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In March 2013 it was reported by the World Health Organization (WHO) the first cases of human infections with avian influenza virus A (H7N9). From 2013 to December 2019, 1568 cases have been reported with 616 deaths. H7N9 infection has been associated with high morbidity and mortality rates, and vaccination is currently the most effective way to prevent infections and consequently flu-related severe illness. Developing and producing vaccines against pandemic influenza viruses is the main strategy for a response to a possible pandemic. This study aims to present the production of three industrial lots under current Good Manufacturing Practices (cGMP) of the active antigen used to produce the pandemic influenza vaccine candidate against A(H7N9). These batches were characterized and evaluated for quality standards and tested for immunogenicity in mice. The average yield was 173.50 ± 7.88 μg/mL of hemagglutinin and all the preparations met all the required specifications. The formulated H7N9 vaccine is poorly immunogenic and needs to be adjuvanted with an oil in water emulsion adjuvant (IB160) to achieve a best immune response, in a prime and in a boost scheme. These data are important for initial production planning and preparedness in the case of a H7N9 pandemic., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Introduction : Influenza Virus-like Particles (VLPs) are one of the most promising vaccine strategies to complement traditional egg-based processes and contribute to shortening the response time when facing future pandemics. Research programs have taken advantage of the potential of this approach to produce influenza VLPs on a variety of cellular platforms, reaching the industrial level of development and recent commercialization. Area covered : This review aims to give an overview of available strategies for influenza-VLP production and their respective stages of development, from small-scale preclinical studies to large-scale industrial processes. Recent trends and fulfillments in purification schemes of influenza VLP were also reviewed with regards to quality and potency requirements that go along with influenza vaccine manufacturing. Expert opinion : In the next five years, it is expected that there will be licensing of new influenza vaccine products based on VLP strategy. Few VLP upstream processes are mature enough and close to fully complement or seriously concurrence the ovoculture process. Nevertheless, many improvements have yet to be achieved in downstream processes. In the next few years, research efforts in this field are expected to provide purification strategies and tools to achieve higher recovery yields and improve the cost-effectiveness of VLP processes.

Continuous cell culture-based influenza vaccine production could significantly reduce footprint and manufacturing costs compared to current batch processing. However, yields of influenza virus in continuous mode can be affected by oscillations in virus titers caused by periodic accumulation of defective interfering particles. The generation of such particles has also been observed previously in cascades of continuous stirred tank reactors (CSTRs) and is known as the "von Magnus effect". To improve virus yields and to avoid these oscillations, we have developed a novel continuous tubular bioreactor system for influenza A virus production. It was built using a 500 mL CSTR for cell growth linked to a 105 m long tubular plug-flow bioreactor (PFBR). Virus propagation took place only in the PFBR with a nominal residence time of 20 h and a production capacity of 0.2 mL/min. The bioreactor was first tested with suspension MDCK cells at different multiplicities of infection (MOI), and then with suspension avian AGE1.CR.pIX cells at a fixed nominal MOI of 0.02. Maximum hemagglutinin (HA) titers of 2.4 and 1.6 log10(HA units/100 μL) for suspension MDCK cells and AGE1.CR.pIX cells, respectively, were obtained. Flow cytometric analysis demonstrated that 100% infected cells with batch-like HA titers can be obtained at a MOI of at least 0.1. Stable HA and TCID50 titers over 18 days of production were confirmed using the AGE1.CR.pIX cell line, and PCR analysis demonstrated stable production of full-length genome. The contamination level of segments with deletions (potentially defective interfering particles), already present in the virus seed, was low and did not increase. Control experiments using batch and semi-continuous cultures confirmed these findings. A comparison showed that influenza virus production can be achieved with the tubular bioreactor system in about half the time with a space-time-yield up to two times higher than for typical batch cultures. In summary, a novel continuous tubular bioreactor system for cell culture-based influenza virus production was developed. One main advantage, an essentially single-passage amplification of viruses, should enable efficient production of vaccines as well as vectors for gene and cancer therapy., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: This work was part of the doctoral project of FT and was funded by the Max Planck Institute (MPI). FT, YG and UR are employed by the MPI. FT, YG and UR are authors on patent application WO/2017/190790 entitled “Plug-flow bioreactor, system containing the same, and method for production of virus” which covers the tubular bioreactor described in this work. The AGE1.CR.pIX cell line is a proprietary product of ProBioGen AG, who authorized its use in this project. IJ and VS are employed by ProBioGen AG. IJ and VS are authors on patents WO/2005/042728 and WO/2007/054516 covering the AGE1.CR.pIX cell line. No additional funding was provided by ProBioGen AG to the work described in this manuscript. There are no further patents, products in development or marketed products to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Novel low-pathogenic avian influenza (LPAI) H5N2 viruses hit poultry farms in Taiwan in 2003, and evolved into highly pathogenic avian influenza (HPAI) viruses in 2010. These viruses are reassortant viruses containing HA and NA genes from American-lineage H5N2 and six internal genes from local H6N1 viruses. According to a serological survey, the Taiwan H5N2 viruses can cause asymptomatic infections in poultry workers. Therefore, a development of influenza H5N2 vaccines is desirable for pandemic preparation. In this study, we employed reverse genetics to generate a vaccine virus having HA and NA genes from A/Chicken/CY/A2628/2012 (E7, LPAI) and six internal genes from a Vero cell-adapted high-growth H5N1 vaccine virus (Vero-15). The reassortant H5N2 vaccine virus, E7-V15, presented high-growth efficiency in Vero cells (512 HAU, 10 7.6 TCID 50 /mL), and passed all tests for qualification of candidate vaccine viruses. In ferret immunization, two doses of inactivated whole virus antigens (3 μg of HA protein) adjuvanted with alum could induce robust antibody response (HI titre 113.14). In conclusion, we have established reverse genetics to generate a qualified reassortant H5N2 vaccine virus for further development.

H7N9, a novel strain of avian origin influenza was the first recorded incidence where a human was transited by a N9 type influenza virus. Effective vaccination against influenza A (H7N9) is a major concern, since it has emerged as a life threatening viral pathogen. Here, an in silico reverse vaccinology strategy was adopted to design a unique chimeric subunit vaccine against avian influenza A (H7N9). Induction of humoral and cell-mediated immunity is the prime concerned characteristics for a peptide vaccine candidate, hence both T cell and B cell immunity of viral proteins were screened. Antigenicity testing, transmembrane topology screening, allergenicity and toxicity assessment, population coverage analysis and molecular docking approach were adopted to generate the most antigenic epitopes of avian influenza A (H7N9) proteome. Further, a novel subunit vaccine was designed by the combination of highly immunogenic epitopes along with suitable adjuvant and linkers. Physicochemical properties and secondary structure of the designed vaccine were assessed to ensure its thermostability, h ydrophilicity, theoretical PI and structural behavior. Homology modeling, refinement and validation of the designed vaccine allowed to construct a three dimensional structure of the predicted vaccine, further employed to molecular docking analysis with different MHC molecules and human immune TLR8 receptor present on lymphocyte cells. Moreover, disulfide engineering was employed to lessen the high mobility region of the designed vaccine in order to extend its stability. Furthermore, we investigated the molecular dynamic simulation of the modeled subunit vaccine and TLR8 complexed molecule to strengthen our prediction. Finally, the suggested vaccine was reverse transcribed and adapted for E. coli strain K12 prior to insertion within pET28a(+) vector for checking translational potency and microbial expression., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objectives: Novel approaches to advance the field of vaccinology must be investigated, and are particularly of importance for influenza in order to produce a more effective vaccine. A systematic review of human challenge studies for influenza was performed, with the goal of assessing safety and ethics and determining how these studies have led to therapeutic and vaccine development. A systematic review of systems biology approaches for the study of influenza was also performed, with a focus on how this technology has been utilized for influenza vaccine development. Methods: The PubMed database was searched for influenza human challenge studies, and for systems biology studies that have addressed both influenza infection and immunological effects of vaccination. Results: Influenza human challenge studies have led to important advancements in therapeutics and influenza immunization, and can be performed safely and ethically if certain criteria are met. Many studies have investigated the use of systems biology for evaluating immune response to influenza vaccine, and several promising molecular signatures may help advance our understanding of pathogenesis and be used as targets for influenza interventions. Combining these methodologies has the potential to lead to significant advances in the field of influenza vaccinology and therapeutics. Conclusions: Human challenge studies and systems biology approaches are important tools that should be used in concert to advance our understanding of influenza infection and provide targets for novel therapeutics and immunizations.

