Egg-adapted Strain Research Articles

A rapid and flexible microneutralization assay for serological assessment of influenza viruses.

Serological responses from influenza vaccination or infection are typically measured by hemagglutinin inhibition (HAI) or microneutralization (MN). Both methods are limited in feasibility, standardization, and generalizability to recent strains. We developed a luciferase MN (LMN) assay that combines the advantages of the conventional MN assay with the ease of the HAI assay. Sera were obtained from the HIVE study, a Michigan household cohort. Reverse genetics was used to generate recombinant influenza viruses expressing the hemagglutinin and neuraminidase of test strains, all other viral proteins from an A/WSN/1933 backbone, and a NanoLuc reporter. Serum neutralization of luciferase-expressing targets was quantified as a reduction in light emission from infected cells. Neutralization titers were measured for cell- and egg-adapted versions of A/Hong Kong/4801/2014 and A/Singapore/INFIMH-16-0019/2016 and compared to HAI titers against egg-grown antigens. Three hundred thirty-three sera were collected from 259 participants between May 2016 and July 2018. Sampled participants were 7-68 years of age, and >80% were vaccinated against influenza. HAI and LMN titers were correlated for A/Hong Kong/4801/2014 (ρ= 0.52, p≤ 0.01) and A/Singapore/INFIMH-16-0019/2016 (ρ= 0.79, p≤ 0.01). LMN titers were lower for cell strains compared to egg strains (A/Hong Kong/4801/2014 mean log2 fold change = -2.66, p≤ 0.01 and A/Singapore/INFIMH-16-0019/2016 mean log2 fold change = -3.15, p≤ 0.01). The LMN assay was feasible using limited sample volumes and able to differentiate small antigenic differences between egg-adapted and cell-derived strains. The correspondence of these results with the commonly used HAI confirms the utility of this assay for high-throughput studies of correlates of protection and vaccine response.

Complete Genome Sequencing of an Embryonated Chicken Egg-Adapted Duck atadenovirus A.

Egg drop syndrome (EDS) is prevalent in industrial poultry globally. This disease is caused by Duck atadenovirus A or EDS virus (EDSV), a member of the genus Atadenovirus under the family Adenoviridae. The disease is attributed to significant economic losses in the poultry industry worldwide due to a drop in egg production, reduction in egg quality, and failure to reach maximum egg production. Oil-adjuvant inactivated vaccines, which are widely used in the poultry industry, provide good protection for immunized chickens against EDS. This study aimed to genetically and phylogenetically analyze the full-length genome of an embryonated chicken egg-adapted EDSV strain 127. After extraction of viral DNA from the allantoic fluid, overlapping fragments of the viral genome sequence were generated by polymerase chain reaction (PCR) using 25 pairs of primers. Purified PCR products were subjected to complete genome sequencing by the next-generation sequencing (NGS) approach. The nucleotide homology observed between genomes of the studied strain and that of the original strain 127 (NC_001813) of laying chickens was 99.9%. Its genome was 33,213 bp in length, with a G + C content of 43.01%. A comparison of the genome sequence of the egg-adapted virus with strain 127 revealed only three non-synonymous single-nucleotide polymorphisms (SNPs) between these viral genome sequences. Two mutations of S320G and I62K out of these SNPs were found within the coding regions of fiber and hypothetical proteins which may play a role in the adaptation of EDSV in the embryonated chicken eggs. The full genome sequencing of EDSV using NGS techniques provides insights into the discovery of genetic variants. Moreover, the genome sequence information of the EDSV provides valuable data for vaccine development in near future.

Cost-effectiveness of the cell-based quadrivalent versus the standard egg-based quadrivalent influenza vaccine in Germany

