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BACKGROUND: Influenza vaccine viruses grown in eggs may acquire egg-adaptive mutations that may reduce antigenic similarity between vaccine and circulating influenza viruses and decrease vaccine effectiveness. We compared cell- and egg-based quadrivalent influenza vaccines (QIVc and QIVe, respectively) for preventing test-confirmed influenza over 3 US influenza seasons (2017-2020). METHODS: Using a retrospective test-negative design, we estimated the relative vaccine effectiveness (rVE) of QIVc vs QIVe among individuals aged 4 to 64 years who had an acute respiratory or febrile illness and were tested for influenza in routine outpatient care. Exposure, outcome, and covariate data were obtained from electronic health records linked to pharmacy and medical claims. Season-specific rVE was estimated by comparing the odds of testing positive for influenza among QIVc vs QIVe recipients. Models were adjusted for age, sex, geographic region, influenza test date, and additional unbalanced covariates. A doubly robust approach was used combining inverse probability of treatment weights with multivariable regression. RESULTS: The study included 31 824, 33 388, and 34 398 patients in the 2017-2018, 2018-2019, and 2019-2020 seasons, respectively; ∼10% received QIVc and ∼90% received QIVe. QIVc demonstrated superior effectiveness vs QIVe in prevention of test-confirmed influenza: rVEs were 14.8% (95% CI, 7.0%-22.0%) in 2017-2018, 12.5% (95% CI, 4.7%-19.6%) in 2018-2019, and 10.0% (95% CI, 2.7%-16.7%) in 2019-2020. CONCLUSIONS: This study demonstrated consistently superior effectiveness of QIVc vs QIVe in preventing test-confirmed influenza over 3 seasons characterized by different circulating viruses and degrees of egg adaptation.

The immune response to inactivated influenza vaccines (IIV) is influenced by multiple factors, including hemagglutinin content and egg-based manufacturing. Only two US-licensed vaccines are manufactured without egg passage: cell culture-based inactivated vaccine (ccIIV) and recombinant vaccine (RIV). We conducted a randomized open-label trial in central Wisconsin during the 2018–19 and 2019–20 seasons to compare immunogenicity of sequential vaccination. Participants 18–64 years old were randomized 1:1:1 to receive RIV, ccIIV or IIV in strata defined by number of influenza vaccine doses in the prior 3 years. They were revaccinated with the same product in year two. Paired serum samples were tested by hemagglutination inhibition against egg-adapted and cell-grown vaccine viruses. Serologic endpoints included geometric mean titer (GMT), mean fold rise, and percent seroconversion. There were 373 participants randomized and vaccinated in 2018–19; 332 were revaccinated in 2019–20. In 2018–19, RIV and ccIIV were not more immunogenic than IIV against A/H1N1. The post-vaccination GMT against the cell-grown 3C.2a A/H3N2 vaccine virus was higher for RIV vs IIV (p = .001) and RIV vs ccIIV (p = .001). The antibody response to influenza B viruses was similar across study arms. In 2019–20, GMT against the cell-grown 3C.3a A/H3N2 vaccine virus was higher for RIV vs IIV (p = .03) and for RIV vs ccIIV (p = .001). RIV revaccination generated significantly greater backboosting to the antigenically distinct 3C.2a A/H3N2 virus (2018–19 vaccine strain) compared to ccIIV or IIV. This study adds to the evidence that RIV elicits a superior immunologic response against A/H3N2 viruses compared to other licensed influenza vaccine products.