Influenza vaccines targeting the highly conserved stem of the hemagglutinin (HA) surface glycoprotein have the potential to protect against pandemic and drifted seasonal influenza viruses not covered by current vaccines. While HA stem-based immunogens derived from group 1 influenza A viruses have been shown to induce intragroup heterosubtypic protection, HA stem-specific antibody lineages originating from group 2 may be more likely to possess broad cross-group reactivity. We report the structure-guided development of mammalian-cell-expressed candidate vaccine immunogens based on influenza A virus group 2 H3 and H7 HA stem trimers displayed on self-assembling ferritin nanoparticles using an iterative, multipronged approach involving helix stabilization, loop optimization, disulfide bond addition, and side-chain repacking. These immunogens were thermostable, formed uniform and symmetric nanoparticles, were recognized by cross-group-reactive broadly neutralizing antibodies (bNAbs) with nanomolar affinity, and elicited protective, homosubtypic antibodies in mice. Importantly, several immunogens were able to activate B cells expressing inferred unmutated common ancestor (UCA) versions of cross-group-reactive human bNAbs from two multidonor classes, suggesting they could initiate elicitation of these bNAbs in humans. IMPORTANCE Current influenza vaccines are primarily strain specific, requiring annual updates, and offer minimal protection against drifted seasonal or pandemic strains. The highly conserved stem region of hemagglutinin (HA) of group 2 influenza A virus subtypes is a promising target for vaccine elicitation of broad cross-group protection against divergent strains. We used structure-guided protein engineering employing multiple protein stabilization methods simultaneously to develop group 2 HA stem-based candidate influenza A virus immunogens displayed as trimers on self-assembling nanoparticles. Characterization of antigenicity, thermostability, and particle formation confirmed structural integrity. Group 2 HA stem antigen designs were identified that, when displayed on ferritin nanoparticles, activated B cells expressing inferred unmutated common ancestor (UCA) versions of human antibody lineages associated with cross-group-reactive, broadly neutralizing antibodies (bNAbs). Immunization of mice led to protection against a lethal homosubtypic influenza virus challenge. These candidate vaccines are now being manufactured for clinical evaluation.

The alarming rise of morbidity and mortality caused by influenza pandemics and epidemics has drawn attention worldwide since the last few decades. This life-threatening problem necessitates the development of a safe and effective vaccine to protect against incoming pandemics. The currently available flu vaccines rely on inactivated viral particles, M2e-based vaccine, live attenuated influenza vaccine (LAIV) and virus like particle (VLP). While inactivated vaccines can only induce systemic humoral responses, LAIV and VLP vaccines stimulate both humoral and cellular immune responses. Yet, these vaccines have limited protection against newly emerging viral strains. These strains, however, can be targeted by universal vaccines consisting of conserved viral proteins such as M2e and capable of inducing cross-reactive immune response. The lack of viral genome in VLP and M2e-based vaccines addresses safety concern associated with existing attenuated vaccines. With the emergence of new recombinant viral strains each year, additional effort towards developing improved universal vaccine is warranted. Besides various types of vaccines, microRNA and exosome-based vaccines have been emerged as new types of influenza vaccines which are associated with new and effective properties. Hence, development of a new generation of vaccines could contribute to better treatment of influenza., (© 2018 John Wiley & Sons, Ltd.)

The aim of this study was to construct a vaccine peptide candidate against pandemic Influenza H1N1 hemagglutinin and to test its structure. With the help of bioinformatic algorithms we showed that the sequence encoding the second polypeptide of pandemic Influenza H1N1 hemagglutinin (HA2) is protected from nonsynonymous mutations better than the sequence encoding its first polypeptide (HA1). With the help of secondary and ternary structure predicting algorithms we found the fragment of HA2 with the most reproducible secondary structure and synthesized the NY25 peptide corresponding to the residues Asn117 - Tyr141 of HA2. According to the circular dichroism spectra analysis, the peptide has short helix and beta hairpin. According to the analysis of differential fluorescence quenching results, two tyrosine residues are situated on a long distance from each other. These facts taken together with the positive results of affine chromatography with the serum of a person immunized by full-length hemagglutinin confirm that the structure of the fragment of viral full-length protein has been reproduced in the synthetic NY25 peptide. Amino acid sequence of the NY25 peptide (NLYEKVRSQLKNNAKEIGNGCFEFY) is relatively conserved in 18 subtypes of Influenza A virus hemagglutinin., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The H7N9 viruses that emerged in China in 2013 were nonpathogenic in chickens but mutated to a highly pathogenic form in early 2017 and caused severe disease outbreaks in chickens. The H7N9 influenza viruses have caused five waves of human infection, with almost half of the total number of human cases (766 of 1,567) being reported in the fifth wave, raising concerns that even more human infections could occur in the sixth wave. In September 2017, an H5/H7 bivalent inactivated vaccine for chickens was introduced, and the H7N9 virus isolation rate in poultry dropped by 93.3% after vaccination. More importantly, only three H7N9 human cases were reported between October 1, 2017 and September 30, 2018, indicating that vaccination of poultry successfully eliminated human infection with H7N9 virus. These facts emphasize that active control of animal disease is extremely important for zoonosis control and human health protection.

A major challenge in influenza research is the selection of an appropriate animal model that accurately reflects the disease and the protective immune response observed in humans. A workshop organised by the EDUFLUVAC consortium, a European Union funded project coordinated by the European Vaccine Initiative, brought together experts from the influenza vaccine community with the aim to discuss the current knowledge and future perspectives for testing broadly reactive influenza vaccines in animal models. The programme included a diversity of models from well-established and publicly accepted models to cutting edge, newly developed animal models as well as ex-vivo approaches and human models. The audience concluded that different vaccine approaches may require evaluation in different animal models, depending on the type of immune response induced by the vaccine. Safety is the main concern for transition to clinical development and influenza vaccine associated enhanced disease was specifically emphasised. An efficient animal model to evaluate this aspect of safety still needs to be identified. Working with animal models requires ethical compliance and consideration of the 3R principles. Development of alternative approaches such as ex-vivo techniques is progressing but is still at an early stage and these methods are not yet suitable for broader application for vaccine evaluation. The human challenge is the ultimate model to assess influenza vaccines. However this model is expensive and not largely applicable. The currently used pre-clinical models are not yet specifically focused on studying unique aspects of a universal influenza vaccine. Further collaboration, communication and effective networking are needed for success in establishment of harmonised and standardised pre-clinical models for evaluation of new influenza vaccines. This report does not provide a complete review of the field but discusses the data presented by the speakers and discussion points raised during the meeting., (Copyright © 2018.)

