Innovations in cell culture-based influenza vaccine manufacturing - from static cultures to high cell density cultivations.

Influenza remains a serious global health concern, causing significant morbidity and mortality each year. Vaccination is crucial to mitigate its impact, but requires rapid and efficient manufacturing strategies to handle timing and supply. Traditionally relying on egg-based production, the field has witnessed a paradigm shift toward cell culture-based methods offering enhanced flexibility, scalability, and process safety. This review provides a concise overview of available cell substrates and technological advancements. We summarize crucial steps toward process intensification - from roller bottle production to dynamic cultures on carriers and from suspension cultures in batch mode to high cell density perfusion using various cell retention devices. Moreover, we compare single-use and conventional systems and address challenges including defective interfering particles. Taken together, we describe the current state-of-the-art in cell culture-based influenza virus production to sustainably meet vaccine demands, guarantee a timely supply, and keep up with the challenges of seasonal epidemics and global pandemics.

Development of Robust Freeze-Drying Process for Long-Term Stability of rVSV-SARS-CoV-2 Vaccine.

The thermostability of vaccines, particularly enveloped viral vectored vaccines, remains a challenge to their delivery wherever needed. The freeze-drying of viral vectored vaccines is a promising approach but remains challenging due to the water removal process from the outer and inner parts of the virus. In the case of enveloped viruses, freeze-drying induces increased stress on the envelope, which often leads to the inactivation of the virus. In this study, we designed a method to freeze-dry a recombinant vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike glycoprotein. Since the envelope of VSV is composed of 50% lipids and 50% protein, the formulation study focused on both the protein and lipid portions of the vector. Formulations were prepared primarily using sucrose, trehalose, and sorbitol as cryoprotectants; mannitol as a lyoprotectant; and histidine as a buffer. Initially, the infectivity of rVSV-SARS-CoV-2 and the cake stability were investigated at different final moisture content levels. High recovery of the infectious viral titer (~0.5 to 1 log loss) was found at 3-6% moisture content, with no deterioration in the freeze-dried cakes. To further minimize infectious viral titer loss, the composition and concentration of the excipients were studied. An increase from 5 to 10% in both the cryoprotectants and lyoprotectant, together with the addition of 0.5% gelatin, resulted in the improved recovery of the infectious virus titer and stable cake formation. Moreover, the secondary drying temperature of the freeze-drying process showed a significant impact on the infectivity of rVSV-SARS-CoV-2. The infectivity of the vector declined drastically when the temperature was raised above 20 °C. Throughout a long-term stability study, formulations containing 10% sugar (sucrose/trehalose), 10% mannitol, 0.5% gelatin, and 10 mM histidine showed satisfactory stability for six months at 2-8 °C. The development of this freeze-drying process and the optimized formulation minimize the need for a costly cold chain distribution system.

Plant Expression of Trans-Encapsidated Chimeric Viral Vaccines with Animal RNA Replicons: An Update.

In this protocol, we outline how to produce a chimeric viral vaccine in a biosafety level 1 (BSL1) environment. An animal viral vector RNA encapsidated with tobacco mosaic virus (TMV) coat protein can be fully assembled in planta. Agrobacterium cultures containing each component are inoculated together into tobacco leaves and the self-assembled hybrid chimeric viral vaccine is harvested 4 days later and purified with a simple PEG precipitation. The viral RNA delivery vector is derived from the BSL1 insect virus, Flock House virus (FHV), and replicates in human and animal cells but does not spread systemically. A polyethylene glycol purification protocol is also provided to collect and purify these vaccines for immunological tests. In this update, we also provide a protocol for in trans co-inoculation of a modified FHV protein A, which significantly increased the yield of in planta chimeric viral vaccine.

From single-cell cloning to high-yield influenza virus production - implementing advanced technologies in vaccine process development.