Background Standard influenza vaccines are produced using egg-based manufacturing methods. Through the process, the resulting egg-adapted viral strains may differ from the selected vaccine strain. Cell-derived influenza vaccine manufacturing prevents egg-adaptation of the antigen which can improve vaccine effectiveness. We evaluated the cost-effectiveness of quadrivalent cell-derived influenza vaccine (QIVc) versus an egg-based quadrivalent influenza vaccine (QIVe) in preventing seasonal influenza from German societal and payer perspectives. Methods Adapted version of the individual-based dynamic 4Flu transmission model was combined with a decision-tree to calculate the impact of QIVc versus QIVe on influenza over 20 seasons in Germany. Egg-adaptation, resulting in lower effectiveness of QIVe versus QIVc towards the H3N2 influenza strain, is sourced from a US retrospective study and assumed in 100% (base case) or 55% (conservative scenario) of years. Influenza-related probabilities of outpatient visits, hospitalizations, productivity loss, and mortality, with associated (dis)utilities/costs, were extracted from literature. Costs and outcomes were discounted 3.0%/year. Results Replacing QIVe with QIVc in subjects aged ≥ 9 years can annually prevent 167,265 symptomatic cases, 51,114 outpatient visits, 2,091 hospitalizations, and 103 deaths in Germany. The annual number of quality-adjusted life-years (QALYs) increased by 1,628 and healthcare costs decreased by €178 M from societal perspective. From payer perspective, the incremental cost-effectiveness ratio was €2,285 per QALY. Scenario analyses confirmed results robustness. Conclusions The use of QIVc compared to QIVe, in the German Immunization Program, could significantly prevent outpatient visits and hospitalizations and would enable substantial savings from a societal perspective.

Establishment of a mouse- and egg-adapted strain for the evaluation of vaccine potency against H3N2 variant influenza virus in mice.

Sporadic spreads of swine-origin influenza H3N2 variant (H3N2v) viruses were reported in humans, resulting in 437 human infections between 2011 and 2021 in the USA. Thus, an effective vaccine is needed to better control a potential pandemic for these antigenically distinct viruses from seasonal influenza. In this study, a candidate vaccine strain with efficient growth capacity in chicken embryos was established through serial blind passaging of A/Indiana/08/2011 (H3N2)v in mice and chicken embryos. Seven amino acid substitutions (M21I in PA; A138T, N165K, and V226A in HA; S312L in NP; T167I in M1; G62A in NS1 proteins) were found in the passaged viruses without a major change in the antigenicity. This mouse- and egg-adapted virus was used as a vaccine and challenge strain in mice to evaluate the efficacy of the H3N2v vaccine in different doses. Antibodies with high neutralizing titers were induced in mice immunized with 100 µg of inactivated whole-virus particles, and those mice were significantly protected from the challenge of homologous strain. The findings indicated that the established strain in the study was useful for vaccine study in mouse models.

Segment 2 from influenza A(H1N1) 2009 pandemic viruses confers temperature-sensitive haemagglutinin yield on candidate vaccine virus growth in eggs that can be epistatically complemented by PB2 701D.

Candidate vaccine viruses (CVVs) for seasonal influenza A virus are made by reassortment of the antigenic virus with an egg-adapted strain, typically A/Puerto Rico/8/34 (PR8). Many 2009 A(H1N1) pandemic (pdm09) high-growth reassortants (HGRs) selected this way contain pdm09 segment 2 in addition to the antigenic genes. To investigate this, we made CVV mimics by reverse genetics (RG) that were either 6 : 2 or 5 : 3 reassortants between PR8 and two pdm09 strains, A/California/7/2009 (Cal7) and A/England/195/2009, differing in the source of segment 2. The 5 : 3 viruses replicated better in MDCK-SIAT1 cells than the 6 : 2 viruses, but the 6 : 2 CVVs gave higher haemagglutinin (HA) antigen yields from eggs. This unexpected phenomenon reflected temperature sensitivity conferred by pdm09 segment 2, as the egg HA yields of the 5 : 3 viruses improved substantially when viruses were grown at 35 °C compared with 37.5 °C, whereas the 6 : 2 virus yields did not. However, the authentic 5 : 3 pdm09 HGRs, X-179A and X-181, were not markedly temperature sensitive despite their PB1 sequences being identical to that of Cal7, suggesting compensatory mutations elsewhere in the genome. Sequence comparisons of the PR8-derived backbone genes identified polymorphisms in PB2, NP, NS1 and NS2. Of these, PB2 N701D affected the temperature dependence of viral transcription and, furthermore, improved and drastically reduced the temperature sensitivity of the HA yield from the 5 : 3 CVV mimic. We conclude that the HA yield of pdm09 CVVs can be affected by an epistatic interaction between PR8 PB2 and pdm09 PB1, but that this can be minimized by ensuring that the backbones used for vaccine manufacture in eggs contain PB2 701D.