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Aim: Seasonal influenza poses a significant burden of disease, affecting not only older adults but also individuals under the age of 60. It carries a high economic burden, mainly driven by influenza-associated productivity losses in the working population. Conventional egg-based influenza vaccines may have reduced effectiveness due to antigen adaptation in eggs. In contrast, cell-based influenza vaccines are less likely to be affected by such antigen adaptation. This review aims to present real-world data (RWD) comparing the effectiveness of quadrivalent cell-based (QIVc) and egg-based (QIVe) influenza vaccines over three consecutive seasons.Methods: A comprehensive review was conducted, analyzing RWD from retrospective cohort and case-control studies on the relative vaccine effectiveness (rVE) of QIVc versus QIVe during the 2017/18–2019/20 seasons.Results: This study included six retrospective cohort studies and one case-control study, with a combined total of approximately 29 million participants. A cohort study involving people aged ≥4 years during the 2017/18 season showed a statistically significant rVE of QIVc compared to QIVe in preventing influenza-like illness, with a value of 36.2%. QIVc demonstrated statistically significant superiority over QIVe in preventing outpatient and inpatient medical encounters as observed in two cohort studies conducted during the 2018/19 and 2019/20 seasons. The rVE of QIVc compared to QIVe was found to be 7.6% in individuals aged ≥ears and 9.5% in individuals aged ≥18 years. Three additional cohort studies conducted between 2017/18–2019/20 reported a statistically significant improvement in rVE (5.3–14.4%) of QIVc compared to QIVe in preventing influenza-related hospitalizations and emergency department visits due to influenza in individuals aged 4–64 years. In a case-control study across all three seasons, QIVc showed statistically significantly higher effectiveness compared to QIVe in preventing test-confirmed influenza, with rVEs of 10.0–14.8%.Conclusions: RWD from the 2017/18–2019/20 seasons demonstrated that QIVc is more effective than QIVe in preventing influenza-related outcomes in individuals aged 4–64 years. Preferential use of cell-based influenza vaccines, as opposed to conventional egg-based vaccines, could reduce the burden of influenza-related symptoms on individuals and alleviate the economic impact on the German population under 60 years of age.

To ensure that vaccination offers the best protection against an infectious disease, sequence identity between the vaccine and the circulating strain is paramount. During replication of nucleic acid, random mutations occur due to the level of polymerase fidelity. In traditional influenza vaccine manufacture, vaccine viruses are propagated in fertilized chicken eggs, which can result in egg-adaptive mutations in the antigen-encoding genes. Whilst this improves infection and replication in eggs, mutations may reduce the effectiveness of egg-based influenza vaccines against circulating human viruses. In contrast, egg-adaptive mutations are avoided when vaccine viruses are propagated in Madin-Darby canine kidney (MDCK) cell lines during manufacture of cell-based inactivated influenza vaccines. The first mammalian cell-only strain was included in Flucelvax® Quadrivalent in 2017. A sequence analysis of the viruses selected for inclusion in this vaccine (n = 15 vaccine strains, containing both hemagglutinin and neuraminidase) demonstrated that no mutations occur in the antigenic sites of either hemagglutinin or neuraminidase, indicating that cell adaptation does not occur during production of this cell-based vaccine. The development of this now entirely mammalian-based vaccine system, which incorporates both hemagglutinin and neuraminidase, ensures that the significant protective antigens are equivalent to the strains recommended by the World Health Organization (WHO) in both amino acid sequence and glycosylation pattern. The inclusion of both proteins in a vaccine may provide an advantage over recombinant vaccines containing hemagglutinin alone. Findings from real world effectiveness studies support the use of cell-based influenza vaccines. [ABSTRACT FROM AUTHOR]

Background: Quadrivalent cell-based influenza vaccines (QIVc) avoid egg-adaptive mutations and can be more effective than traditional quadrivalent egg-based influenza vaccines (QIVe). This analysis compared the cost-effectiveness of QIVc and QIVe in Argentinian populations < 65 years old from the payer and societal perspectives. Methods: A static decision tree model compared the costs and health benefits of vaccination with QIVc vs. QIVe using a one-year time horizon. The relative vaccine effectiveness of QIVc vs. QIVe was assumed to be 8.1% for children and 11.4% for adults. An alternative high egg-adaptation scenario was also assessed. Model inputs were sourced from Argentina or the international literature. Deterministic and probabilistic sensitivity analyses were performed. Results: Compared to QIVe, QIVc would prevent 17,857 general practitioner visits, 2418 complications, 816 hospitalizations, and 12 deaths per year. From the payers' perspective, the incremental cost-effectiveness ratio per quality-adjusted life years gained was USD12,214 in the base case and USD2311 in the high egg-adaptation scenario. QIVc was cost-saving from the societal perspective in both scenarios. Conclusions: QIVc in Argentina would be cost-effective relative to QIVe. The potential health benefits and savings would be even higher in high egg-adaptation seasons. [ABSTRACT FROM AUTHOR]

