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.

Inactivation methods for whole influenza vaccine production.

Despite tremendous efforts toward vaccination, influenza remains an ongoing global threat. The induction of strain-specific neutralizing antibody responses is a common phenomenon during vaccination with the current inactivated influenza vaccines, so the protective effect of these vaccines is mostly strain-specific. There is an essential need for the development of next-generation vaccines, with a broad range of immunogenicity against antigenically drifted or shifted influenza viruses. Here, we evaluate the potential of whole inactivated vaccines, based on chemical and physical methods, as well as new approaches to generate cross-protective immune responses. We also consider the mechanisms by which some of these vaccines may induce CD8+ T-cells cross-reactivity with different strains of influenza. In this review, we have focused on conventional and novel methods for production of whole inactivated influenza vaccine. As well as chemical modification, using formaldehyde or ß-propiolactone and physical manipulation by ultraviolet radiation or gamma-irradiation, novel approaches, including visible ultrashort pulsed laser, and low-energy electron irradiation are discussed. These two latter methods are considered to be attractive approaches to design more sophisticated vaccines, due to their ability to maintain most of the viral antigenic properties during inactivation and potential to produce cross-protective immunity. However, further studies are needed to validate them before they can replace traditional methods for vaccine manufacturing.

Enhancing enterovirus A71 vaccine production yield by microcarrier profusion bioreactor culture.

Hand, foot and mouth diseases (HFMD) are mainly caused by Enterovirus A71 (EV-A71) infections. Clinical trials in Asia conducted with formalin-inactivated EV-A71 vaccine candidates produced from serum-free Vero cell culture using either roller bottle or cell factory technology, are found to be safe and highly efficacious. To increase vaccine yields and reduce the production costs, the bioprocess improvement for EV-A71 vaccine manufacturing is currently being investigated. The parameters that could affect and enhance the production yields of EV-A71 virus growth in the microcarrier bioreactor were investigated. The medium replacement culture strategy included a multi-harvested semi-batch process and perfusion technology and was found to increase the production yields more than 7-14 folds. Based on the western blot and cryo-EM analyses of the EV-A71 virus particles produced from either the multi-harvested semi-batch (MHSBC) or perfusion cultures were found to be similar to those virus particles obtained from the single batch culture. Mouse immunogenicity studies indicate that the EV-A71 vaccine candidates produced from the perfusion culture have similar potency to those obtained from single batch bioprocess. The physical structures of the EV-A71 particles revealed by the cryo-EM analysis were found to be spherical capsid particles. These results provide feasible technical bioprocesses for increasing virus yields and the scale up of EV-A71 vaccine manufacturing using the bioreactor cell culture methods.

Influenza Vaccine Manufacturing: Effect of Inactivation, Splitting and Site of Manufacturing. Comparison of Influenza Vaccine Production Processes.

The aim of this study was to evaluate the impact of different inactivation and splitting procedures on influenza vaccine product composition, stability and recovery to support transfer of process technology. Four split and two whole inactivated virus (WIV) influenza vaccine bulks were produced and compared with respect to release criteria, stability of the bulk and haemagglutinin recovery. One clarified harvest of influenza H3N2 A/Uruguay virus prepared on 25.000 fertilized eggs was divided equally over six downstream processes. The main unit operation for purification was sucrose gradient zonal ultracentrifugation. The inactivation of the virus was performed with either formaldehyde in phosphate buffer or with beta-propiolactone in citrate buffer. For splitting of the viral products in presence of Tween®, either Triton™ X-100 or di-ethyl-ether was used. Removal of ether was established by centrifugation and evaporation, whereas removal of Triton-X100 was performed by hydrophobic interaction chromatography. All products were sterile filtered and subjected to a 5 months real time stability study. In all processes, major product losses were measured after sterile filtration; with larger losses for split virus than for WIV. The beta-propiolactone inactivation on average resulted in higher recoveries compared to processes using formaldehyde inactivation. Especially ether split formaldehyde product showed low recovery and least stability over a period of five months.

Inactivated Enterovirus 71 Vaccine Produced by 200-L Scale Serum-Free Microcarrier Bioreactor System Provides Cross-Protective Efficacy in Human SCARB2 Transgenic Mouse.

