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Influenza remains a public health threat, partly due to suboptimal effectiveness of vaccines. One factor impacting vaccine effectiveness is strain mismatch, occurring when vaccines no longer match circulating strains due to antigenic drift or the incorporation of inadvertent (eg, egg-adaptive) mutations during vaccine manufacturing. In this review, we summarize the evidence for antigenic drift of circulating viruses and/or egg-adaptive mutations occurring in vaccine strains during the 2011-2020 influenza seasons. Evidence suggests that antigenic drift led to vaccine mismatch during four seasons and that egg-adaptive mutations caused vaccine mismatch during six seasons. These findings highlight the need for alternative vaccine development platforms. Recently, vaccines based on mRNA technology have demonstrated efficacy against SARS-CoV-2 and respiratory syncytial virus and are under clinical evaluation for seasonal influenza. We discuss the potential for mRNA vaccines to address strain mismatch, as well as new multi-component strategies using the mRNA platform to improve vaccine effectiveness.

Modified vaccinia virus Ankara is a versatile vaccine vector, well suited for transgene delivery, with an excellent safety profile. However, certain transgenes render recombinant MVA (rMVA) genetically unstable, leading to the accumulation of mutated rMVA with impaired transgene expression. This represents a major challenge for upscaling and manufacturing of rMVA vaccines. To prevent transgene-mediated negative selection, the continuous avian cell line AGE1.CR pIX (CR pIX) was modified to suppress transgene expression during rMVA generation and amplification. This was achieved by constitutively expressing a tetracycline repressor (TetR) together with a rat-derived shRNA in engineered CR pIX PRO suppressor cells targeting an operator element (tetO) and 3\' untranslated sequence motif on a chimeric poxviral promoter and the transgene mRNA, respectively. This cell line was instrumental in generating two rMVA (isolate CR19) expressing a Macaca fascicularis papillomavirus type 3 (MfPV3) E1E2E6E7 artificially-fused polyprotein following recombination-mediated integration of the coding sequences into the DelIII (CR19 M-DelIII) or TK locus (CR19 M-TK), respectively. Characterization of rMVA on parental CR pIX or engineered CR pIX PRO suppressor cells revealed enhanced replication kinetics, higher virus titers and a focus morphology equaling wild-type MVA, when transgene expression was suppressed. Serially passaging both rMVA ten times on parental CR pIX cells and tracking E1E2E6E7 expression by flow cytometry revealed a rapid loss of transgene product after only few passages. PCR analysis and next-generation sequencing demonstrated that rMVA accumulated mutations within the E1E2E6E7 open reading frame (CR19 M-TK) or deletions of the whole transgene cassette (CR19 M-DelIII). In contrast, CR pIX PRO suppressor cells preserved robust transgene expression for up to 10 passages, however, rMVAs were more stable when E1E2E6E7 was integrated into the TK as compared to the DelIII locus. In conclusion, sustained knock-down of transgene expression in CR pIX PRO suppressor cells facilitates the generation, propagation and large-scale manufacturing of rMVA with transgenes hampering viral replication.

Influenza A virus (IAV) is a negative-sense RNA virus that causes seasonal infections and periodic pandemics, inflicting huge economic and human costs on society. The current production of influenza virus for vaccines is initiated by generating a seed virus through the transfection of multiple plasmids in HEK293 cells followed by the infection of seed viruses into embryonated chicken eggs or cultured mammalian cells. We took a system design and synthetic biology approach to engineer cell lines that can be induced to produce all viral components except hemagglutinin (HA) and neuraminidase (NA), which are the antigens that specify the variants of IAV. Upon the transfection of HA and NA, the cell line can produce infectious IAV particles. RNA-Seq transcriptome analysis revealed inefficient synthesis of viral RNA and upregulated expression of genes involved in host response to viral infection as potential limiting factors and offered possible targets for enhancing the productivity of the synthetic cell line. Overall, we showed for the first time that it was possible to create packaging cell lines for the production of a cytopathic negative-sense RNA virus. The approach allows for the exploitation of altered kinetics of the synthesis of viral components and offers a new method for manufacturing viral vaccines.