We tested a new A/H1N1 inactivated influenza vaccine (IIV) manufactured by Institute of Vaccines and Medical Biologics (IVAC), Vietnam in 48 adults in a Phase 1, double-blinded, randomized, placebo-controlled trial. Two doses of unadjuvanted vaccine or placebo were administered three weeks apart. The vaccine was well tolerated with only transient mild local reactions and low-grade fever in a small proportion of the subjects. One serious adverse event considered unrelated to the study product was reported. The IVAC vaccine proved to be highly immunogenic with 91 percent (95% CI: 0.78, 1) of the subjects developing a ≥4 fold immune responses by hemagglutination inhibition (HAI) assay, and 96 percent (95% CI: 0.78, 1) by the microneutralization (MN) assay. Post-vaccination geometric mean titers (GMTs) were 283.7 (95% CI: 161.7, 497.5) in the HAI and 725.7 (95% CI: 411.3, 1280.3) in the MN assay. These promising results merit further development of the vaccine. ClinicalTrials.gov number: NCT01507779., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Cold adapted influenza virus A/Leningrad/134/17/57 (H2N2) is a reliable master donor virus (Len/17-MDV) for preparing live attenuated influenza vaccines (LAIV). LAIVs are 6:2 reasortants that contain 6 segments of Len/17-MDV and the hemagglutinin (HA) and neuraminidase (NA) of contemporary circulating influenza A viruses. The problem with the classical reassortment procedure used to generate LAIVs is that there is limited selection pressure against NA of the Len/17-MDV resulting in 7:1 reassortants with desired HA only, which are not suitable LAIVs. The monoclonal antibodies (mAb) directed against the N2 of Len/17-MDV were generated. 10C4-8E7 mAb inhibits cell-to-cell spread of viruses containing the Len/17-MDV N2, but not viruses with the related N2 from contemporary H3N2 viruses. 10C4-8E7 antibody specifically inhibited the Len/17-MDV replication in vitro and in ovo but didn't inhibit replication of H3N2 or H1N1pdm09 reassortants. Our data demonstrate that addition of 10C4-8E7 in the classical reassortment improves efficiency of LAIV production., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A priority for the National Institute of Allergy and Infectious Diseases is development of a universal influenza vaccine providing durable protection against multiple influenza strains. NIAID will use this strategic plan as a foundation for future investments in influenza research.

As shown during the 2009 pandemic H1N1 (A(H1N1)pdm09) outbreak, egg-based influenza vaccine production technology is insufficient to meet global demands during an influenza pandemic. Therefore, there is a need to adapt cell culture-derived vaccine technology using suspended cell lines for more rapid and larger-scale vaccine production. In this study, we attempted to generate a high-growth influenza vaccine strain in MDCK cells using an A/Puerto/8/1934 (H1N1) vaccine seed strain. Following 48 serial passages with four rounds of virus plaque purification in MDCK cells, we were able to select several MDCK-adapted plaques that could grow over 10⁸ PFU/ml. Genetic characterization revealed that these viruses mainly had amino acid substitutions in internal genes and exhibited higher polymerase activities. By using a series of Rg viruses, we demonstrated the essential residues of each gene and identified a set of high-growth strains in MDCK cells (PB1 D153N , M1 A137T , and NS1 N176S ). In addition, we confirmed that in the context of the high-growth A/PR/8/34 backbone, A/California/7/2009 (H1N1), A/Perth/16/2009 (H3N2), and A/environment/Korea/deltaW150/2006 (H5N1) also showed significantly enhanced growth properties (more than 10⁷ PFU/ml) in both attached- and suspended-MDCK cells compared with each representative virus and the original PR8 vaccine strain. Taken together, this study demonstrates the feasibility of a cell culture-derived approach to produce seed viruses for influenza vaccines that are cheap and can be grown promptly and vigorously as a substitute for egg-based vaccines. Thus, our results suggest that MDCK cell-based vaccine production is a feasible option for producing large-scale vaccines in case of pandemic outbreaks.

Influenza vaccines are the most effective intervention to prevent the substantial public health burden of seasonal and pandemic influenza. The capability of hemagglutinin (HA), the main antigen in inactivated influenza vaccines (IIVs), to elicit functional neutralizing antibodies determines IIV effectiveness. When HA is subjected to environmental stress during manufacturing or while stored prior to administration, such as low pH and temperature excursions, the HA immunological activity can be affected. Single-radial immunodiffusion (SRID), the standard in vitro potency assay for IIVs, is believed to specifically detect immunologically active HA and has been applied to evaluate HA stability against stress. Here we report that transient low pH treatment and freeze/thaw cycles with HA in PBS abolish SRID-quantified in vitro potency for all HAs of multiple influenza strains. Raised temperature substantially decreases in vitro potency with more extensive HA structural changes. Chemical stress and mechanical stress moderately change SRID in vitro potency values in a strain-dependent manner. Trypsin digestion, which selectively degrades stressed HA, followed by RP-HPLC quantification as a candidate alternative in vitro potency assay yields results comparable to SRID. Mouse immunogenicity studies confirm that HA stressed by transient low pH treatment does not elicit functional antibodies in vivo, nor does it have a measureable SRID value. However, HA stressed by raised temperature elicits high titers of functional antibodies in vivo despite substantial loss of SRID in vitro potency. This discrepancy between SRID in vitro potency and vaccine immunogenicity suggests that SRID may not reliably indicate IIV potency under all conditions. Further efforts to develop alternate potency assays that can better predict in vivo immunogenicity should continue along with additional studies exploring HA conformation, SRID values and consequent immunogenicity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Human adenoviruses (HAdVs) are prevalent in pediatric and adult patients with severe acute respiratory disease (ARD). To date, there have been no widely used HAdV vaccines available. In this report, we developed a cold-adapted attenuated influenza virus, termed rg HAdV-Flu ca, carrying epitopes from HAdV hexon protein in the backbone of the ca influenza vaccine neuraminidase (NA) gene using reverse genetics. Rg HAdV-Flu ca virus exhibited a cold-adapted (ca) phenotype, and its morphological characteristics were observed using electron microscopy. Moreover, BALB/c mice were immunized intranasally (i.n.) with 10 5 , 10 6 or 10 7 TCID50 rg HAdV-Flu ca. Results showed a specific, robust antibody response against influenza and HAdV in a dose-dependent manner. More importantly, potent humoral, mucosal and cellular immune responses protected against subsequent wild-type HAdV-3 or HAdV-7 challenges, as determined by a significant decrease in viral titers and a noticeable alleviation of histopathological alterations in the lung tissue of challenged mice. These findings demonstrate that rg HAdV-Flu ca warrants attention as a potential vaccine candidate against HAdV infection., (Copyright © 2018. Published by Elsevier B.V.)