Epidemics and outbreaks caused by infections of several subgenotypes of EV71 and other serotypes of coxsackie A viruses have raised serious public health concerns in the Asia-Pacific region. These concerns highlight the urgent need to develop a scalable manufacturing platform for producing an effective and sufficient quantity of vaccines against deadly enteroviruses. In this report, we present a platform for the large-scale production of a vaccine based on the inactivated EV71(E59-B4) virus. The viruses were produced in Vero cells in a 200 L bioreactor with serum-free medium, and the viral titer reached 10(7) TCID50/mL 10 days after infection when using an MOI of 10(-4). The EV71 virus particles were harvested and purified by sucrose density gradient centrifugation. Fractions containing viral particles were pooled based on ELISA and SDS-PAGE. TEM was used to characterize the morphologies of the viral particles. To evaluate the cross-protective efficacy of the EV71 vaccine, the pooled antigens were combined with squalene-based adjuvant (AddaVAX) or aluminum phosphate (AlPO4) and tested in human SCARB2 transgenic (Tg) mice. The Tg mice immunized with either the AddaVAX- or AlPO4-adjuvanted EV71 vaccine were fully protected from challenges by the subgenotype C2 and C4 viruses, and surviving animals did not show any degree of neurological paralysis symptoms or muscle damage. Vaccine treatments significantly reduced virus antigen presented in the central nervous system of Tg mice and alleviated the virus-associated inflammatory response. These results strongly suggest that this preparation results in an efficacious vaccine and that the microcarrier/bioreactor platform offers a superior alternative to the previously described roller-bottle system.

Development and preclinical testing of HNVAC, a cell culture-based H1N1 pandemic influenza vaccine from India.

Several limitations of the use of embryonated eggs and the threat of pandemics have highlighted the need for other platforms for the production of influenza vaccines. We report the indigenous development and pre-clinical testing of an MDCK-based H1N1 pandemic influenza vaccine HNVAC from India. The cell bank and virus seed were characterized extensively. The cells were characterized by PCR, electron microscopy, and karyotyping, and found to be of female canine epithelial origin. The virus was confirmed by neutralization, haemagglutination inhibition, neuraminidase inhibition, and PCR and nucleotide sequencing. Adventitious agent testing was performed by both in vitro and in vivo studies. The in vitro studies included culturing, haemadsorption, haemagglutination, PCR and RT-PCR, whereas in vivo studies included passage in embryonated eggs and in laboratory animals. Both cell bank and virus seed were free of adventitious agents. MDCK cell lysates as well as cellular DNA did not produce tumours in newborn or adult laboratory animals. The bioprocess parameters were standardized to recover antigen with minimal levels of process-related impurities. The vaccine bulk was tested for the presence of specific antigen, and quantified by single radial immunodiffusion. Finally, non-adjuvanted and aluminium hydroxide adjuvanted vaccine formulations were found to be safe in preclinical toxicity studies in mice, rats, guinea pigs and rabbits, and immunogenic in mice and rabbits. This is the first and only cell culture-based influenza vaccine platform developed in any developing country.

Production of inactivated influenza H5N1 vaccines from MDCK cells in serum-free medium.

BACKGROUND: Highly pathogenic influenza viruses pose a constant threat which could lead to a global pandemic. Vaccination remains the principal measure to reduce morbidity and mortality from such pandemics. The availability and surging demand for pandemic vaccines needs to be addressed in the preparedness plans. This study presents an improved high-yield manufacturing process for the inactivated influenza H5N1 vaccines using Madin-Darby canine kidney (MDCK) cells grown in a serum-free (SF) medium microcarrier cell culture system. PRINCIPAL FINDING: The current study has evaluated the performance of cell adaptation switched from serum-containing (SC) medium to several commercial SF media. The selected SF medium was further evaluated in various bioreactor culture systems for process scale-up evaluation. No significant difference was found in the cell growth in different sizes of bioreactors studied. In the 7.5 L bioreactor runs, the cell concentration reached to 2.3 × 10(6) cells/mL after 5 days. The maximum virus titers of 1024 Hemagglutinin (HA) units/50 µL and 7.1 ± 0.3 × 10(8) pfu/mL were obtained after 3 days infection. The concentration of HA antigen as determined by SRID was found to be 14.1 µg/mL which was higher than those obtained from the SC medium. A mouse immunogenicity study showed that the formalin-inactivated purified SF vaccine candidate formulated with alum adjuvant could induce protective level of virus neutralization titers similar to those obtained from the SC medium. In addition, the H5N1 viruses produced from either SC or SF media showed the same antigenic reactivity with the NIBRG14 standard antisera. CONCLUSIONS: The advantages of this SF cell-based manufacturing process could reduce the animal serum contamination, the cost and lot-to-lot variation of SC medium production. This study provides useful information to manufacturers that are planning to use SF medium for cell-based influenza vaccine production.

Development and optimisation of a procedure for the production of Parapoxvirus ovis by large-scale microcarrier cell culture in a non-animal, non-human and non-plant-derived medium.