Antibodies Against Egg- and Cell-Grown Influenza A(H3N2) Viruses in Adults Hospitalized During the 2017-2018 Influenza Season.

Influenza vaccine effectiveness was low in 2017-2018, yet circulating influenza A(H3N2) viruses were antigenically similar to cell-grown vaccine strains. Notably, most influenza vaccines are egg propagated. Serum specimens were collected shortly after illness onset from 15 influenza A(H3N2) virus-infected cases and 15 uninfected hospitalized adults. Geometric mean titers against egg- and cell-grown influenza A/Hong Kong/4801/2014(H3N2) virus vaccine strains and representative circulating viruses (including A/Washington/16/2017) were determined by a microneutralization (MN) assay. Independent effects of strain-specific titers on susceptibility were estimated by logistic regression. MN titers against egg-grown influenza A/Hong Kong virus were significantly higher among vaccinated individuals (173 vs 41; P = 0.01). In unadjusted models, a 2-fold increase in titers against egg-grown influenza A/Hong Kong virus was not significantly protective (29% reduction; P = .09), but a similar increase in the cell-grown influenza A/Washington virus antibody titer (3C.2a2) was protective (60% reduction; P = .02). Higher egg-grown influenza A/Hong Kong virus titers were not significantly associated with infection, when adjusted for antibody titers against influenza A/Washington virus (15% reduction; P = .61). A 54% reduction in the odds of infection was observed with a 2-fold increase in titer against influenza A/Washington virus (P = not significant), adjusted for the titer against egg-grown influenza A/Hong Kong virus titer. Individuals vaccinated in 2017-2018 had high antibody titers against the egg-adapted vaccine strain and lower titers against circulating viruses. Titers against circulating but not egg-adapted strains were correlated with protection.

Identification of human vaccinees that possess antibodies targeting the egg-adapted hemagglutinin receptor binding site of an H1N1 influenza vaccine strain

Human influenza viruses passaged in eggs often acquire mutations in the hemagglutinin (HA) receptor binding site (RBS). To determine if egg-adapted H1N1 vaccines commonly elicit antibodies targeting the egg-adapted RBS of HA, we completed hemagglutinin-inhibition assays with A/California/7/2009 HA and egg-adapted A/California/7/2009-X-179A HA using sera collected from 159 humans vaccinated with seasonal influenza vaccines during the 2015–16 season. We found that ∼5% of participants had ≥4-fold higher antibody titers to the egg-adapted viral strain compared to wild type viral strain. We used reverse-genetics to demonstrate that a single egg-adapted HA RBS mutation (Q226R) was responsible for this phenotype.

Contemporary H3N2 influenza viruses have a glycosylation site that alters binding of antibodies elicited by egg-adapted vaccine strains

H3N2 viruses continuously acquire mutations in the hemagglutinin (HA) glycoprotein that abrogate binding of human antibodies. During the 2014-2015 influenza season, clade 3C.2a H3N2 viruses possessing a new predicted glycosylation site in antigenic site B of HA emerged, and these viruses remain prevalent today. The 2016-2017 seasonal influenza vaccine was updated to include a clade 3C.2a H3N2 strain; however, the egg-adapted version of this viral strain lacks the new putative glycosylation site. Here, we biochemically demonstrate that the HA antigenic site B of circulating clade 3C.2a viruses is glycosylated. We show that antibodies elicited in ferrets and humans exposed to the egg-adapted 2016-2017 H3N2 vaccine strain poorly neutralize a glycosylated clade 3C.2a H3N2 virus. Importantly, antibodies elicited in ferrets infected with the current circulating H3N2 viral strain (that possesses the glycosylation site) and humans vaccinated with baculovirus-expressed H3 antigens (that possess the glycosylation site motif) were able to efficiently recognize a glycosylated clade 3C.2a H3N2 virus. We propose that differences in glycosylation between H3N2 egg-adapted vaccines and circulating strains likely contributed to reduced vaccine effectiveness during the 2016-2017 influenza season. Furthermore, our data suggest that influenza virus antigens prepared via systems not reliant on egg adaptations are more likely to elicit protective antibody responses that are not affected by glycosylation of antigenic site B of H3N2 HA.