Innovations in viral vaccine manufacturing are crucial for pandemic preparedness and to meet ever-rising global demands. For influenza, however, production still mainly relies on technologies established decades ago. Although modern production shifts from egg-based towards cell culture technologies, the full potential has not yet been fully exploited. Here, we evaluate whether implementation of state-of-the-art technologies for cell culture-based recombinant protein production are capable to challenge outdated approaches in viral vaccine process development. For this, a fully automated single-cell cloning strategy was established to generate monoclonal suspension Madin-Darby canine kidney (MDCK) cells. Among selected cell clones, we could observe distinct metabolic and growth characteristics, with C59 reaching a maximum viable cell concentration of 17.3 × 106 cells/mL and low doubling times in batch mode. Screening for virus production using a panel of human vaccine-relevant influenza A and B viruses in an ambr15 system revealed high titers with yields competing or even outperforming available MDCK cell lines. With C113, we achieved cell-specific virus yields of up to 25,000 virions/cell, making this cell clone highly attractive for vaccine production. Finally, we confirmed process performance at a 50-fold higher working volume. In summary, we present a scalable and powerful approach for accelerated development of high-yield influenza virus production in chemically defined medium starting from a single cell.

Safety and Immunogenicity of a Messenger RNA-Based Cytomegalovirus Vaccine in Healthy Adults: Results From a Phase 1, Randomized, Clinical Trial.

BACKGROUND: This phase 1 trial evaluated the safety, reactogenicity, and immunogenicity of mRNA-1647, an mRNA-based cytomegalovirus (CMV) vaccine, in CMV-seronegative and -seropositive adults. METHODS: Participants were randomly assigned to receive 30, 90, 180, or 300 µg of mRNA-1647 or placebo on a 0-, 2-, and 6-month schedule and followed for 12 months after the last dose. RESULTS: A total of 154 (80 CMV-seronegative and 74 CMV-seropositive) participants were enrolled; 118 participants were randomized to mRNA-1647 and 36 to placebo. Mean (SD) age was 32.5 (8.6) and 35.1 (8.9) years in the placebo and mRNA-1647 groups, respectively, in phase B (63% and 64% female) and 42.5 (6.2) and 33.3 (8.7) years, respectively, in phase C (2% and 16% female). No deaths, related serious adverse events, or adverse events of special interest were reported. Most adverse reactions were grade ≤2 severity. Increased neutralizing antibody, binding antibody, and antigen-specific cell-mediated responses were observed across mRNA-1647 treatment groups, regardless of CMV serostatus. CONCLUSIONS: This phase 1, first-in-human trial demonstrated mRNA-1647 has an acceptable safety profile in adults and elicits humoral and cellular immune responses. TRIALS REGISTRATION: NCT03382405; https://clinicaltrials.gov/ct2/show/NCT03382405.

The Development of a Rabies Virus-Vectored Vaccine against Borrelia burgdorferi, Targeting BBI39.

Lyme disease (LD) is the most common tick-borne illness in the United States (U.S.), Europe, and Asia. Borrelia burgdorferi, a spirochete bacterium transmitted by the tick vector Ixodes scapularis, causes LD in the U.S. If untreated, Lyme arthritis, heart block, and meningitis can occur. Given the absence of a human Lyme disease vaccine, we developed a vaccine using the rabies virus (RABV) vaccine vector BNSP333 and an outer surface borrelial protein, BBI39. BBI39 was previously utilized as a recombinant protein vaccine and was protective in challenge experiments; therefore, we decided to utilize this protective antigen in a rabies virus-vectored vaccine against Borrelia burgdorferi. To incorporate BBI39 into the RABV virion, we generated a chimeric BBI39 antigen, BBI39RVG, by fusing BBI39 with the final amino acids of the RABV glycoprotein by molecular cloning and viral recovery with reverse transcription genetics. Here, we have demonstrated that the BBI39RVG antigen was incorporated into the RABV virion via immunofluorescence and Western blot analysis. Mice vaccinated with our BPL inactivated RABV-BBI39RVG (BNSP333-BBI39RVG) vaccine induced high amounts of BBI39-specific antibodies, which were maintained long-term, up to eight months post-vaccination. The BBI39 antibodies neutralized Borrelia in vaccinated mice when challenged with Borrelia burgdorferi by either syringe injection or infected ticks and they reduced the Lyme disease pathology of arthritis in infected mouse joints. Overall, the RABV-based LD vaccine induced more and longer-term antibodies compared to the recombinant protein vaccine. This resulted in lower borrelial RNA in RABV-based vaccinated mice compared to recombinant protein vaccinated mice. The results of this study indicate the successful use of BBI39 as a vaccine antigen and RABV as a vaccine vector for LD.