The immune response to inactivated influenza vaccines (IIV) is influenced by multiple factors, including hemagglutinin content and egg-based manufacturing. Only two US-licensed vaccines are manufactured without egg passage: cell culture-based inactivated vaccine (ccIIV) and recombinant vaccine (RIV). We conducted a randomized open-label trial in central Wisconsin during the 2018-19 and 2019-20 seasons to compare immunogenicity of sequential vaccination. Participants 18-64 years old were randomized 1:1:1 to receive RIV, ccIIV or IIV in strata defined by number of influenza vaccine doses in the prior 3 years. They were revaccinated with the same product in year two. Paired serum samples were tested by hemagglutination inhibition against egg-adapted and cell-grown vaccine viruses. Serologic endpoints included geometric mean titer (GMT), mean fold rise, and percent seroconversion. There were 373 participants randomized and vaccinated in 2018-19; 332 were revaccinated in 2019-20. In 2018-19, RIV and ccIIV were not more immunogenic than IIV against A/H1N1. The post-vaccination GMT against the cell-grown 3C.2a A/H3N2 vaccine virus was higher for RIV vs IIV ( p  = .001) and RIV vs ccIIV ( p  = .001). The antibody response to influenza B viruses was similar across study arms. In 2019-20, GMT against the cell-grown 3C.3a A/H3N2 vaccine virus was higher for RIV vs IIV ( p  = .03) and for RIV vs ccIIV ( p  = .001). RIV revaccination generated significantly greater backboosting to the antigenically distinct 3C.2a A/H3N2 virus (2018-19 vaccine strain) compared to ccIIV or IIV. This study adds to the evidence that RIV elicits a superior immunologic response against A/H3N2 viruses compared to other licensed influenza vaccine products.

To ensure that vaccination offers the best protection against an infectious disease, sequence identity between the vaccine and the circulating strain is paramount. During replication of nucleic acid, random mutations occur due to the level of polymerase fidelity. In traditional influenza vaccine manufacture, vaccine viruses are propagated in fertilized chicken eggs, which can result in egg-adaptive mutations in the antigen-encoding genes. Whilst this improves infection and replication in eggs, mutations may reduce the effectiveness of egg-based influenza vaccines against circulating human viruses. In contrast, egg-adaptive mutations are avoided when vaccine viruses are propagated in Madin-Darby canine kidney (MDCK) cell lines during manufacture of cell-based inactivated influenza vaccines. The first mammalian cell-only strain was included in Flucelvax® Quadrivalent in 2017. A sequence analysis of the viruses selected for inclusion in this vaccine (n = 15 vaccine strains, containing both hemagglutinin and neuraminidase) demonstrated that no mutations occur in the antigenic sites of either hemagglutinin or neuraminidase, indicating that cell adaptation does not occur during production of this cell-based vaccine. The development of this now entirely mammalian-based vaccine system, which incorporates both hemagglutinin and neuraminidase, ensures that the significant protective antigens are equivalent to the strains recommended by the World Health Organization (WHO) in both amino acid sequence and glycosylation pattern. The inclusion of both proteins in a vaccine may provide an advantage over recombinant vaccines containing hemagglutinin alone. Findings from real world effectiveness studies support the use of cell-based influenza vaccines.

Background: Quadrivalent cell-based influenza vaccines (QIVc) avoid egg-adaptive mutations and can be more effective than traditional quadrivalent egg-based influenza vaccines (QIVe). This analysis compared the cost-effectiveness of QIVc and QIVe in Argentinian populations < 65 years old from the payer and societal perspectives. Methods: A static decision tree model compared the costs and health benefits of vaccination with QIVc vs. QIVe using a one-year time horizon. The relative vaccine effectiveness of QIVc vs. QIVe was assumed to be 8.1% for children and 11.4% for adults. An alternative high egg-adaptation scenario was also assessed. Model inputs were sourced from Argentina or the international literature. Deterministic and probabilistic sensitivity analyses were performed. Results: Compared to QIVe, QIVc would prevent 17,857 general practitioner visits, 2418 complications, 816 hospitalizations, and 12 deaths per year. From the payers’ perspective, the incremental cost-effectiveness ratio per quality-adjusted life years gained was USD12,214 in the base case and USD2311 in the high egg-adaptation scenario. QIVc was cost-saving from the societal perspective in both scenarios. Conclusions: QIVc in Argentina would be cost-effective relative to QIVe. The potential health benefits and savings would be even higher in high egg-adaptation seasons.

