H7N9 pandemic preparedness: A large-scale production of a split inactivated vaccine.

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.

Advances in influenza virus-like particles bioprocesses.

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 influenza virus production in a tubular bioreactor system provides stable titers and avoids the "von Magnus effect".

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.

Development of American-Lineage Influenza H5N2 Reassortant Vaccine Viruses for Pandemic Preparedness.

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, 107.6 TCID50/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.

The Future of Flu: A Review of the Human Challenge Model and Systems Biology for Advancement of Influenza Vaccinology.

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.

Reverse vaccinology approach to design a novel multi-epitope subunit vaccine against avian influenza A (H7N9) virus.

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.

Design of Nanoparticulate Group 2 Influenza Virus Hemagglutinin Stem Antigens That Activate Unmutated Ancestor B Cell Receptors of Broadly Neutralizing Antibody Lineages.

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.

Influenza vaccine: Where are we and where do we go?

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.

Vaccination of poultry successfully eliminated human infection with H7N9 virus in China.

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.

Safety and immunogenicity of inactivated monovalent influenza A/H1N1 vaccine candidate manufactured in Vietnam.

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.

Workshop report: Experimental animal models for universal influenza vaccines.

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.

Selection and structural analysis of the NY25 peptide - A vaccine candidate from hemagglutinin of swine-origin Influenza H1N1.

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.

Monoclonal antibody against N2 neuraminidase of cold adapted A/Leningrad/134/17/57 (H2N2) enables efficient generation of live attenuated influenza vaccines.

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.

The Human Vaccines Project: Towards a comprehensive understanding of the human immune response to immunization.

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.

Inactivated influenza vaccine stress can affect in vitro potency assay relationship to immunogenicity.

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.

Cell culture-derived flu vaccine: Present and future.

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.

Generation of a High-Growth Influenza Vaccine Strain in MDCK Cells for Vaccine Preparedness.

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 (PB1D153N, M1A137T, and NS1N176S). 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.

A Universal Influenza Vaccine: The Strategic Plan for the National Institute of Allergy and Infectious Diseases.

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.