Numerous modern technological and scientific advances have changed the vaccine industry. However, nearly 70 years of influenza vaccine usage have passed without substantial changes in the underlying principles of the vaccine. The challenge of vaccinating against influenza lies in the constantly changing nature of the virus itself. Influenza viruses undergo antigenic evolution through antigenic drift and shift in their surface glycoproteins. This has forced frequent updates of vaccine antigens to ensure that the somewhat narrowly focused vaccine-induced immune responses defend against circulating strains. Few vaccine production systems have been developed that can entertain such constant changes. Although influenza virus infection induces long-lived immunologic memory to the same or similar strains, most people do not encounter the same strain repeatedly in their lifespan, suggesting that enhancement of natural immunity is required to improve influenza vaccines. It is clear that transformative change of influenza vaccines requires a rethink of how we immunize. In this study, we review the problems associated with the changing nature of the virus, and highlight some of the approaches being employed to improve influenza vaccines.

Empirically derived vaccines have in the past relied on the isolation and growth of disease-causing microorganisms that are then inactivated or attenuated before being administered. This is often done without prior knowledge of the mechanisms involved in conferring protective immunity. Recent advances in scientific technologies and in our knowledge of how protective immune responses are induced enable us to rationally design novel and safer vaccination strategies. Such advances have accelerated the development of inactivated whole-organism- and subunit-based vaccines. In this review, we discuss ideal attributes and criteria that need to be considered for the development of vaccines and some existing vaccine platforms. We focus on inactivated vaccines against influenza virus and ways by which vaccine efficacy can be improved with the use of adjuvants and Toll-like receptor-2 signaling.

Although the success of vaccination to date has been unprecedented, our inadequate understanding of the details of the human immune response to immunization has resulted in several recent vaccine failures and significant delays in the development of high-need vaccines for global infectious diseases and cancer. Because of the need to better understand the immense complexity of the human immune system, the Human Vaccines Project was launched in 2015 with the mission to decode the human immune response to accelerate development of vaccines and immunotherapies for major diseases. The Project currently has three programs: 1) The Human Immunome Program, with the goal of deciphering the complete repertoire of B and T cell receptors across the human population, termed the Human Immunome, 2) The Rules of Immunogenicity Program, with the goal of understanding the key principles of how a vaccine elicits a protective and durable response using a system immunology approach, and 3) The Universal Influenza Vaccine Initiative (UIVI), with the goal of conducting experimental clinical trials to understand the influence of influenza pre-exposures on subsequent influenza immunization and the mechanisms of protection. Given the dramatic advances in computational and systems biology, genomics, immune monitoring, bioinformatics and machine learning, there is now an unprecedented opportunity to unravel the intricacies of the human immune response to immunization, ushering in a new era in vaccine development.

The benefit of influenza vaccines is difficult to estimate due to the complexity of accurately assessing the burden of influenza. To improve the efficacy of influenza vaccines, vaccine manufacturers have developed quadrivalent influenza vaccine (QIV) formulations for seasonal vaccination by including both influenza B lineages. Three parallel approaches for producing influenza vaccines are attracting the interest of many vaccine manufacturing companies. The first and oldest is the conventional egg-derived influenza vaccine, which is used by the current licensed influenza vaccines. The second approach is a cell culture-derived influenza vaccine, and the third and most recent is synthetic vaccines. Here, we analyze the difficulties with vaccines production in eggs and compare this to cell culture-derived influenza vaccines and discuss the future of cell culture-derived QIVs.

The overwhelming majority of influenza vaccines are prepared with the use of chicken embryo allantoic fluid. The presence of ovalbumin (this protein constitutes >60% total protein in the allantoic fluid) in the vaccine can lead to severe allergy. Hence, effective reduction of ovalbumin content is of crucial importance for vaccine production. We compared two methods of purification and concentration of influenza virus: zonal gradient ultracentrifugation and combined ultrafiltration/diafiltration and exclusion chromatography protocol, used for fabrication of seasonal vaccines. Combined chromatography is comparable with zonal centrifugation protocol by the results of ovalbumin removal (to meet standard requirements).

Background: The lack of a universal influenza vaccine is a global health problem. Interest is now focused on structurally conserved protein domains capable of eliciting protection against a broad range of influenza virus strains. The long alpha helix (LAH) is an attractive vaccine component since it is one of the most conserved influenza hemagglutinin (HA) stalk regions. For an improved immune response, the LAH domain from H3N2 strain has been incorporated into virus-like particles (VLPs) derived from hepatitis B virus core protein (HBc) using recently developed tandem core technology., Results: Fermentation conditions for recombinant HBc-LAH were established in yeast Pichia pastoris and a rapid and efficient purification method for chimeric VLPs was developed to match the requirements for industrial scale-up. Purified VLPs induced strong antibody responses against both group 1 and group 2 HA proteins in mice., Conclusion: Our results indicate that the tandem core technology is a useful tool for incorporation of highly hydrophobic LAH domain into HBc VLPs. Chimeric VLPs can be successfully produced in bioreactor using yeast expression system. Immunologic data indicate that HBc VLPs carrying the LAH antigen represent a promising universal influenza vaccine component.

Introduction: Annual vaccination is one of the most efficient and cost-effective strategies to prevent and control influenza epidemics. Most of the currently available influenza vaccines are strong inducers of antibody responses against viral surface proteins, hemagglutinin (HA) and neuraminidase (NA), but are poor inducers of cell-mediated immune responses against conserved internal proteins. Moreover, due to the high variability of viral surface proteins because of antigenic drift or antigenic shift, many of the currently licensed vaccines confer little or no protection against drift or shift variants. Areas covered: Next generation influenza vaccines that can induce humoral immune responses to receptor-binding epitopes as well as broadly neutralizing conserved epitopes, and cell-mediated immune responses against highly conserved internal proteins would be effective against variant viruses as well as a novel pandemic influenza until circulating strain-specific vaccines become available. Here we discuss vaccine approaches that have the potential to provide broad spectrum protection against influenza viruses. Expert commentary: Based on current progress in defining cross-protective influenza immunity, it seems that the development of a universal influenza vaccine is feasible. It would revolutionize the strategy for influenza pandemic preparedness, and significantly impact the shelf-life and protection efficacy of seasonal influenza vaccines.

Despite all the advantages that cell-cultured influenza vaccines have over egg-based influenza vaccines, the inferior productivity of cell-culture systems is a major drawback that must be addressed. BST-2 (tetherin) is a host restriction factor which inhibits budding-out of various enveloped viruses from infected host cells. We developed BST-2-deficient MDCK and Vero cell lines to increase influenza virus release in cell culture. BST-2 gene knock-out resulted in increased release of viral particles into the culture medium, by at least 2-fold and up to 50-fold compared to release from wild-type counterpart cells depending on cell line and virus type. The effect was not influenza virus/MDCK/Vero-specific, but was also present in a broad range of host cells and virus families; we observed similar results in murine, human, canine, and monkey cell lines with viruses including MHV-68 (Herpesviridae), influenza A virus (Orthomyxoviridae), porcine epidemic diarrhea virus (Coronaviridae), and vaccinia virus (Poxviridae). Our results suggest that the elimination of BST-2 expression in virus-producing cell lines can enhance the production of viral vaccines. Biotechnol. Bioeng.2017;114: 2289-2297. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Background: It was to generate a new Vero and cold-adapted live attenuated influenza B vaccine with enough safety and immunogenicity., Methods: According to modified classical reassortment method, the donor strain was B/Yunnan/2/2005Vca(B), and the parental virus strain was B/Brisbane/60/2008wt. After co-infection in Vero cells, the prepared antibody serum inhibited the donor strain growth, and screening conditions inhibited the parental virus growth, which induced the growth of the new reassortant virus B/Brisbane/60/2008Vca(B) grow. Through intraperitoneal injection (i.j.) and intranasal injection (n.j.) we evaluated the safety and immunogenicity of the vaccine., Results: A high-yield of the reassortant virus was produced in Vero cells at 25°C, similar to the donor strains. After sequencing, it was found that B/Brisbane/60/2008Vca(B) Hemagglutinin (HA) and Neuraminidase (NA) gene fragments were from B/Brisbane/60/2008wt, while the other 6 gene fragments were from B/Yunnan/2/2005Vca(B). The n.j. immune pathway experiments showed no significant differences between the treatment and the PBS control group with respect to weight changes (P > 0.5). Furthermore, the new strain had a sufficient geometric mean titter (GMT) against B/Brisbane/60/2008wt., Conclusion: The new reassortant live attenuated influenza B vaccine was safe and having enough immune stimulating ability.