Background: An antigenic mismatch between the vaccine and circulating H3N2 strains was hypothesized to contribute to the severity of the 2017-2018 season in North America.Methods: Serum and nasal washes were collected from influenza positive and negative patients during the 2017-2018 season to determine neutralizing antibody (nAb) titers and for influenza virus sequencing, respectively.Results: The circulating and vaccine H3N2 virus strains were different clades, with the vaccine strain being clade 3C.2a and the circulating viruses being 3C.2a2 or 3C.3a. At enrollment, both the H3N2 negative and positive patients had greater nAb titers to the egg-adapted vaccine virus compared to the cell-grown vaccine but the H3N2-negative population had significantly greater titers to the circulating 3C.2a2. Among H3N2-positive patients, vaccination, younger age, and female sex were associated with greater nAb responses to the egg-adapted vaccine H3N2 virus but not to the cell-grown vaccine or circulating viruses.Conclusions: For the 2017-2018 circulating viruses, mutations introduced by egg adaptation decreased vaccine efficacy. No increased protection was afforded by vaccination, younger age, or female sex against 2017-2018 circulating H3N2 viruses. [ABSTRACT FROM AUTHOR]

Purpose: When influenza viruses are cultured in eggs, amino acid mutations of the hemagglutinin may occur through egg adaptation. On the other hand, when influenza viruses are cultured in animal cells, no antigenic mutation occurs unlike in eggs. Therefore, we examined whether the antigenic mutations actually occurred after passage of H3N2 (A/Texas/50/2012) virus up to 15 times in eggs and MDCK-Sky3851 cells. Materials and Methods: Prototype A/Texas/50/2012 (H3N2) influenza virus which was isolated from clinical patient, not passaged in egg, was obtained and propagated using the specific pathogen free egg and the MDCK-Sky3851 cell line up to 15 passage, and the changes in the antigen sequence of the influenza viruses were confirmed by gene sequencing and protein structure analysis. Results: In term of the hemagglutination titer of influenza virus, the reactivity to chicken and guinea pig red blood cell showed different results between egg propagated and cell propagated viruses. In the sequence analysis results for hemagglutinin and neuraminidase, no antigenic mutation was observed throughout all passages when cultured in MDCK-Sky3851 cells. On the other hand, mutations occurred in three amino acid sequences (H156R, G186S, S219F) in hemagglutinin up to 15 passages when cultured in eggs. Conclusion: H3N2 influenza virus cultured in eggs could lead mutations in amino acid sequence of hemagglutinin, distinct from the corresponding virus cultured in cells for which no antigenic mutation was observed. These findings suggest that cell culture is a more stable and effective way of production with lower risk of antigenic mutations for the manufacture of influenza vaccines. [ABSTRACT FROM AUTHOR]

The population < 60 years of age is also affected by a significant disease burden from seasonal influenza. It carries a high economic burden, mainly driven by influenza-associated productivity losses in the working population. Conventional egg-based influenza vaccines may experience reduced effectiveness due to antigen adaptation in eggs. In contrast, cell-based influenza vaccines are less likely to be affected by antigenic adaptations to the host system and showed better effectiveness in individuals 4-64 years old over several seasons compared to conventional egg-based influenza vaccines under real-world conditions. Preferential use of cell-based influenza vaccines, as opposed to conventional egg-based vaccines, could reduce the burden of influenza-related symptoms on individuals and alleviate the economic impact on the German population < 60 years of age., (© 2024. The Author(s).)

Background: 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.Methods: 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.Results: 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.Conclusion: 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. [ABSTRACT FROM AUTHOR]

An H9N2 avian influenza A virus (AIV), A/chicken/Korea/01310/2001 (01310-CE20), was established after 20 passages of influenza A/chicken/Korea/01310/2001 (01310-CE2) virus through embryonated chicken eggs (ECEs). As a result of this process, the virus developed highly replicative and pathogenic traits within the ECEs through adaptive mutations in hemagglutinin (HA: T133N, V216G, and E439D) and neuraminidase (NA: 18-amino acid deletion and E54D). Here, we also established that 01310-CE20 acquired resistance to innate inhibitors present in the egg white during these passages. To investigate the role of egg-adapted mutations in resistance to innate inhibitors, we generated four PR8-derived recombinant viruses using various gene combinations of HA and NA from 01310-CE2 and 01310-CE20 (rH2N2, rH2N20, rH20N2, and rH20N20). As expected, rH20N20 showed significantly higher replication efficiency in MDCK cells and mouse lungs, and demonstrated greater pathogenicity in mice. In addition, rH20N20 showed higher resistance to innate inhibitors than the other viruses. By using a loss-of-function mutant and receptor-binding assay, we demonstrated that a T133N site directed mutation created an additional N-glycosite at position 133 in rH20N20. Further, this mutation played a crucial role in viral replication and resistance to innate inhibitors by modulating the binding affinities to avian-like and mammalian-like receptors on the host cells and inhibitors. Thus, egg-adapted HA and NA may exacerbate the mammalian pathogenicity of AIVs by defying host innate inhibitors as well as by increasing replication efficiency in mammalian cells.