[Advances of human oral viral vaccine development].

Development of a vaccine that can simultaneously induce effective mucosal immunity and systemic immunity is an ideal goal to prevent mucosal pathogenic infections. The digestive tract has many sites for inducing mucosal immunity, including the mouth, stomach and small intestine. An ideal oral viral vaccine can not only induce better local and distal mucosal immunity, but also produce better systemic immunity. The oral viral vaccine has also attracted much attention because of its painless vaccination, self-administration and other advantages. Due to the complexity of human digestive tract environment and mucosal immunity, only three oral attenuated live vaccines have been successfully marketed for human use. This review summarizes the characteristics of gastrointestinal mucosal immunity, the current types and research status of oral viral vaccines, and the challenges faced by oral viral vaccines, with the hope to facilitate the research and development of oral viral vaccines for human use in China.

Evaluation of Antibody Response and Adverse Effects following Heterologous COVID-19 Vaccine Booster with mRNA Vaccine among Healthcare Workers in Indonesia.

Background: The administration of the third (or booster) dose of COVID-19 vaccine is important in maintaining protection against SARS-CoV-2 infection or the severity of the disease. In Indonesia, health care workers (HCWs) are among the first to receive a booster dose of the COVID-19 vaccine. In this study, we evaluated the antibody response and adverse events following heterologous booster vaccine using mRNA-1273 among HCWs that were fully vaccinated with inactivated viral vaccine as the priming doses. Methods: 75 HCWs at Dr. Soetomo General Hospital in Surabaya, Indonesia, participated in this study. The level of antibody against the SARS-CoV-2 receptor binding domain was analyzed at 1, 3, and 5 months following the second priming dose and at 1, 3, and 5 months after the booster dose. Results: We found a significantly higher level of antibody response in subjects receiving a booster dose of the mRNA-1273 vaccine compared to those receiving an inactivated viral vaccine as a booster. Interestingly, participants with hypertension and a history of diabetes mellitus showed a lower antibody response following the booster dose. There was a higher frequency of adverse events following injection with the mRNA-1273 vaccine compared to the inactivated viral vaccine, although the overall adverse events were considered minor. Conclusions: A heterologous booster dose using mRNA vaccine resulted in a high antibody response; however, participants with hypertension and diabetes mellitus displayed a lower antibody response.

Investigation into the use of gamma irradiated Cytodex-1 microcarriers to produce a human cytomegalovirus (HCMV) vaccine candidate in epithelial cells.

V160 is a viral vaccine candidate against human cytomegalovirus (HCMV) that is manufactured using Adult Retinal Pigment Epithelial cells (ARPE-19) grown on Cytodex-1 microcarriers. The microcarriers are generally hydrated, washed, and autoclaved prior to use, which can be limiting at large production scales. To minimize microcarrier preparation and sterilization, the use of gamma irradiated Cytodex-1 was investigated. Similar ARPE-19 cell growth was observed on heat-sterilized and gamma irradiated Cytodex-1; however, significantly reduced virus production was observed in cultures exposed to gamma irradiated Cytodex-1. Additional experiments suggest that infection inhibition is not exclusive to ARPE-19 but is most directly linked to HCMV V160, as evidenced by similar inhibition of V160 with Vero cells and no inhibition of Measles virus with either cell type. These observations suggest a putative impact on HCMV infection from the presence of extractable(s)/leachable(s) in the gamma irradiated microcarriers. Thorough aseptic rinsing of gamma irradiated Cytodex-1 prior to use can mitigate this impact and enable comparable process performance to heat-sterilized Cytodex-1. Though not fully a "ready-to-use" product for the HCMV V160 production process, utilization of Cytodex-1 microcarriers was possible without requiring heat sterilization, suggesting a potential path forward for large scale production of V160.

Purification of recombinant vesicular stomatitis virus-based HIV vaccine candidate.