Influenza immunization elicits antibodies specific for an egg-adapted vaccine strain.

For broad protection against infection by viruses such as influenza or HIV, vaccines should elicit antibodies that bind conserved viral epitopes, such as the receptor-binding site (RBS). RBS-directed antibodies have been described for both HIV and influenza virus, and the design of immunogens to elicit them is a goal of vaccine research in both fields. Residues in the RBS of influenza virus hemagglutinin (HA) determine a preference for the avian or human receptor, α-2,3-linked sialic acid and α-2,6-linked sialic acid, respectively. Transmission of an avian-origin virus between humans generally requires one or more mutations in the sequences encoding the influenza virus RBS to change the preferred receptor from avian to human, but passage of a human-derived vaccine candidate in chicken eggs can select for reversion to avian receptor preference. For example, the X-181 strain of the 2009 new pandemic H1N1 influenza virus, derived from the A/California/07/2009 isolate and used in essentially all vaccines since 2009, has arginine at position 226, a residue known to confer preference for an α-2,3 linkage in H1 subtype viruses; the wild-type A/California/07/2009 isolate, like most circulating human H1N1 viruses, has glutamine at position 226. We describe, from three different individuals, RBS-directed antibodies that recognize the avian-adapted H1 strain in current influenza vaccines but not the circulating new pandemic 2009 virus; Arg226 in the vaccine-strain RBS accounts for the restriction. The polyclonal sera of the three donors also reflect this preference. Therefore, when vaccines produced from strains that are never passaged in avian cells become widely available, they may prove more capable of eliciting RBS-directed, broadly neutralizing antibodies than those produced from egg-adapted viruses, extending the established benefits of current seasonal influenza immunizations.

Effectiveness of Influenza Vaccine in Adults Using A Test-negative, Case-control Design ―2013/2014 and 2014/2015 Seasons―

The influenza vaccine forms the basis of efforts to prevent the occurrence of influenza virus infection. However, vaccine effectiveness (VE) differs every season, which complicates efforts to combat the spread of infection. To develop a robust method to analyse variations in VE, we assessed VE among adult patients with influenza using a test-negative, case-control study design that evaluated vaccination records and the corresponding results of rapid influenza diagnostic tests during the 2013/14 and 2014/15 influenza seasons. During the 2013/14season, the adjusted VEs against influenza A and B viruses were 54.9% (95% confidence interval [CI] = 24.2% - 73.2%) and 56.6% (95% CI = 19.1% - 76.7%), respectively. In contrast, during the 2014/15season, the adjusted VE against the influenza A (H3N2), virus was -2% (95% CI = -66% - 37.5%). Moreover, only a few patients were infected with the influenza B virus, thus, the VE against influenza B could not be assessed. The low VE during the 2014/15 season could be attributed to antigenic drift in the circulating influenza A (H3N2) viruses and mutations in the egg-adapted vaccine strains. Estimation of the VE against the influenza virus using this test-negative, case-control study design was simple and easy, and this study design had a precision similar to that of a randomized control trial. Therefore, this study design could be employed to predict VE through out the influenza season and may be used as the basis of influenza prophylaxis.

Protein-Carbohydrate Interactions, and Beyond …

Carbohydrates are ubiquitous and play an intriguing role inside the cell as well as on the cell surface.[...]

Molecular Characterisation of the Haemagglutinin Glycan-Binding Specificity of Egg-Adapted Vaccine Strains of the Pandemic 2009 H1N1 Swine Influenza A Virus.