Background: Although ferret antisera used in influenza surveillance did not detect antigenic drift of A(H1N1)pdm09 viruses during the 2015-2016 season, low vaccine effectiveness was reported in adults. We investigated the immune basis of low responses to circulating A(H1N1)pdm09 viruses after vaccination.Methods: Prevaccination and postvaccination serum samples collected from >300 adults (aged 18-49 years) in 6 seasons (2010-2011 to 2015-2016) were analyzed using hemagglutination inhibition assays to evaluate the antibody responses to 13 A(H1N1) viruses circulated from 1977 to 2016. Microneutralization and serum adsorption assays were used to verify the 163K and 223R specificity of antibodies.Results: Individual antibody profiles to A(H1N1) viruses revealed 3 priming patterns: USSR/77, TW/86, or NC/99 priming. More than 20% of adults had reduced titers to cell-propagated circulating 6B.1 and 6B.2 A(H1N1)pdm09 viruses compared with the A/California/07/2009 vaccine virus X-179A. Significantly reduced antibody reactivity to circulating viruses bearing K163Q was observed only in the USSR/77-primed cohort, whereas significantly lower reactivity caused by egg-adapted Q223R change was detected across all 3 cohorts.Conclusion: Both 163K specificity driven by immune priming and 223R specificity from egg-adapted changes in the vaccine contributed to low responses to circulating A(H1N1)pdm09 viruses after vaccination. Our study highlights the need to incorporate human serology in influenza surveillance and vaccine strain selection. [ABSTRACT FROM AUTHOR]

An H9N2 avian influenza A virus (AIV), A/chicken/Korea/01310/2001 (01310-CE20), was established after 20 passages of influenza A/chicken/Korea/01310/2001 (01310-CE2) virus through embryonated chicken eggs (ECEs). As a result of this process, the virus developed highly replicative and pathogenic traits within the ECEs through adaptive mutations in hemagglutinin (HA: T133N, V216G, and E439D) and neuraminidase (NA: 18-amino acid deletion and E54D). Here, we also established that 01310-CE20 acquired resistance to innate inhibitors present in the egg white during these passages. To investigate the role of egg-adapted mutations in resistance to innate inhibitors, we generated four PR8-derived recombinant viruses using various gene combinations of HA and NA from 01310-CE2 and 01310-CE20 (rH2N2, rH2N20, rH20N2, and rH20N20). As expected, rH20N20 showed significantly higher replication efficiency in MDCK cells and mouse lungs, and demonstrated greater pathogenicity in mice. In addition, rH20N20 showed higher resistance to innate inhibitors than the other viruses. By using a loss-of-function mutant and receptor-binding assay, we demonstrated that a T133N site directed mutation created an additional N-glycosite at position 133 in rH20N20. Further, this mutation played a crucial role in viral replication and resistance to innate inhibitors by modulating the binding affinities to avian-like and mammalian-like receptors on the host cells and inhibitors. Thus, egg-adapted HA and NA may exacerbate the mammalian pathogenicity of AIVs by defying host innate inhibitors as well as by increasing replication efficiency in mammalian cells. [ABSTRACT FROM AUTHOR]

Background Influenza vaccination aims to prevent infection by influenza virus and reduce associated morbidity and mortality; however, vaccine effectiveness (VE) can be modest, especially for subtype A(H3N2). Low VE has been attributed to mismatches between the vaccine and circulating influenza strains and to the vaccine’s elicitation of protective immunity in only a subset of the population. The low H3N2 VE in the 2012–2013 season was attributed to egg-adaptive mutations that created antigenic mismatch between the actual vaccine strain (IVR-165) and both the intended vaccine strain (A/Victoria/361/2011) and the predominant circulating strains (clades 3C.2 and 3C.3). Methods We investigated the basis of low VE in 2012–2013 by determining whether vaccinated and unvaccinated individuals were infected by different viral strains and by assessing the serologic responses to IVR-165, A/Victoria/361/2011, and 3C.2 and 3C.3 strains in an adult cohort before and after vaccination. Results We found no significant genetic differences between the strains that infected vaccinated and unvaccinated individuals. Vaccination increased titers to A/Victoria/361/2011 and 3C.2 and 3C.3 representative strains as much as to IVR-165. These results are consistent with the hypothesis that vaccination boosted cross-reactive immune responses instead of specific responses against unique vaccine epitopes. Only approximately one-third of the cohort achieved a ≥4-fold increase in titer. Conclusions In contrast to analyses based on ferret studies, low H3N2 VE in 2012–2013 in adults does not appear to be due to egg adaptation of the vaccine strain. Instead, low VE might have been caused by low vaccine immunogenicity in a subset of the population. [ABSTRACT FROM AUTHOR]