In this work, laboratory- and large-scale methods were tested for purification of a human immunodeficiency virus (HIV) vaccine candidate, based on recombinant vesicular stomatitis virus (rVSV). First step of the purification, the clarification of the rVSVs produced in serum-free cell culture medium, was tested by centrifugation and filtration using different filtration media and pore sizes (0.45 to 30 µm). To reduce the supernatant volume and process time, the clarified sample was concentrated by ultrafiltration either using tangential flow filtration or centrifugal-based filtration units, depending on the process scale. The final purification step at laboratory-scale, was carried out by density gradient ultracentrifugation, the recovery of which was compared with chromatographic purification at large-scale. The virus preparations were analyzed using dynamic light scattering to verify the virus size and transmission electron microscopy for purity and virus morphology. Density gradient ultracentrifugation allowed the recovery of ≥ 80% infectious particles and reduced the contaminant DNA and host cell proteins relatively to standard ultracentrifugation pelleting using a sucrose cushion. At large-scale, weak and strong anion-exchangers were tested and compared. The best columns allowed infectious virus recoveries as high as 77% and eliminated 92% of host cell proteins.

Longitudinal neutralization activities on authentic Omicron variant provided by three doses of BBIBP-CorV vaccination during one year.

The majority of people in China have been immunized with the inactivated viral vaccine BBIBP-CorV. The emergence of the Omicron variant raised the concerns about protection efficacy of the inactivated viral vaccine in China. However, longitudinal neutralization data describing protection efficacy against Omicron variant is still lacking. Here we present one-year longitudinal neutralization data of BBIBP-CorV on authentic Omicron, Delta, and wild-type strains using 224 sera collected from 14 volunteers who have finished three doses BBIBP-CorV. The sera were also subjected for monitoring the SARS-CoV-2 specific IgG, IgA, and IgM responses on protein and peptide microarrays. The neutralization titers showed different protection efficacies against the three strains. By incorporating IgG and IgA signals of proteins and Spike protein derived peptide on microarray, panels as potential surrogate biomarkers for rapid estimation of neutralization titers were established. These data support the necessity of the 3rd dose of BBIBP-CorV vaccination. After further validation and assay development, the panels could be used for reliable, convenient and fast evaluation of the efficacy of vaccination.

Advances of human oral viral vaccine development / 生物工程学报

Development of a vaccine that can simultaneously induce effective mucosal immunity and systemic immunity is an ideal goal to prevent mucosal pathogenic infections. The digestive tract has many sites for inducing mucosal immunity, including the mouth, stomach and small intestine. An ideal oral viral vaccine can not only induce better local and distal mucosal immunity, but also produce better systemic immunity. The oral viral vaccine has also attracted much attention because of its painless vaccination, self-administration and other advantages. Due to the complexity of human digestive tract environment and mucosal immunity, only three oral attenuated live vaccines have been successfully marketed for human use. This review summarizes the characteristics of gastrointestinal mucosal immunity, the current types and research status of oral viral vaccines, and the challenges faced by oral viral vaccines, with the hope to facilitate the research and development of oral viral vaccines for human use in China.

mRNA nanomedicine: Design and recent applications.

The rational design and application of mRNA-based medicine have recently yielded some key successes in the clinical management of human diseases. mRNA technology allows for the facile and direct production of proteins in vivo, thus circumventing the need for lengthy drug development cycles and complex production workflows. As such, mRNA formulations can significantly improve upon the biological therapies that have become commonplace in modern medicine. Despite its many advantages, mRNA is inherently fragile and has specific delivery requirements. Leveraging the engineering flexibility of nanobiotechnology, mRNA payloads can be incorporated into nanoformulations such that they do not invoke unwanted immune responses, are targeted to tissues of interest, and can be delivered to the cytosol, resulting in improved safety while enhancing bioactivity. With the rapidly evolving landscape of nanomedicine, novel technologies that are under development have the potential to further improve the clinical utility of mRNA medicine. This review covers the design principles relevant to engineering mRNA-based nanomedicine platforms. It also details the current research on mRNA nanoformulations for addressing viral infections, cancers, and genetic diseases. Given the trends in the field, future mRNA-based nanomedicines have the potential to change how many types of diseases are managed in the clinic.

Application of bioreactor technology for cell culture-based viral vaccine production: Present status and future prospects.