The haemagglutinin (HA) glycan binding selectivity of H1N1 influenza viruses is an important determinant for the host range of the virus and egg-adaption during vaccine production. This study integrates glycan binding data with structure-recognition models to examine the impact of the K123N, D225G and Q226R mutations (as seen in the HA of vaccine strains of the pandemic 2009 H1N1 swine influenza A virus). The glycan-binding selectivity of three A/California/07/09 vaccine production strains, and purified recombinant A/California/07/09 HAs harboring these mutations was examined via a solid-phase ELISA assay. Wild-type A/California/07/09 recombinant HA bound specifically to α2,6-linked sialyl-glycans, with no affinity for the α2,3-linked sialyl-glycans in the array. In contrast, the vaccine virus strains and recombinant HA harboring the Q226R HA mutation displayed a comparable pattern of highly specific binding to α2,3-linked sialyl-glycans, with a negligible affinity for α2,6-linked sialyl-glycans. The D225G A/California/07/09 recombinant HA displayed an enhanced binding affinity for both α2,6- and α2,3-linked sialyl-glycans in the array. Notably its α2,6-glycan affinity was generally higher compared to its α2,3-glycan affinity, which may explain why the double mutant was not naturally selected during egg-adaption of the virus. The K123N mutation which introduces a glycosylation site proximal to the receptor binding site, did not impact the α2,3/α2,6 glycan selectivity, however, it lowered the overall glycan binding affinity of the HA; suggesting glycosylation may interfere with receptor binding. Docking models and ‘per residues’ scoring were employed to provide a structure-recognition rational for the experimental glycan binding data. Collectively, the glycan binding data inform future vaccine design strategies to introduce the D225G or Q226R amino acid substitutions into recombinant H1N1 viruses.

Influenza virus PB1 and neuraminidase gene segments can cosegregate during vaccine reassortment driven by interactions in the PB1 coding region.

Egg-grown influenza vaccine yields are maximized by infection with a seed virus produced by "classical reassortment" of a seasonal isolate with a highly egg-adapted strain. Seed viruses are selected based on a high-growth phenotype and the presence of the seasonal hemagglutinin (HA) and neuraminidase (NA) surface antigens. Retrospective analysis of H3N2 vaccine seed viruses indicated that, unlike other internal proteins that were predominantly derived from the high-growth parent A/Puerto Rico/8/34 (PR8), the polymerase subunit PB1 could be derived from either parent depending on the seasonal strain. We have recently shown that A/Udorn/307/72 (Udorn) models a seasonal isolate that yields reassortants bearing the seasonal PB1 gene. This is despite the fact that the reverse genetics-derived virus that includes Udorn PB1 with Udorn HA and NA on a PR8 background has inferior growth compared to the corresponding virus with PR8 PB1. Here we use competitive plasmid transfections to investigate the mechanisms driving selection of a less fit virus and show that the Udorn PB1 gene segment cosegregates with the Udorn NA gene segment. Analysis of chimeric PB1 genes revealed that the coselection of NA and PB1 segments was not directed through the previously identified packaging sequences but through interactions involving the internal coding region of the PB1 gene. This study identifies associations between viral genes that can direct selection in classical reassortment for vaccine production and which may also be of relevance to the gene constellations observed in past antigenic shift events where creation of a pandemic virus has involved reassortment. Influenza vaccine must be produced and administered in a timely manner in order to provide protection during the winter season, and poor-growing vaccine seed viruses can compromise this process. To maximize vaccine yields, manufacturers create hybrid influenza viruses with gene segments encoding the surface antigens from a seasonal virus isolate, important for immunity, and others from a virus with high growth properties. This involves coinfection of cells with both parent viruses and selection of dominant progeny bearing the seasonal antigens. We show that this method of creating hybrid viruses does not necessarily select for the best yielding virus because preferential pairing of gene segments when progeny viruses are produced determines the genetic makeup of the hybrids. This not only has implications for how hybrid viruses are selected for vaccine production but also sheds light on what drives and limits hybrid gene combinations that arise in nature, leading to pandemics.

Transplacental transmission of field and rescued strains of BTV-2 and BTV-8 in experimentally infected sheep

Transplacental transmission of bluetongue virus has been shown previously for the North European strain of serotype 8 (BTV-8) and for tissue culture or chicken egg-adapted vaccine strains but not for field strains of other serotypes. In this study, pregnant ewes (6 per group) were inoculated with either field or rescued strains of BTV-2 and BTV-8 in order to determine the ability of these viruses to cross the placental barrier. The field BTV-2 and BTV-8 strains was passaged once in Culicoides KC cells and once in mammalian cells. All virus inoculated sheep became infected and seroconverted against the different BTV strains used in this study. BTV RNA was detectable in the blood of all but two ewes for over 28 days but infectious virus could only be detected in the blood for a much shorter period. Interestingly, transplacental transmission of BTV-2 (both field and rescued strains) was demonstrated at high efficiency (6 out of 13 lambs born to BTV-2 infected ewes) while only 1 lamb of 12 born to BTV-8 infected ewes showed evidence of in utero infection. In addition, evidence for horizontal transmission of BTV-2 between ewes was observed. As expected, the parental BTV-2 and BTV-8 viruses and the viruses rescued by reverse genetics showed very similar properties to each other. This study showed, for the first time, that transplacental transmission of BTV-2, which had been minimally passaged in cell culture, can occur; hence such transmission might be more frequent than previously thought.