Human influenza viruses preferentially bind to sialic acid-α2,6-galactose (SAα2,6Gal) receptors, which are predominant in human upper respiratory epithelia, whereas avian influenza viruses preferentially bind to SAα2,3Gal receptors. However, variants with amino acid substitutions around the receptor-binding sites of the hemagglutinin (HA) protein can be selected after several passages of human influenza viruses from patients’ respiratory samples in the allantoic cavities of embryonated chicken eggs. In this study, we detected an egg-adapted HA S190R mutation in the pandemic H1N1 virus 2009 (pdmH1N1), and evaluated the effects of this mutation on receptor binding affinity and pathogenicity in mice. Our results revealed that residue 190 is located within the pocket structure of the receptor binding site. The single mutation to arginine at position 190 slightly increased the binding affinity of the virus to the avian receptor and decreased its binding to the long human α2,6-linked sialic acid receptor. Our study demonstrated that the S190R mutation resulted in earlier death and higher weight loss in mice compared with the wild-type virus. Higher viral titers at 1 dpi (days post infection) and diffuse damage at 4 dpi were observed in the lung tissues of mice infected with the mutant virus. [ABSTRACT FROM AUTHOR]

In 2013, a novel avian-origin H7N9 influenza A virus causing severe lower respiratory tract disease in humans emerged in China, with continued sporadic cases. An effective vaccine is needed for this virus in case it acquires transmissibility among humans; however, PR8-based A/Anhui/1/2013 (Anhui/1, H7N9), a WHO-recommended H7N9 candidate vaccine virus (CVV) for vaccine production, does not replicate well in chicken eggs, posing an obstacle to egg-based vaccine production. To address this issue, we explored the possibility that PR8’s hemagglutinin (HA) and neuraminidase (NA) packaging signals mediate improvement of Anhui/1 CVV yield in eggs. We constructed chimeric HA and NA genes having the coding region of Anhui/1 HA and NA flanked by the 5′ and 3′ packaging signals of PR8’s HA and NA, respectively. The growth of CVVs containing the chimeric HA was not affected, but that of those containing the chimeric NA gene grew in embryonated chicken eggs with a more than 2-fold higher titer than that of WT CVV. Upon 6 passages in eggs further yield increase was achieved although this was not associated with any changes in the chimeric NA gene. The HA of the passaged CVV, did, however, exhibit egg-adaptive mutations and one of them (HA-G218E) improved CVV growth in eggs without significantly changing antigenicity. The HA-G218E substitution and a chimeric NA, thus, combine to provide an Anhui/1 CVV with properties more favorable for vaccine manufacture. [ABSTRACT FROM AUTHOR]

The recently detected zoonotic H3N2 variant influenza A (H3N2v) viruses have caused 343 documented cases of human infection linked to contact with swine. An effective vaccine is needed for these viruses, which may acquire transmissibility among humans. However, viruses isolated from human cases do not replicate well in embryonated chicken eggs, posing an obstacle to egg-based vaccine production. To address this issue, we sought to identify egg-adaptive mutations in surface proteins that increase the yield of candidate vaccine viruses (CVVs) in eggs while preserving their immunizing effectiveness. After serial passage of a representative H3N2v isolate (A/Indiana/08/2011), we identified several egg-adaptive combinations of HA mutations and assessed the egg-based replication, antigenicity, and immunogenicity of A/Puerto Rico/8/34 (H1N1, PR8)-based 6 + 2 reverse genetics CVVs carrying these mutations. Here we demonstrate that the respective combined HA substitutions G186 1 V + N246 1 K, N165 1 K + G186 1 V, T128 1 N + N165 1 K + R76 2 G, and T128 1 N + N165 1 K + I10 2 M, all identified after egg passage, enhanced the replication of the CVVs in eggs without substantially affecting their antigenicity or immunogenicity. The mutations were stable, and the mutant viruses acquired no additional substitutions during six subsequent egg passages. We found two crucial mutations, G186V, which was previously defined, and N246K, which in combination improved virus yield in eggs without significantly impacting antigenicity or immunogenicity. This combination of egg-adaptive mutations appears to most effectively generate high egg-based yields of influenza A/Indiana/08/2011-like CVVs. [ABSTRACT FROM AUTHOR]