Influenza virus dominant antigens presentation using virus like particle (VLP) approach is attractive for the development of new generation of influenza vaccines. Mammalian cell platform offers many advantages for VLP production. However, limited attention has been paid to the processing of mammalian cell produced VLPs. Better understanding of the production system could contribute to increasing the yields and making large-scale VLP vaccine manufacturing feasible. In a previous study, we have generated a human embryonic kidney HEK-293 inducible cell line expressing Hemagglutinin (HA) and Neuraminidase (NA), which was used to produce VLPs upon transient transfection with a plasmid containing HIV-1 Gag. In this work, to streamline the production process, we have developed a new HEK-293 inducible cell line adapted to suspension growth expressing the three proteins HA, NA (H1N1 A/PR/8/1934) and the Gag fused to GFP for monitoring the VLP production. The process was optimized to reach higher volumetric yield of VLPs by increasing the cell density at the time of induction without sacrificing the cell specific productivity. A 5-fold improvement was achieved by doing media evaluation at small scale. Furthermore, a 3-L perfusion bioreactor mirrored the performance of small-scale shake flask cultures with sequential medium replacement. The cell density was increased to 14×10 6 cells/ml at the time of induction which augmented by 60-fold the volumetric yield to 1.54×10 10 Gag-GFP fluorescent events/ml, as measured by flow cytometry. The 9.5-L harvest from the perfusion bioreactor was concentrated by tangential flow filtration at low shear rate. The electron micrographs revealed the presence of VLPs of 100-150nm with the characteristic dense core of HIV-1 particles. The developed process shows the feasibility of producing high quantity of influenza VLPs from an inducible mammalian stable cell line aiming at large scale vaccine manufacturing., (Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.)

We have developed pandemic live attenuated influenza vaccines (pLAIVs) against clade 1 H5N1 viruses on an Ann Arbor cold-adapted ( ca ) backbone that induced long-term immune memory. In 2015, many human infections caused by a new clade (clade 2.2.1.1) of goose/Guangdong (gs/GD) lineage H5N1 viruses were reported in Egypt, which prompted updating of the H5N1 pLAIV. We explored two strategies to generate suitable pLAIVs. The first approach was to modify the hemagglutinin gene of a highly pathogenic wild-type ( wt ) clade 2.2.1.1 virus, A/Egypt/N03434/2009 (Egy/09) (H5N1), with its unmodified neuraminidase (NA) gene; this virus was designated Egy/09 ca The second approach was to select a low-pathogenicity avian influenza H5 virus that elicited antibodies that cross-reacted with a broad range of H5 viruses, including the Egypt H5N1 viruses, and contained a novel NA subtype for humans. We selected the low-pathogenicity A/duck/Hokkaido/69/2000 (H5N3) (dk/Hok/00) virus for this purpose. Both candidate vaccines were attenuated and immunogenic in ferrets, inducing antibodies that neutralized homologous and heterologous H5 viruses with different degrees of cross-reactivity; Egy/09 ca vaccine antisera were more specific for the gs/GD lineage viruses but did not neutralize recent North American isolates (clade 2.3.4.4), whereas antisera from dk/Hok/69 ca -vaccinated ferrets cross-reacted with clade 2.3.4.4 and 2.2.1 viruses but not clade 1 or 2.1 viruses. When vaccinated ferrets were challenged with homologous and heterologous H5 viruses, challenge virus replication was reduced in the respiratory tract. Thus, the two H5 pLAIV candidates are suitable for clinical development to protect humans from infection with different clades of H5 viruses. IMPORTANCE In response to the continuing evolution of H5N1 avian influenza viruses and human infections, new candidate H5 live attenuated vaccines were developed by using two different approaches: one targeted a specific circulating strain in Egypt, and the other was based on a virus that elicits broadly cross-reactive antibodies against a wide range of H5 viruses. Both candidate vaccines were immunogenic and exhibited protective efficacy in ferrets. Our study permits a comparison of the two approaches, and the data support the further development of both vaccine viruses to optimally prepare for the further spread of clade 2.2.1 or 2.3.4.4 viruses., (Copyright © 2017 American Society for Microbiology.)

A universal vaccine that provides long-lasting protection from both epidemic and pandemic influenza viruses remains the "holy grail" of influenza vaccine research. Though virus neutralization assays are the current benchmark of measuring vaccine effectiveness, it is clear that Fc-receptor functions can drastically improve the effectiveness of antibodies and vaccines in vivo. Antibodies that kill virus-infected cells and/or elicit an antiviral environment, termed antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies, provide a link between the innate and adaptive immune response. New technologies allowing the rapid isolation and characterization of monoclonal antibodies (mAb) have yielded a plethora of mAbs which target conserved regions of influenza virus, such as the hemagglutinin (HA) stem region. Many such mAbs have been used to gain a better understanding of Fc-receptor functions in vivo. In parallel, several studies have characterized the induction of polyclonal ADCC following influenza vaccination and infection in humans. Taken together, these studies suggest that ADCC-mediating antibodies (ADCC-Abs) significantly contribute to host immunity against influenza virus and may be a mechanism to exploit for rational vaccine and therapeutic design. We discuss recent research on influenza-specific ADCC and potential future avenues to extend our understanding.

Timely vaccine supply is critical during influenza pandemics but is impeded by current virus-based manufacturing methods. The 2009 H1N1/2009pdm 'swine flu' pandemic reinforced the need for innovation in pandemic vaccine design. We report on insights gained during rapid development of a pandemic vaccine based on recombinant haemagglutinin (rHA) formulated with Advax™ delta inulin adjuvant (Panblok-H1/Advax). Panblok-H1/Advax was designed and manufactured within 1 month of the pandemic declaration by WHO and successfully entered human clinical testing in under 3 months from first isolation and sequencing of the novel pandemic virus, requiring several major challenges to be overcome. Panblok-H1/Advax successfully induced neutralising antibodies against the pandemic strain, but also induced cross-neutralising antibodies in a subset of subjects against an H1N1 strain (A/Puerto Rico/8/34) derived from the 1918 Spanish flu, highlighting the possibility to use Advax to induce more broadly cross-protective antibody responses. Interestingly, the rHA from H1N1/2009pdm exhibited variants in the receptor binding domain that had a major impact on receptor binding and hemagglutination ability. We used an in silico structural modeling approach to better understand the unusual behavior of the novel hemagglutinin, thereby demonstrating the power of computational modeling approaches for rapid characterization of new pandemic viruses. While challenges remain in ensuring ultrafast vaccine access for the entire population in response to future pandemics, the adjuvanted recombinant Panblok-H1/Advax vaccine proved its utility during a real-life pandemic situation.