For the production of a chemically inactivated Parapoxvirus ovis (PPVO), an adherent bovine kidney cell line was cultivated on Cytodex-3 microcarriers in suspension culture. The inactivated and purified virus particles have shown immune modulatory activity in several animal models. PPVO was produced by a biphasic batch process at the 3.5 and 10 L scale. Aeration was realised by bubble-free membrane oxygenation via a tube stator with a central two-blade anchor impeller. In order to increase efficiency, process robustness and safety, the established process was optimised. The cell line was adapted to a protein-free medium (except recombinant insulin) in order to increase biosafety. A scale up to a 50 L pilot plant with direct cell expansion was performed successfully. In parallel, the biphasic batch process was optimised with special emphasis on different operating conditions (cell number, Multiplicity of Infection (MOI), etc.) and process management (fed-batch, dialysis, etc.). The quality and concentration of the purified virus particles was assessed by quantitative electron microscopy, residual host cell protein and DNA-content and, finally, biologic activity in a transgenic mouse model. This integrated approach led to a new, safe, robust and highly productive large-scale production process, called "Volume-Expanded-Fed" Batch with cell densities up to 6-7e06 cells/mL. By subsequent dilution of infected cells into the next process scale, an increase in total productivity by a factor of 40 (related to an established biphasic batch process) was achieved.

Establishment of a mink enteritis vaccine production process in stirred-tank reactor and Wave Bioreactor microcarrier culture in 1-10 L scale.

A scale-up and process optimization scheme for the growth of adherent embryonic feline lung fibroblasts (E-FL) on microcarriers and the propagation of a mink enteritis virus (MEV) strain for the production of an inactivated vaccine is shown. Stirred-tank cultivations are compared with results obtained from Wave Bioreactors. Transfer from a roller bottle-based production process into large-scale microcarrier culture with starting concentrations of 2g/L Cytodex 1 microcarriers and 2.0 x 10(5)cells/mL was successful. A maximum cell yield of 1.2 x 10(6)cells/mL was obtained in stirred-tank microcarrier batch culture while cell numbers in the Wave Bioreactor could not be determined accurately due to the fast sedimentation of microcarriers under non-rocking conditions required for sampling. Detailed off-line analysis was carried out to understand the behaviour of the virus-host cell system in both cultivation systems. Metabolic profiles for glucose, lactate, glutamine, and ammonium showed slight differences for both systems. E-FL cell growth was on the same level in stirred-tank and Wave Bioreactor with a higher volumetric cell yield compared to roller bottles. Propagation of MEV, which can only replicate efficiently in mitotic cells, was characterized in the Wave Bioreactor using a multiple harvest strategy. Maximum virus titres of 10(6.6) to 10(6.8) TCID(50)/mL were obtained, which corresponds to an increase in virus yield by a factor of about 10 compared to cultivations in roller bottles. As a consequence, a single Wave Bioreactor cultivation of appropriate scale can replace hundreds of roller bottles. Thus, the Wave Bioreactor proved to be a suitable system for large-scale production of an inactivated MEV vaccine.

Influenza vaccines: recent advances in production technologies.

In spite of ongoing annual vaccination programs, the seasonal influenza epidemics remain a major cause of high morbidity and mortality. The currently used "inactivated" vaccines provide very short-term and highly specific humoral immunity due to the frequent antigenic variations in the influenza virion. These intra-muscularly administered vaccines also fail to induce protective mucosal immunity at the portal of viral entry and destruction of the virally infected cells by induction of cytotoxic T lymphocytes. Therefore, it is necessary to develop immunologically superior vaccines. This article highlights some of the recent developments in investigational influenza vaccines. The most notable recent developments of interest include the use of immunopotentiators, development of DNA vaccines, use of reverse genetics, and the feasibility of mammalian cell-based production processes. Presently, due to their safety and efficacy, the cold-adapted "live attenuated" vaccines are seen as viable alternatives to the "inactivated vaccines". The DNA vaccines are gaining importance due to the induction of broad-spectrum immunity. In addition, recent advances in recombinant technologies have shown the possibility of constructing pre-made libraries of vaccine strains, so that adequately preparations can be made for epidemics and pandemics.

[Present situation regarding development of an HIV vaccine]. / Situación actual en el desarrollo de una vacuna frente al virus de la inmunodeficiencia humana.

The AIDS epidemic continues to advance, and the development of a preventive HIV vaccine has become a major objective for scientific research. An effective vaccine against this virus is not available and complete protection still has not been achieved in animal models. In this review the major challenges related to the development of a vaccine against HIV are analyzed, particularly the mechanisms involved in viral escape from the immune response, and the results obtained with the various therapeutic and preventive vaccine prototypes are summarized. Finally, the social, economic and health aspects related to research on HIV vaccines and the current controversy around the performance of clinical trials with these agents is discussed.

A purified inactivated Japanese encephalitis virus vaccine made in Vero cells.