To ensure that vaccination offers the best protection against an infectious disease, sequence identity between the vaccine and the circulating strain is paramount. During replication of nucleic acid, random mutations occur due to the level of polymerase fidelity. In traditional influenza vaccine manufacture, vaccine viruses are propagated in fertilized chicken eggs, which can result in egg-adaptive mutations in the antigen-encoding genes. Whilst this improves infection and replication in eggs, mutations may reduce the effectiveness of egg-based influenza vaccines against circulating human viruses. In contrast, egg-adaptive mutations are avoided when vaccine viruses are propagated in Madin-Darby canine kidney (MDCK) cell lines during manufacture of cell-based inactivated influenza vaccines. The first mammalian cell-only strain was included in Flucelvax ® Quadrivalent in 2017. A sequence analysis of the viruses selected for inclusion in this vaccine ( n = 15 vaccine strains, containing both hemagglutinin and neuraminidase) demonstrated that no mutations occur in the antigenic sites of either hemagglutinin or neuraminidase, indicating that cell adaptation does not occur during production of this cell-based vaccine. The development of this now entirely mammalian-based vaccine system, which incorporates both hemagglutinin and neuraminidase, ensures that the significant protective antigens are equivalent to the strains recommended by the World Health Organization (WHO) in both amino acid sequence and glycosylation pattern. The inclusion of both proteins in a vaccine may provide an advantage over recombinant vaccines containing hemagglutinin alone. Findings from real world effectiveness studies support the use of cell-based influenza vaccines.

The article presents a study on the molecular changes involved in the adaptation of equine influenza H3N8 virus in embryonated chicken eggs (ECEs). The researchers determined the virus titers after conducting a hemagglutination test using the red bloods cells of chicken based on the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals published by the World Organization for Animal Health. The found that the adaptation of the equine influenza virus H3N8 to ECEs resulted in six amino acid substitutions in the internal proteins PA, NP and M1 aside from the surface protein HA.

Abstract: Influenza H3N2 viruses have recently drifted from A/Wisconsin/67/05-like to A/Brisbane/10/07-like viruses. The effect of key amino acid substitutions in the hemagglutinin (HA) protein on the replication, antigenicity and immunogenicity of cold-adapted, live attenuated vaccine strains was examined. A/Brisbane/10/07 egg isolate contained a unique combination of G186 and P194 which were required for efficient virus growth in Madin–Darby Canine Kidney (MDCK) cells and embryonated chicken eggs, but the virus induced low level of serum antibody response in ferrets. Substitution of the G186 and P194 in the HA of A/Brisbane/10/07 by V186 and L194 that were present in the HA of A/Wisconsin/67/05-like viruses significantly impaired virus replication but greatly improved the immunogenicity of the vaccine virus to a level comparable to that elicited by the A/Wisconsin/67/05 vaccine. The replication of the variants with impaired growth could be improved by amino acid substitutions at the 195 or 226 residues. The viruses with the G186 and P194 residues were antigenically distinct from the viruses with V186 and L194. Sequence analysis of the HA sequences of the H3N2 viruses from the database and sequencing of the HA gene of a cell-derived A/Brisbane/10/07-like virus before and after egg passage indicated that the P194 residue was most likely derived from egg adaptation. Our results demonstrated the importance of careful evaluation of vaccine strains to ensure that the selected vaccines not only replicate well in eggs, but also retain their antigenicity and are immunogenic in the host. [Copyright &y& Elsevier]

Summary: The currently circulating influenza B viruses from both antigenic lineages contain an N-linked glycosylation site in the hemagglutinin (HA) protein at positions of 196 or 197. However, egg adaptation caused the loss of the glycosylation site that could impact virus antigenicity and vaccine efficacy. The effect of the 196/197 glycosylation site on influenza B virus growth and antigenicity was systemically evaluated in this study by the molecular approach. Paired recombinant 6:2 reassortant influenza B vaccine strains, with or without the 196/197 glycosylation site, were generated by reverse genetics and the glycosylation site was retained in MDCK cells. In contrast, all the viruses that contained the introduced glycosylation site were unable to grow in eggs and rapidly lost the glycosylation site once adapted to grow in eggs. We showed that glycosylation affected virus binding to the α-2,3-linked sialic acid receptor and affected virus antigenicity as tested by postinfected ferret sera. We have further identified that the Arginine residue at amino acid position 141 (141R) can stabilize the 196/197 glycosylation site without affecting virus antigenicity. Thus, the 141R could be introduced into vaccine strains to retain the 196/197 glycosylation site for influenza B vaccines. [Copyright &y& Elsevier]