New influenza vaccines that provide effective and broad protection are desperately needed. Live attenuated viruses are attractive vaccine candidates because they can elicit both humoral and cellular immune responses. However, recent formulations of live attenuated influenza vaccines (LAIVs) have not been protective. We combined high-coverage transposon mutagenesis of influenza virus with a rapid high-throughput screening for attenuation to generate W7-791, a live attenuated mutant virus strain. W7-791 produced only a transient asymptomatic infection in adult and neonatal mice even at doses 100-fold higher than the LD 50 of the parent strain. A single administration of W7-791 conferred full protection to mice against lethal challenge with H1N1, H3N2, and H5N1 strains, and improved viral clearance in ferrets. Adoptive transfer of T cells from W7-791-immunized mice conferred heterologous protection, indicating a role for T cell-mediated immunity. These studies present an LAIV development strategy to rapidly generate and screen entire libraries of viral clones., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Subtype H2 Influenza A viruses were the cause of a severe pandemic in the winter of 1957. However, this subtype no longer circulates in humans and is no longer included in seasonal vaccines. As a result, individuals under 50years of age are immunologically naïve. H2 viruses persist in aquatic birds, which were a contributing source for the 1957 pandemic, and have also been isolated from swine. Reintroduction of the H2 via zoonotic transmission has been identified as a pandemic risk, so pre-pandemic planning should include preparation and testing of vaccine candidates against this subtype. We evaluated the immunogenicity of two inactivated, whole virus influenza vaccines (IVV) in mice: a monovalent IVV containing human pandemic virus A/Singapore/1/1957 (H2N2), and a multivalent IVV containing human A/Singapore/1/1957, avian A/Duck/HongKong/319/1978 (H2N2), and swine A/Swine/Missouri/2124514/2006 (H2N3) viruses. While both vaccines induced protective immunity compared to naïve animals, the multivalent formulation was advantageous over the monovalent in terms of level and breadth of serological responses, neutralization of infectious virus, and reduction of clinical disease and respiratory tissue replication in mice. Therefore, multivalent pandemic H2 vaccines containing diverse viruses from animal reservoirs, are a potential option to improve the immune responses in a pre-pandemic scenario where antigenic identity cannot be predicted., (Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Antigenic drift of seasonal influenza viruses and the occasional introduction of influenza viruses of novel subtypes into the human population complicate the timely production of effective vaccines that antigenically match the virus strains that cause epidemic or pandemic outbreaks. The development of game-changing vaccines that induce broadly protective immunity against a wide variety of influenza viruses is an unmet need, in which recombinant viral vectors may provide. Use of viral vectors allows the delivery of any influenza virus antigen, or derivative thereof, to the immune system, resulting in the optimal induction of virus-specific B- and T-cell responses against this antigen of choice. This systematic review discusses results obtained with vectored influenza virus vaccines and advantages and disadvantages of the currently available viral vectors.

Recombinant vaccines based on virus-like particles (VLPs) or nanoparticles have been successful in their safety and efficacy in preclinical and clinical studies. The technology of expressing enveloped VLP vaccines has combined with molecular engineering of proteins in membrane-anchor and immunogenic forms mimicking the native conformation of surface proteins on the enveloped viruses. This review summarizes recent developments in influenza VLP vaccines against seasonal, pandemic, and avian influenza viruses from the perspective of use in humans. The immunogenicity and efficacies of influenza VLP vaccine in the homologous and cross-protection were reviewed. Discussions include limitations of current influenza vaccination strategies and future directions to confer broadly cross protective new influenza vaccines as well as vaccination., Competing Interests: Declaration of Interests This work was supported by NIH/NIAID grants AI105170 (S-M K), AI119366 (S-M K), and AI093772 (S-M K), and supported by a grant from the National Research Foundation of Korea (NRF) (NRF-2014R1A2A2A01004899) (F-S Quan), a grant from the Agri-Bio Industry Technology Development Program (315030-03-1-HD020) (F-S Quan), IPET, MAFRA, KHIDI, and a grant from the Ministry of Health & Welfare, Republic of Korea (HI15C2928) (F-S Quan). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Size exclusion chromatography is a standard method in quality control of biopharmaceutical proteins. In contrast, vaccine analysis is often based on activity assays. The hemagglutination assay is a widely accepted influenza quantification method, providing no insight in the size distribution of virus particles. Capabilities of size exclusion chromatography to complement the hemagglutination assay are investigated. The presented method is comparatively robust regarding different buffer systems, ionic strength and additive concentrations. Addition of 200mM arginine or sodium chloride is necessary to obtain complete virus particle recovery. 0.5 and 1.0M arginine increase the hydrodynamic radius of the whole virus particles by 5nm. Sodium citrate induces virus particle aggregation. Results are confirmed by dynamic light scattering. Retention of a H1N1v strain correlates with DNA contents between 5ng/mL and 670ng/mL. Quantitative elution of the virus preparations is verified on basis of hemagglutination activity. Elution of hemagglutination inducing compounds starts at a flow channel diameter of 7000nm. The universal applicability is demonstrated with three different influenza virus samples, including an industrially produced, pandemic vaccine strain. Size distribution of the pandemic H1N1v 5258, H1N1 PR/8/34, and H3N2 Aichi/2/68 preparations spreads across inter- and intra-particle volume and extends to the secondary interaction dominated range. Thus, virus particle debris seems to induce hemagglutination. Fragments generated by 0.5% Triton™ X-100 treatment increase overall hemagglutination activity., (Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.)

Quadrivalent influenza vaccines (QIVs), which include both B lineage strains, are expected to provide broader protection than trivalent influenza vaccines (TIVs). The non-inferiority, immunogenicity, and safety of a cell culture-based investigational QIVc and 2 TIVs (TIV1c, TIV2c), in adults (≥18 y), were evaluated in this Phase III, double-blind, multicenter study. A total of 2680 age-stratified subjects were randomized (2:1:1) to receive 1 dose of QIVc (n = 1335), TIV1c (n = 676), or TIV2c (n = 669). TIV1c (B/Yamagata) and TIV2c (B/Victoria) differed only in B strain lineage. The primary objective was to demonstrate non-inferiority of the hemagglutinin-inhibition antibody responses of QIVc against TIVc, 22 d post-vaccination. Secondary objectives included the evaluation of immunogenicity of QIVc and TIVc in younger (≥18 - <65 y) and older (≥65 y) adults. Hemagglutinin inhibition assays were performed at days 1 and 22. Solicited local and systemic adverse events (AEs) were monitored for 7 d post-vaccination, and unsolicited AEs and serious AEs until day 181. QIVc met the non-inferiority criteria for all 4 vaccine strains and demonstrated superiority for both influenza B strains over the unmatched B strain included in the TIV1c and TIV2c, when geometric mean titers and seroconversion rates with TIVc were compared at day 22. Between 48%-52% of subjects experienced ≥1 solicited AE, the most common being injection-site pain and headache. Serious AEs were reported by ≤1% of subjects, none were vaccine-related. The results indicate that QIVc is immunogenic and well tolerated in both younger and older adults. The immunogenicity and safety profiles of QIVc and TIVc were comparable at all ages evaluated.