The Source of the PB1 Gene in Influenza Vaccine Reassortants Selectively Alters the Hemagglutinin Content of the Resulting Seed Virus

The yields of egg-grown influenza vaccines are maximized by the production of a seed strain using a reassortment of the seasonal influenza virus isolate with a highly egg-adapted strain. The seed virus is selected based on high yields of viral hemagglutinin (HA) and expression of the surface antigens from the seasonal isolate. The remaining proteins are usually derived from the high-growth parent. However, a retrospective analysis of vaccine seeds revealed that the seasonal PB1 gene was selected in more than 50% of reassortment events. Using the model seasonal H3N2 virus A/Udorn/307/72 (Udorn) virus and the high-growth A/Puerto Rico/8/34 (PR8) virus, we assessed the influence of the source of the PB1 gene on virus growth and vaccine yield. Classical reassortment of these two strains led to the selection of viruses that predominantly had the Udorn PB1 gene. The presence of Udorn PB1 in the seed virus, however, did not result in higher yields of virus or HA compared to the yields in the corresponding seed virus with PR8 PB1. The 8-fold-fewer virions produced with the seed virus containing the Udorn PB1 were somewhat compensated for by a 4-fold increase in HA per virion. A higher HA/nucleoprotein (NP) ratio was found in past vaccine preparations when the seasonal PB1 was present, also indicative of a higher HA density in these vaccine viruses. As the HA viral RNA (vRNA) and mRNA levels in infected cells were similar, we propose that PB1 selectively alters the translation of viral mRNA. This study helps to explain the variability of vaccine seeds with respect to HA yield.

Cloned cDNA of A/swine/Iowa/15/1930 internal genes as a candidate backbone for reverse genetics vaccine against influenza A viruses

Reverse genetics viruses for influenza vaccine production usually utilize the internal genes of the egg-adapted A/Puerto Rico/8/34 (PR8) strain. This egg-adapted strain provides high production yield in embryonated eggs but does not necessarily give the best yield in mammalian cell culture. In order to generate a reverse genetics viral backbone that is well-adapted to high growth in mammalian cell culture, a swine influenza isolate A/swine/Iowa/15/30 (H1N1) (rg1930) that was shown to give high yield in Madin-Darby canine kidney (MDCK) cells was used as the internal gene donor for reverse genetics plasmids. In this report, the internal genes from rg1930 were used for construction of reverse genetics viruses carrying a cleavage site-modified hemagglutinin (HA) gene and neuraminidase (NA) gene from a highly pathogenic H5N1 virus. The resulting virus (rg1930H5N1) was low pathogenic in vivo. Inactivated rg1930H5N1 vaccine completely protected chickens from morbidity and mortality after challenge with highly pathogenic H5N1. Protective immunity was obtained when chickens were immunized with an inactivated vaccine consisting of at least 2(9) HA units of the rg1930H5N1 virus. In comparison to the PR8-based reverse genetics viruses carrying the same HA and NA genes from an H5N1 virus, rg1930 based viruses yielded higher viral titers in MDCK and Vero cells. In addition, the reverse genetics derived H3N2 and H5N2 viruses with the rg1930 backbone replicated in MDCK cells better than the cognate viruses with the rgPR8 backbone. It is concluded that this newly established reverse genetics backbone system could serve as a candidate for a master donor strain for development of inactivated influenza vaccines in cell-based systems.