Receptor-binding specificity of HA. the major surface glycoprotein of influenza virus, primarily determines the host ranges that the virus can infect. Influenza type B virus almost exclusively infects humans and contributes to the annual ‘flu’ sickness. Here we report the structures of influenza B virus HA in complex with human and avian receptor analogs, respectively. These structures provide a structural basis for the different receptor-binding properties of influenza A and B virus HA molecules and for the ability of influenza B virus HA to distinguish human and avian receptors. The structure of influenza B virus HA with avian receptor analog also reveals how mutations in the region of residues 194 to 196, which are frequently observed in egg-adapted and naturally occurring variants, directly affect the receptor binding of the resultant virus strains. Furthermore, these structures of influenza B virus HA are compared with known structures of influenza A virus HAs, which suggests the role of the residue at 222 as a key and likely a universal determinant for the different binding modes of human receptor analogs by different HA molecules. [ABSTRACT FROM AUTHOR]

Seasonal influenza vaccines prevent influenza-related illnesses, hospitalizations, and deaths. However, these vaccines are not as effective as other viral vaccines, and there is clearly room for improvement. Here, we review the history of seasonal influenza vaccines, describe challenges associated with producing influenza vaccine antigens, and discuss the inherent difficulties of updating influenza vaccine strains each influenza season. We argue that seasonal influenza vaccines can be dramatically improved by modernizing antigen production processes and developing models that are better at predicting viral evolution. Resources should be specifically dedicated to improving seasonal influenza vaccines while developing entirely new vaccine platforms.

Purpose: When influenza viruses are cultured in eggs, amino acid mutations of the hemagglutinin may occur through egg adaptation. On the other hand, when influenza viruses are cultured in animal cells, no antigenic mutation occurs unlike in eggs. Therefore, we examined whether the antigenic mutations actually occurred after passage of H3N2 (A/Texas/50/2012) virus up to 15 times in eggs and MDCK-Sky3851 cells., Materials and Methods: Prototype A/Texas/50/2012 (H3N2) influenza virus which was isolated from clinical patient, not passaged in egg, was obtained and propagated using the specific pathogen free egg and the MDCK-Sky3851 cell line up to 15 passage, and the changes in the antigen sequence of the influenza viruses were confirmed by gene sequencing and protein structure analysis., Results: In term of the hemagglutination titer of influenza virus, the reactivity to chicken and guinea pig red blood cell showed different results between egg propagated and cell propagated viruses. In the sequence analysis results for hemagglutinin and neuraminidase, no antigenic mutation was observed throughout all passages when cultured in MDCK-Sky3851 cells. On the other hand, mutations occurred in three amino acid sequences (H156R, G186S, S219F) in hemagglutinin up to 15 passages when cultured in eggs., Conclusion: H3N2 influenza virus cultured in eggs could lead mutations in amino acid sequence of hemagglutinin, distinct from the corresponding virus cultured in cells for which no antigenic mutation was observed. These findings suggest that cell culture is a more stable and effective way of production with lower risk of antigenic mutations for the manufacture of influenza vaccines., Competing Interests: No potential conflict of interest relevant to this article was reported., (© Korean Vaccine Society.)

Antigenic changes in the hemagglutinin protein of recent A(H3N2) viruses often arise when these viruses adapt to their egg host. By serial egg passages of a cell-propagated virus, we successfully isolated an egg-adapted influenza A(H3N2) virus, A/Saitama/103/2014, without amino acid substitutions at the antigenic sites of its hemagglutinin protein but with multiple substitutions in its neuraminidase protein. Antigenic analysis of this egg-adapted A/Saitama/103/2014 virus indicated that its antigenicity did not differ from that of the World Health Organization prototype cell-propagated vaccine virus: A/Hong Kong/4801/2014. Our results suggest that this strategy may facilitate egg-based vaccine production without antigenic alterations in hemagglutinin by egg adaptation.