Newcastle disease (ND) remains a critical disease affecting poultry in sub-Saharan Africa. In some countries, repeated outbreaks have a major impact on local economies and food security. Recently, we developed an adenovirus-vectored vaccine encoding the Fusion protein from an Ethiopian isolate of Newcastle disease virus (NDV). The adenoviral vector was designed, and a manufacturing process was developed in the context of the Livestock Vaccine Innovation Fund initiative funded by the International Development Research Centre (IDRC) of Canada. The industrially relevant recombinant vaccine technology platform is being transferred to the National Veterinary Institute (Ethiopia) for veterinary applications. Here, a manufacturing process using HEK293SF suspension cells cultured in stirred-tank bioreactors for the vaccine production is proposed. Taking into consideration supply chain limitations, options for serum-free media selection were evaluated. A streamlined downstream process including a filtration, an ultrafiltration, and a concentration step was developed. With high volumetric yields (infectious titers up to 5 × 109 TCID50/mL) in the culture supernatant, the final formulations were prepared at 1010 TCID50/mL, either in liquid or lyophilized forms. The liquid formulation was suitable and safe for mucosal vaccination and was stable for 1 week at 37 °C. Both the liquid and lyophilized formulations were stable after 6 months of storage at 4 °C. We demonstrate that the instillation of the adenoviral vector through the nasal cavity can confer protection to chickens against a lethal challenge with NDV. Overall, a manufacturing process for the adenovirus-vectored vaccine was developed, and protective doses were determined using a convenient route of delivery. Formulation and storage conditions were established, and quality control protocols were implemented.

There is a continued need for sarbecovirus vaccines that can be manufactured and distributed in low- and middle-income countries (LMICs). Subunit protein vaccines are manufactured at large scales at low costs, have less stringent temperature requirements for distribution in LMICs, and several candidates have shown protection against SARS-CoV-2. We previously reported an engineered variant of the SARS-CoV-2 Spike protein receptor binding domain antigen (RBD-L452K-F490W; RBD-J) with enhanced manufacturability and immunogenicity compared to the ancestral RBD. Here, we report a second-generation engineered RBD antigen (RBD-J6) with two additional mutations to a hydrophobic cryptic epitope in the RBD core, S383D and L518D, that further improved expression titers and biophysical stability. RBD-J6 retained binding affinity to human convalescent sera and to all tested neutralizing antibodies except antibodies that target the class IV epitope on the RBD core. K18-hACE2 transgenic mice immunized with three doses of a Beta variant of RBD-J6 displayed on a virus-like particle (VLP) generated neutralizing antibodies (nAb) to nine SARS-CoV-2 variants of concern at similar levels as two doses of Comirnaty. The vaccinated mice were also protected from challenge with Alpha or Beta SARS-CoV-2. This engineered antigen could be useful for modular RBD-based subunit vaccines to enhance manufacturability and global access, or for further development of variant-specific or broadly acting booster vaccines.

According to data from the World Health Organization (WHO) every year, millions of people are affected by flu. Flu is a disease caused by influenza viruses. For preventing this, seasonal influenza vaccinations are widely considered the most efficient way to protect against the negative effects of the flu. To date, there is no \"one-size-fits-all\" vaccine that can be effective all over the world to protect against all seasonal or pandemic influenza virus types. Because influenza virus transforms its genetic structure and it can emerges as immunogenically new (antigenic drift) which causes epidemics or new virus subtype (antigenic shift) which causes pandemics. As a result, annual revaccination or new subtype viral vaccine development is required. Currently, three types of vaccines (inactivated, live attenuated, and recombinant) are approved in different countries. These can be named \"conventional influenza vaccines\" and their production are based on eggs or cell culture. Although, there is good effort to develop new influenza vaccines for broader and longer period of time protection. In this sense these candidate vaccines are called \"universal influenza vaccines\". In this article, after we mentioned the short history of flu then virus morphology and infection, we explained the diseases caused by the influenza virus in humans. Afterward, we explained in detail the production methods of available influenza vaccines, types of bioreactors used in cell culture based production, conventional and new vaccine types, and development strategies for better vaccines.