Developing effective vaccines is of vital importance for protecting public health by preventing potential pandemics or by controlling ongoing ones. However, there is a threshold of rapidly design and develop effective vaccines to prevent virus infection. Inspired by the natural budding processes associated with cell membrane scission when enveloped viruses invade host cells and replicate themselves, a similar strategy was applied to achieve virus-mimetic nanovesicles (VMVs). This strategy loaded genetically engineered viral antigens onto mammalian cell membranes to produce antigen-loaded vesicles, and then used surfactants to optimize their size and stability. The VMVs resemble natural viruses in size, shape and specific immune function and have protein antigens in the correct conformation on their exterior to elicit robust immunogenicity. This was confirmed in animal models against influenza A (H1N1) virus, demonstrating that VMVs could be a versatile platform for vaccine development.

The unprecedented epizootic of avian influenza viruses, such as H5N1, H5N6, H7N1 and H10N8, has continued to cause disease in humans in recent years. In 2013, another novel influenza A (H7N9) virus emerged in China, and 30% of those patients died. Pregnant women are particularly susceptible to avian influenza and are more likely to develop severe complications and to die, especially when infection occurs in the middle and late trimesters. Viremia is believed to occur infrequently, and thus vertical transmission induced by avian influenza appears to be rare. However, avian influenza increases the risk of adverse pregnancy outcomes, including spontaneous abortion, preterm birth and fatal distress. This review summarises 39 cases of pregnant women and their fetuses from different countries dating back to 1997, including 11, 15 and 13 infections with H7N9, H5N1 and the 2009 pandemic influenza (H1N1), respectively. We analysed the epidemic features, following the geographical, population and pregnancy trimester distributions; underlying diseases; exposure history; medical timelines; human-to-human transmission; pathogenicity and vertical transmission; antivirus treatments; maternal severity and mortality and pregnancy outcome. The common experiences reported in different countries and areas suggest that early identification and treatment are imperative. In the future, vigilant virologic and epidemiologic surveillance systems should be developed to monitor avian influenza viruses during pregnancy. Furthermore, extensive study on the immune mechanisms should be conducted, as this will guide safe, rational immunomodulatory treatment among this high-risk population. Most importantly, we should develop a universal avian influenza virus vaccine to prevent outbreaks of the different subtypes. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Unlabelled: Influenza viral infections represent a serious public health problem, with influenza virus causing a contagious respiratory disease which is most effectively prevented through vaccination. Segments 7 (M) and 8 (NS) of the influenza virus genome encode mRNA transcripts that are alternatively spliced to express two different viral proteins. This study describes the generation, using reverse genetics, of three different recombinant influenza A/Puerto Rico/8/1934 (PR8) H1N1 viruses containing M or NS viral segments individually or modified M or NS viral segments combined in which the overlapping open reading frames of matrix 1 (M1)/M2 for the modified M segment and the open reading frames of nonstructural protein 1 (NS1)/nuclear export protein (NEP) for the modified NS segment were split by using the porcine teschovirus 1 (PTV-1) 2A autoproteolytic cleavage site. Viruses with an M split segment were impaired in replication at nonpermissive high temperatures, whereas high viral titers could be obtained at permissive low temperatures (33°C). Furthermore, viruses containing the M split segment were highly attenuated in vivo, while they retained their immunogenicity and provided protection against a lethal challenge with wild-type PR8. These results indicate that influenza viruses can be effectively attenuated by the rearrangement of spliced segments and that such attenuated viruses represent an excellent option as safe, immunogenic, and protective live-attenuated vaccines. Moreover, this is the first time in which an influenza virus containing a restructured M segment has been described. Reorganization of the M segment to encode M1 and M2 from two separate, nonoverlapping, independent open reading frames represents a useful tool to independently study mutations in the M1 and M2 viral proteins without affecting the other viral M product., Importance: Vaccination represents our best therapeutic option against influenza viral infections. However, the efficacy of current influenza vaccines is suboptimal, and novel approaches are necessary for the prevention of disease caused by this important human respiratory pathogen. In this work, we describe a novel approach to generate safer and more efficient live-attenuated influenza virus vaccines (LAIVs) based on recombinant viruses whose genomes encode nonoverlapping and independent M1/M2 (split M segment [Ms]) or both M1/M2 and NS1/NEP (Ms and split NS segment [NSs]) open reading frames. Viruses containing a modified M segment were highly attenuated in mice but were able to confer, upon a single intranasal immunization, complete protection against a lethal homologous challenge with wild-type virus. Notably, the protection efficacy conferred by our viruses with split M segments was better than that conferred by the current temperature-sensitive LAIV. Altogether, these results open a new avenue for the development of safer and more protective LAIVs on the basis of the reorganization of spliced viral RNA segments in the genome., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Influenza A virus (subtype H3N2) causes seasonal human influenza and is included as a component of influenza vaccines. The majority of vaccine viruses are isolated and propagated in eggs, which commonly results in amino acid substitutions in the haemagglutinin (HA) glycoprotein. These substitutions can affect virus receptor-binding and alter virus antigenicity, thereby, obfuscating the choice of egg-propagated viruses for development into candidate vaccine viruses. To evaluate the effects of egg-adaptive substitutions seen in H3N2 vaccine viruses on sialic acid receptor-binding, we carried out quantitative measurement of virus receptor-binding using surface biolayer interferometry with haemagglutination inhibition (HI) assays to correlate changes in receptor avidity with antigenic properties. Included in these studies was a panel of H3N2 viruses generated by reverse genetics containing substitutions seen in recent egg-propagated vaccine viruses and corresponding cell culture-propagated wild-type viruses. These assays provide a quantitative approach to investigating the importance of individual amino acid substitutions in influenza receptor-binding. Results show that viruses with egg-adaptive HA substitutions R156Q, S219Y, and I226N, have increased binding avidity to α2,3-linked receptor-analogues and decreased binding avidity to α2,6-linked receptor-analogues. No measurable binding was detected for the viruses with amino acid substitution combination 156Q+219Y and receptor-binding increased in viruses where egg-adaptation mutations were introduced into cell culture-propagated virus. Substitutions at positions 156 and 190 appeared to be primarily responsible for low reactivity in HI assays with post-infection ferret antisera raised against 2012-2013 season H3N2 viruses. Egg-adaptive substitutions at position 186 caused substantial differences in binding avidity with an insignificant effect on antigenicity.

Virus-like particles (VLPs) are promising molecular structures for the design and construction of novel vaccines, diagnostic tools, and gene therapy vectors. Size, oligomer assembly and repetitiveness of epitopes are optimal features to induce strong immune responses. Several VLP-based vaccines are currently licensed and commercialized, and many vaccine candidates are now under preclinical and clinical studies. In recent years, the development of genetically engineered recombinant VLPs has accelerated the need for new, improved downstream processes. In particular, a rapid low cost purification process has been identified as a remaining key challenge in manufacturing process development. In the present study we set up a size-exclusion chromatography-based, scalable purification protocol for the purification of a VLP-based influenza A vaccine produced in Escherichia coli. Recombinant VLPs derived from the RNA bacteriophage MS2 displaying an epitope from the ectodomain of Matrix 2 protein from influenza A virus were produced and purified. The 3 steps purification protocol uses a recently developed multimodal size-exclusion chromatography medium (Capto™ Core 700) in combination with detergent extraction and size-exclusion polishing to reach a 89% VLP purity with a 19% yield. The combination of this downstream strategy following production in E. coli would be suited for production of VLP-based veterinary vaccines targeting livestock and companion animals where large amounts of doses must be produced at an affordable price., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Seasonal influenza vaccines are unique because they are regularly reformulated and prepared in anticipation of the upcoming influenza season. Selection of vaccine strains occurs in advance of the influenza season, allowing time for vaccine production. Influenza viruses constantly evolve, and mismatches between vaccine strains and circulating strains have occurred in the past, impacting on vaccine effectiveness. The public health impact of a mismatch depends on multiple factors including strain virulence and transmission dynamics, pre-existing population immunity to the drift strain, and cross-reactivity induced by vaccination. Influenza vaccine effectiveness thus varies unpredictably from year to year, and may differ across European and northern American regions. Here we highlight the unpredictability associated with influenza virus circulation and present a comprehensive picture of circulating influenza strains in the northern hemisphere as compared to WHO recommendations for vaccine strains over the last 15 y. In years when vaccine mismatch occurs, such as the 2014-15 influenza season, public health agencies continue to recommend influenza vaccination as the preferred means by which to protect against influenza and influenza-associated complications. Research is on-going to optimise strain selection and vaccine composition to improve effectiveness.