Bioreactors are widely used in cell culture-based viral vaccine production, especially during the coronavirus disease 2019 (COVID-19) pandemic. In this context, the development and application of bioreactors can provide more efficient and cost-effective vaccine production to meet the global vaccine demand. The production of viral vaccines is inseparable from the development of upstream biological processes. In particular, exploration at the laboratory-scale is urgently required for further development. Therefore, it is necessary to evaluate the existing upstream biological processes, to enable the selection of pilot-scale conditions for academic and industrial scientists to maximize the yield and quality of vaccine development and production. Reviewing methods for optimizing the upstream process of virus vaccine production, this review discusses the bioreactor concepts, significant parameters and operational strategies related to large-scale amplification of virus. On this basis, a comprehensive analysis and evaluation of the various process optimization methods for the production of various viruses (SARS-CoV-2, Influenza virus, Tropical virus, Enterovirus, Rabies virus) in bioreactors is presented. Meanwhile, the types of viral vaccines are briefly introduced, and the established animal cell lines for vaccine production are described. In addition, it is emphasized that the co-development of bioreactor and computational biology is urgently needed to meet the challenges posed by the differences in upstream production scales between the laboratory and industry.

Hypertension is associated with antibody response and breakthrough infection in health care workers following vaccination with inactivated SARS-CoV-2.

Several types of vaccines have been developed to prevent the coronavirus disease 2019 (COVID-19). It is important to understand whether demographic and clinical variables affect the effectiveness of various types of vaccines. This study analysed the association between demographic/clinical factors, antibody response and vaccine effectiveness in healthcare workers vaccinated with inactivated virus. We enrolled 101 healthcare workers who received two doses of inactivated viral vaccine (CoronaVac). Blood samples were analysed at 1, 3, and 5 months after the second dose of vaccination. Data regarding demographic characteristics, medical histories, and clinical parameters were collected by interview and medical examination. In a separate retrospective study, we analysed the incidence of vaccine breakthrough infection on 2714 healthcare workers who received two doses of inactivated viral vaccine. Medical histories and demographic data were collected using a structured self-reported questionnaire. We found that antibody titres markedly increased at 1 month after vaccination but gradually decreased at 3-5 months post-vaccination. We observed a significant association between age (≥40 years) and antibody level, whereas sex and body mass index (BMI) exhibited no effect on antibody titres. Amongst clinical variables analysed, high blood pressure and history of hypertension were significantly correlated with lower antibody titres. Consistently, we found a significant association in the retrospective study between hypertension and the incidence of breakthrough infection. In conclusion, our results showed that hypertension is associated with lower antibody titres and breakthrough infection following COVID-19 vaccination. Thus, blood pressure control might be important to improve the efficacy of inactivated virus vaccine.

Evaluation of Recombinant Live-Attenuated Respiratory Syncytial Virus (RSV) Vaccines RSV/ΔNS2/Δ1313/I1314L and RSV/276 in RSV-Seronegative Children.

BACKGROUND: This United States-based study compared 2 candidate vaccines: RSV/ΔNS2/Δ1313/I1314L, attenuated by NS2 gene-deletion and temperature-sensitivity mutation in the polymerase gene; and RSV/276, attenuated by M2-2 deletion. METHODS: RSV-seronegative children aged 6-24 months received RSV/ΔNS2/Δ1313/I1314L (106 plaque-forming units [PFU]), RSV/276 (105 PFU), or placebo intranasally. Participants were monitored for vaccine shedding, reactogenicity, and RSV serum antibodies, and followed over the subsequent RSV season. RESULTS: Enrollment occurred September 2017 to October 2019. During 28 days postinoculation, upper respiratory illness and/or fever occurred in 64% of RSV/ΔNS2/Δ1313/I1314L, 84% of RSV/276, and 58% of placebo recipients. Symptoms were generally mild. Cough was more common in RSV/276 recipients than RSV/ΔNS2/Δ1313/I1314L (48% vs 12%; P = .012) or placebo recipients (17%; P = .084). There were no lower respiratory illness or serious adverse events. Eighty-eight and 96% of RSV/ΔNS2/Δ1313/I1314L and RSV/276 recipients were infected with vaccine (shed vaccine and/or had ≥4-fold rises in RSV antibodies). Serum RSV-neutralizing titers and anti-RSV F IgG titers increased ≥4-fold in 60% and 92% of RSV/ΔNS2/Δ1313/I1314L and RSV/276 vaccinees, respectively. Exposure to community RSV during the subsequent winter was associated with strong anamnestic RSV-antibody responses. CONCLUSIONS: Both vaccines had excellent infectivity and were well tolerated. RSV/276 induced an excess of mild cough. Both vaccines were immunogenic and primed for strong anamnestic responses. CLINICAL TRIALS REGISTRATION: NCT03227029 and NCT03422237.