Bench-to-bedside review: Vaccine protection strategies during pandemic flu outbreaks

Vaccination is the most effective means for the prevention of influenza, including pandemic strains. An ideal pandemic influenza vaccine should provide effective protection with the fewest number of doses in the shortest amount of time, and among the greatest proportion of the population. The current manufacturing processes required for embryonated chicken-egg-based influenza vaccines are limited in their ability to respond to pandemic situations - these limitations include problems with surge capacity, the need for egg-adapted strains, the possibility of contamination, and the presence of trace egg protein. Several vaccine strategies to circumvent the deficiencies intrinsic to an egg-based influenza vaccine are in various phases of development. These include the use of cell-culture-based growth systems, concomitant use of adjuvants, whole virus vaccines, recombinant protein vaccines, plasmid DNA vaccines, virus-like particle vaccines, and universal flu vaccines.

Mismatched hemagglutinin and neuraminidase specificities in recent human H3N2 influenza viruses

The hemagglutinin (HA) of influenza viruses initiates infection by binding to sialic acid on the cell surface via α2,6 (human) or α2,3 (avian) linkage. The influenza neuraminidase (NA) can cleave both α2,3- and α2,6-linked sialic acids, but all influenza NAs have a marked preference for the non-human α2,3 linkage. Recent H3N2 influenza viruses have lost the ability to agglutinate chicken red blood cells. To determine if changes in HA specificity or affinity correlate with NA specificity or activity, we examined red cell binding and elution of a series of H3N2 viruses. We found that the NA activity of many influenza viruses does not release binding by their HA. In some egg-adapted strains, lack of elution correlates with low levels of viral NA activity, and these elute rapidly when bacterial NA is added. However, a Fujian-like virus, A/Oklahoma/323/03, does not elute by its own NA or with Vibrio cholerae sialidase, and it binds to red cells pre-treated with V. cholerae sialidase. It elutes after addition of the broad specificity Micromonospora viridifaciens sialidase. Human glycophorin inhibits A/Oklahoma/323/03 hemagglutination 6-fold better than fetuin. We conclude that specific forms of sialic acid are used as receptor by recent human H3N2 influenza viruses, perhaps involving branched α2,6 sialic acid or α2,8 sialic acid structures on O-linked carbohydrates. The virus itself has no O-linked glycans, so even though the NA is not able to cleave receptors on cells, the viruses will not self-aggregate. It will be important to monitor efficacy of neuraminidase inhibitors in case there are NA-resistant receptors in the human respiratory tract that allow the viruses to be less dependent on NA activity.

Masking of the contribution of V protein to sendai virus pathogenesis in an infection model with a highly virulent field isolate

Sendai virus V protein is not essential for virus replication in cultured cells but is essential for efficient virus replication and pathogenesis in mice, indicating that the V protein has a luxury function to facilitate virus propagation in mice. This was discovered in the Z strain, an egg-adapted avirulent laboratory strain. In the present study, we reexamined the function of Sendai virus V protein by generating a V-knockout Sendai virus derived from the Hamamatsu strain, a virulent field isolate, which is an appropriate model for studying the natural course of Sendai virus infection in mice. We unexpectedly found that the V-knockout virus propagated efficiently in mice and was as virulent as the wild-type virus. Switching of the functionally important V unique region demonstrated that this region of the Hamamatsu strain was also functional in a Z strain background. It thus appears that the V protein is nonsense in a field isolate of Sendai virus. However, the V protein was required for virus growth and pathogenesis of the Hamamatsu strain in mice when the virulence of the virus was attenuated by introducing mutations that had been found in an egg-adapted, avirulent virus. The V protein therefore seems to be potentially functional in the highly virulent Hamamatsu strain and to be prominent if virus replication is restricted.

Egg-adapted replication-restricted virus protects mice against lethal influenza

Infection of mice with the egg-adapted (EA) strain of influenza virus was studied as a murine model of human live attenuated virus vaccine. The growth and spread of the EA virus in the mouse lungs were restricted, and only small inflammatory changes were detected in the respiratory tracts. Deletion and substitutions of amino acids were found in the hemagglutinin molecule of the EA virus, which were attributable to the reduced envelope fusion activity in virus multiplication cycles. Intranasal inoculation of mice with the EA virus induced specific IgG and IgA antibody production together with a specific cytotoxic T lymphocyte response. Immunized mice showed a distinct resistance to subsequent lethal challenge with the virulent influenza virus. These results indicate that the mutant virus loaded with a growth restriction in the respiratory tract is an appropriate candidate for a live attenuated vaccine.