Lately, researchers have made several efforts to improve vaccine production to fight highly contagious respiratory diseases like influenza. One of the most promising options for reducing the impact of this virus is DNA vaccination. However, a large quantity of highly pure plasmid DNA (pDNA) is necessary to attain this goal. The present work describes the production and purification of the plasmid NTC7482-41H-VA2HA expressing influenza virus hemagglutinin using an agmatine monolith. This ligand was chosen to purify supercoiled (sc) pDNA from complex lysates because of its versatile multimodal character. Its natural intervention in several biological systems together with its similarity with the highly studied arginine ligand allowed the development of a simpler and more specific purification process. Agmatine works under two strategies: descending ammonium sulfate gradient and ascending sodium chloride gradient. Furthermore, pH manipulation revealed an important role in pDNA isoforms selectivity. Dynamic binding capacity (DBC) experiments were performed varying different parameters and showed an increase with pDNA concentration, while high flow rate and high pH had the opposite effect. Sc pDNA was purified with high yield and was efficient with respect to cell transfection and cell viability. This monolith showed to be appropriate to purify the plasmid NTC7482-41H-VA2HA, providing a valuable tool for pDNA influenza vaccines preparation., (Copyright © 2016. Published by Elsevier B.V.)

In April 2013, the first three fatal cases of human infection with an avian influenza A virus (H7N9) were reported in China. Because of a pandemic threat by this virus, we have commenced to develop candidate vaccine viruses (CVVs). Three 6:2 genetic reassortant viruses with different hemagglutinin (HA) sequences, NIIDRG-10, -10.1 and -10.2, were generated by a reverse genetics technique between the high egg-growth master virus, A/Puerto Rico/8/34 (H1N1) and A/Anhui/1/2013 (H7N9), kindly provided by the Chinese Center for Disease Control and Prevention. The different HA gene sequences of the three CVVs were derived from the original virus stock. NIIDRG-10 possesses HA, whose sequence is identical to that of the original A/Anhui/1/2013 (H7N9) in the Global Initiative on Sharing Avian Influenza Data (EPI439507), while NIIDRG-10.1 and -10.2 possess amino acid differences, A125T and N123D/N149D, respectively, compared with NIIDRG-10. NIIDRG-10 replicated in embryonated chicken eggs with low hemagglutination titer 128, whereas NIIDRG-10.1 and -10.2 grew well with hemagglutination titer 1024. These viruses reacted well with a ferret antiserum raised against the original A/Anhui/1/2013 virus. Ferret antiserum against NIIDRG-10.1 reacted well with A/Anhui/1/2013 similar to the homologous virus NIIDRG-10.1. These results indicated that NIIDRG-10.1 passed the two-way test of antigenic identity. In contrast, the ferret antiserum against NIIDRG-10.2 reacted with A/Anhui/1/2013 at an 8-fold lower hemagglutination inhibition titer than with the homologous virus NIIDRG-10.2, indicating an antigenic change. The total and HA protein yields of NIIDRG-10.1 were 14.7 and 6.9 μg/ml, respectively, similar to those levels of high-yield seed viruses of seasonal influenza vaccines. NIIDRG-10.1 was approved as one of the CVVs for H7N9 viruses by the WHO in 2013. The candidate vaccine derived from NIIDRG-10.1 is currently being evaluated in a phase II clinical study in Japan., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Avian influenza H5N1 viruses have been enzootic in Egyptian poultry since 2006. Avian influenza H9N2 viruses which have been circulating in Egyptian poultry since 2011 showed high replication rates in embryonated chicken eggs and mammalian cells., Methods: To investigate which gene segment was responsible for increasing replication, we constructed reassortant influenza viruses using the low pathogenic H1N1 PR8 virus as backbone and included individual genes from A/chicken/Egypt/S4456B/2011(H9N2) virus. Then, we invested this finding to improve a PR8-derived H5N1 influenza vaccine strain by incorporation of the NA segment of H9N2 virus instead of the NA of H5N1. The growth properties of this virus and several other forms of reassortant H5 viruses were compared. Finally, we tested the efficacy of this reassortant vaccine strain in chickens., Results: We observed an increase in replication for a reassortant virus expressing the neuraminidase gene (N2) of H9N2 virus relative to that of either parental viruses or reassortant PR8 viruses expressing other genes. Then, we generated an H5N2 vaccine strain based on the H5 from an Egyptian H5N1 virus and the N2 from an Egyptian H9N2 virus on a PR8 backbone. This strain had better replication rates than an H5N2 reassortant strain on an H9N2 backbone and an H5N1 reassortant on a PR8 backbone. This virus was then used to develop a killed, oil-emulsion vaccine and tested for efficacy against H5N1 and H9N2 viruses in chickens. Results showed that this vaccine was immunogenic and reduced mortality and shedding., Discussion: Our findings suggest that an inactivated PR8-derived H5N2 influenza vaccine is efficacious in poultry against H5N1 and H9N2 viruses and the vaccine seed replicates at a high rate thus improving vaccine production., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Influenza vaccine design has changed considerably with advancements in bioinformatics and computational biology. Improved surveillance efforts provide up-to-date information about influenza sequence diversity and assist with monitoring the spread of epidemics and vaccine efficacy rates. The advent of next-generation sequencing, epitope scanning and high-throughput analysis all help decipher influenza-associated protein interactions as well as predict immune responsiveness based on host genetic diversity. Computational approaches are utilized in nearly all aspects of vaccine design, from modeling, compatibility predictions, and optimization of antigens in various platforms. This overview discusses how computational techniques strengthen vaccine efforts against highly diverse influenza species.

Vaccination is considered to be the most effective and economical strategy against pandemic influenza. Vaccine development for multiple highly pathogenic avian influenza viruses, for example, H5N1, is hindered by antigenic drift, especially in the hemagglutinin (HA) sequence, as well as the antigenic shift. Growing efforts have been made to generate universal pandemic influenza vaccines. As mainly shown in animal trials, cross-clade and heterosubtypic protection by these universal vaccines are generally elicited by either a broad antigen-specific antibody response or influenza-specific CD4+ and CD8+ T-cell responses. Strain selection, HA engineering and broad neutralizing antigen determination are major strategies to achieve universal and specific antibody response, while studies on other factors including vectors, adjuvants and administration routes aim for enhanced T-cell responses against diverse influenza subtypes. Prospectively, cost-effective universal vaccines developed based on these combined technologies are promising solutions for broad protection against influenza.