno-oct-2-ulosonic acid) is a fundamental requirement for the development of vaccines against Gram-negative bacteria. Herein, we disclose a short synthetic route to 3-iodo Kdo fluoride donors from Kdo glycal esters that enable efficient α-specific glycosylations and significantly suppress the elimination side reaction. The potency of these donors is demonstrated in a straightforward, six-step synthesis of a branched Chlamydia-related Kdo-trisaccharide ligand without the need for protecting groups at the Kdo glycosyl acceptor. The approach was further extended to include sequential iteration of the basic concept to produce the linear Chlamydia-specific α-Kdo-(2→8)-α-Kdo-(2→4)-α-Kdo trisaccharide in a good overall yield.

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.

First and stereoselective synthesis of an α-(2→5)-linked disaccharide of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo).

Resistance of bacterial pathogens toward antibiotics has revived interest in lipopolysaccharide (LPS) motifs as potential therapeutic targets. The LPS of several pathogenic Acinetobacter strains comprises a 4,5-branched Kdo trisaccharide containing an uncommon (2→5)-linkage. In this contribution the first stereoselective glycosylation method for obtaining an α-Kdo-(2→5)-α-Kdo disaccharide in good yield is highlighted. The synthetic approach used for accessing this linkage type will allow for future studies of the immunoreactivity associated with this unique bacterial Kdo inner core structure.

Scope and Limitations of 3-Iodo-Kdo Fluoride-Based Glycosylation Chemistry using N-Acetyl Glucosamine Acceptors.

The ketosidic linkage of 3-deoxy-d-manno-octulosonic acid (Kdo) to lipid A constitutes a general structural feature of the bacterial lipopolysaccharide core. Glycosylation reactions of Kdo donors, however, are challenging due to the absence of a directing group at C-3 and elimination reactions resulting in low yields and anomeric selectivities of the glycosides. While 3-iodo-Kdo fluoride donors showed excellent glycosyl donor properties for the assembly of Kdo oligomers, glycosylation of N-acetyl-glucosamine derivatives was not straightforward. Specifically, oxazoline formation of a ß-anomeric methyl glycoside, as well as iodonium ion transfer to an allylic aglycon was found. In addition, dehalogenation of the directing group by hydrogen atom transfer proved to be incompatible with free hydroxyl groups next to benzyl groups. In contrast, glycosylation of a suitably protected methyl 2-acetamido-2-deoxy-α-d-glucopyranoside derivative and subsequent deiodination proceeded in excellent yields and α-specificity, and allowed for subsequent 4-O-phosphorylation. This way, the disaccharides α-Kdo-(2→6)-α-GlcNAcOMe and α-Kdo-(2→6)-α-GlcNAcOMe-4-phosphate were obtained in good overall yields.

Synthesis of α-d-glucosyl substituted methyl glycosides of 3-deoxy-α-d-manno- and d-glycero-α-d-talo-oct-2-ulosonic acid (Kdo/Ko) corresponding to inner core fragments of Acinetobacter lipopolysaccharide.

The α-d-glucopyranosyl-(1→5)-substituted methyl glycosides of 3-deoxy-α-d-manno-oct-2-ulosonic acid (Kdo), 3-deoxy-α-d-lyxo-hept-2-ulosonic acid (Kdh), and d-glycero-α-d-talo-oct-2-ulosonic acid (Ko) were prepared using orthogonally protected glycosyl acceptor derivatives via glycosylation with a torsionally disarmed 4,6-O-benzylidene protected trifluoroacetimidate glucosyl donor followed by global deprotection. The related 6-O-phosphoryl-α-d-glucopyranosyl-(1→5)-substituted Kdo and Kdh derivatives were derived from a benzylidene-protected glucosyl intermediate using phosphoramidite and phosphoryl chloride-based phosphorylation steps, respectively. The deprotected disaccharides serve as ligands to study lectin binding of Acinetobacter lipopolysaccharide core oligosaccharides.

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.

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.

The development of vaccine viruses against pandemic A(H1N1) influenza.

Wild type human influenza viruses do not usually grow well in embryonated hens' eggs, the substrate of choice for the production of inactivated influenza vaccine, and vaccine viruses need to be developed specifically for this purpose. In the event of a pandemic of influenza, vaccine viruses need to be created with utmost speed. At the onset of the current A(H1N1) pandemic in April 2009, a network of laboratories began a race against time to develop suitable candidate vaccine viruses. Two approaches were followed, the classical reassortment approach and the more recent reverse genetics approach. This report describes the development and the characteristics of current pandemic H1N1 candidate vaccine viruses.

Receptor specificity of influenza A H3N2 viruses isolated in mammalian cells and embryonated chicken eggs.

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 alpha2-6 and alpha2-3 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 alpha2-6- and alpha2-3-type receptors but retained substantial binding to specific O- and N-linked alpha2-3 glycans, including alpha2-3GalNAc and fucosylated alpha2-3 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.

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 virus's 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.

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.