Vaccines against Major Poultry Viral Diseases: Strategies to Improve the Breadth and Protective Efficacy.

The poultry industry is the largest source of meat and eggs for human consumption worldwide. However, viral outbreaks in farmed stock are a common occurrence and a major source of concern for the industry. Mortality and morbidity resulting from an outbreak can cause significant economic losses with subsequent detrimental impacts on the global food supply chain. Mass vaccination is one of the main strategies for controlling and preventing viral infection in poultry. The development of broadly protective vaccines against avian viral diseases will alleviate selection pressure on field virus strains and simplify vaccination regimens for commercial farms with overall savings in husbandry costs. With the increasing number of emerging and re-emerging viral infectious diseases in the poultry industry, there is an urgent need to understand the strategies for broadening the protective efficacy of the vaccines against distinct viral strains. The current review provides an overview of viral vaccines and vaccination regimens available for common avian viral infections, and strategies for developing safer and more efficacious viral vaccines for poultry.

Development and Scalable Production of Newcastle Disease Virus-Vectored Vaccines for Human and Veterinary Use.

The COVID-19 pandemic has highlighted the need for efficient vaccine platforms that can rapidly be developed and manufactured on a large scale to immunize the population against emerging viruses. Viral-vectored vaccines are prominent vaccine platforms that have been approved for use against the Ebola virus and SARS-CoV-2. The Newcastle Disease Virus is a promising viral vector, as an avian paramyxovirus that infects poultry but is safe for use in humans and other animals. NDV has been extensively studied not only as an oncolytic virus but also a vector for human and veterinary vaccines, with currently ongoing clinical trials for use against SARS-CoV-2. However, there is a gap in NDV research when it comes to process development and scalable manufacturing, which are critical for future approved vaccines. In this review, we summarize the advantages of NDV as a viral vector, describe the steps and limitations to generating recombinant NDV constructs, review the advances in human and veterinary vaccine candidates in pre-clinical and clinical tests, and elaborate on production in embryonated chicken eggs and cell culture. Mainly, we discuss the existing data on NDV propagation from a process development perspective and provide prospects for the next steps necessary to potentially achieve large-scale NDV-vectored vaccine manufacturing.

Sequential Combination of a Strong Interferon Inducer Viral Vector With Low Doses of Nivolumab Plus Ipilimumab Could Provide Functional Cure in Chronic Hepatitis B Virus infections: Technical Report Proposing a New Modality.

Based on the recommendation of the International Coalition to Eliminate hepatitis B virus (ICE-HBV), we intend to mimic the spontaneous resolution of HBV infection to achieve a functional cure of chronic hepatitis B virus (HBV) infection. To this end, we propose sequential targeting of the innate and adaptive host immune responses. Long-term suppression of HBV replication and hepatitis B surface antigen (HbsAg) production will be achieved first by inducing a strong innate immune response. The clinically validated viral superinfection therapy (SIT) will be administered, which employs an attenuated, non-lytic, double-stranded RNA (dsRNA) infectious bursal disease virus (IBDV) that provides an exceptionally strong interferon (IFN) response. Then, the exhausted HBV-specific T cell function will be restored by blocking the cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and programmed cell death protein 1 (PD-1) receptors with immune checkpoint inhibitors (ICIs). In order to minimize any risk of toxicity, off-label low doses of nivolumab (0.5 mg/kg) plus ipilimumab (0.3 mg/kg) will be administered, the safety and efficacy of which has already been demonstrated in 131 unselected stage IV cancer patients. We predict that this combination therapy will provide sustained off-treatment virological and clinical responses during a relatively short treatment period.