Subunit vaccines employing designer antigens such as Computationally Optimized Broadly Reactive Antigen (COBRA) hemagglutinin (HA) hold the potential to direct the immune response toward more effective and broadly-neutralizing targets on the Influenza virus. However, subunit vaccines generally require coadministration with an adjuvant to elicit a robust immune response. One such adjuvant is the stimulator of interferon genes (STING) agonist cyclic dinucleotide 3\'3\'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). We have shown that encapsulation of cGAMP in acetalated dextran (Ace-DEX) microparticles through electrospray results in significantly greater biological activity. Electrospray is a continuous manufacturing process which achieves excellent encapsulation efficiency. However, the throughput of electrospray with a single spray head is limited. Here we report the development of a multiplexed electrospray apparatus with an order of magnitude greater throughput than a single-head apparatus. Physicochemical characterization and evaluation of adjuvant activity in vitro and in vivo indicated that microparticles produced with the higher throughput process are equally suited for use as a potent vaccine adjuvant to induce a balanced immune response to COBRA HA antigens.

With the Corona Virus Disease 2019 (COVID-19) outbreak, vaccination is an urgent need worldwide. Internet of Things (IoT) has a vital role in the smart city for vaccine manufacturing with wearable sensors. According to the advanced services in intelligent manufacturing, the fourth resolution is also changing in Industry 5.0 and utilizes high-definition connectivity sensors. Traditional manufacturing companies rely on trusted third parties, which may act as a single point of failure. Access control, big data, and scalability are also challenging issues in existing systems because of the demand response data (DRD) in advanced manufacturing. To mitigate these challenges, CoVAC: A P2P Smart Contract-based Intelligent Smart City Architecture for Vaccine Manufacturing is proposed with three layers, including connection, conversion, and intelligent cloud layer. Smart contract-based blockchain is utilized at the conversion layer for resolving access control, security, and privacy issues. Deep learning is adopted in the intelligent cloud layer for big data analysis and increasing production for vaccine manufacturing in smart city environments. A case study is carried out wherein access data are collected from the various smart plants for vaccines using smart manufacturing to validate the effectiveness of the proposed architecture. Simulation of the proposed architecture is performed on the collected advanced sensor IoT plants data to address the challenges above, offering scalable production in the vaccine manufacturing for the smart city.

Measles-containing vaccines (MCV), specifically vaccines against measles and rubella (MR), are extremely effective and critical for the eradication of measles and rubella diseases. In developed countries, vaccination rates are high and vaccines are readily available, but continued high prevalence of both diseases in developing countries and surges in measles deaths in recent years have highlighted the need to expand vaccination efforts. To meet demand for additional vaccines at a globally affordable price, it is highly desirable to streamline vaccine production thereby reducing cost and speeding up time to delivery. MR vaccine characterization currently relies on the 50% cell culture infectious dose (CCID50) assay, an endpoint assay with low reproducibility that requires 10-14 days to complete. For streamlining bioprocess analysis and improving measurement precision relative to CCID50, we developed the VaxArray Measles and Rubella assay kit, which is based on a multiplexed microarray immunoassay with a 5-hour time to result. Here we demonstrate vaccine-relevant sensitivity ranging from 345 to 800 IFU/mL up to 100,000 IFU/mL (infectious units per mL) and specificity that allows simultaneous analysis in bivalent vaccine samples. The assay is sensitive to antigen stability and has minimal interference from common vaccine additives. The assay exhibits high reproducibility and repeatability, with 15% CV, much lower than the typical 0.3 log10 error (∼65%) observed for the CCID50 assay. The intact protein concentration measured by VaxArray is reasonably correlated to, but not equivalent to, CCID50 infectivity measurements for harvest samples. However, the measured protein concentration exhibits equivalency to CCID50 for more purified samples, including concentrated virus pools and monovalent bulks, making the assay a useful new tool for same-day analysis of vaccine samples for bioprocess development, optimization, and monitoring.