A second generation, purified, inactivated vaccine (PIV) against Japanese encephalitis (JE) virus was produced and tested in mice where it was found to be highly immunogenic and protective. The JE-PIV was made from an attenuated strain of JE virus propagated in certified Vero cells, purified, and inactivated with formalin. Its manufacture followed current GMP guidelines for the production of biologicals. The manufacturing process was efficient in generating a high yield of virus, essentially free of contaminating host cell proteins and nucleic acids. The PIV was formulated with aluminum hydroxide and administered to mice by subcutaneous inoculation. Vaccinated animals developed high-titered JE virus neutralizing antibodies in a dose dependent fashion after two injections. The vaccine protected mice against morbidity and mortality after challenge with live, virulent, JE virus. Compared with the existing licensed mouse brain-derived vaccine, JE-Vax, the Vero cell-derived JE-PIV was more immunogenic and as effective as preventing encephalitis in mice. The JE-PIV is currently being tested for safety and immunogenicity in volunteers.

Development of a mammalian cell (Vero) derived candidate influenza virus vaccine.

Influenza vaccine production is dependent on the availability of embryonated hen eggs for virus growth. This is an extremely cumbersome system with many disadvantages with respect to selection of virus variants and presence of adventitious viruses. We have developed an alternative cell culture system which allows rapid production of large volumes of vaccine. The World Health Organisation (WHO) approved Vero cell line was used in serum-free culture to grow a multitude of influenza strains to high titre. This system could be scaled-up to allow vaccine production with a 1200 litre fermenter volume. A purification scheme was developed which resulted in a high purity whole virus vaccine. This was demonstrated to be at least as immunogenic as a conventional egg-derived preparation in a mouse model.

[Attempts to develop a vaccine against Ebola fever]. / Popytka polucheniia vaktsiny protiv likhoradki Ebola.

Data on the immunopathogenesis of Ebola fever in laboratory animals are presented and the efficacy of some methods of vaccine prophylaxis discussed. Antiviral immunity induced in guinea pigs by injection of inactivated viral agents did not protect them from infection, whereas injections of a nonlethal strain of the virus in ascending doses led to the formation of immunity preventing the development of disease upon inoculation with a lethal strain in high doses. The role of some viral peptides in the development of immune response is shown and variants of recombinant constructions for the prevention of Ebola fever are offered.

Development and testing of an inactivated feline leukemia virus vaccine.

We assessed an inactivated whole virus feline leukemia virus (FeLV) vaccine developed from a molecularly cloned feline leukemia virus isolate (FeLV-61E-A) for its ability to protect cats against homologous and heterologous virulent virus challenge. The fractions of cats that resisted the induction of persistent viremia after FeLV challenge were the following: (1) FeLV-61E-A vaccine, 95%; (2) adjuvant controls, 26%; and (3) established commercial control FeLV vaccine, 35%. The pre-challenge mean neutralizing antibody titers for each group were (1) FeLV-61E-A vaccine, 1:43; (2) adjuvant controls, <1:8; and (3) established commercial control FeLV vaccine: 1:12. The commercial version of the prototype FeLV-61E-A vaccine (Fel-O-Vax, Fort Dodge Laboratories, Fort Dodge, IA) was developed through use of a proprietary adjuvant and a stable high antigen production cell lines. The efficacy and duration of immunity produced by Fel-O-Vax was studied alone and in multivalent combination with other feline virus vaccines in seven subsequent efficacy trials conducted in over 150 immunized cats. The overall FeLV-resistant fraction in these trials was 87% in vaccinated cats versus 8% in adjuvant controls. The duration of protective immunity induced by the multivalent Fel-O-Vax-LvK IV at 1 year postvaccination was 100% in challenged vaccinees versus 0% in challenged controls. The results of these studies show that an inactivated FeLV vaccine prepared from a molecularly cloned subgroup A FeLV can produce high level protective immunity against FeLV infection. This immunity is durable for at least 1 year without intervening booster immunization or virus exposure.

Development of the formalin-inactivated hepatitis A vaccine, VAQTA from the live attenuated virus strain CR326F.

The development of the formalin-inactivated hepatitis A vaccine, VAQTA, culminates nearly two decades of the basic science studies of VAQTA in hepatitis A virology at the MRL. The master seed virus for production of VAQTA is derived from the F'(P18) variant of the strain CR326F which has been studied in human clinical trials and shown to the highly attenuated. The antigen is highly purified to make possible the consistency and thoroughness of its inactivation by formalin. Phase I clinical studies of VAQTA were initiated in 1989 and have progressed since that time to the recent Phase III clinical trials which demonstrated efficacy of a single dose of the vaccine in preventing clinical hepatitis A disease in pediatric populations in Monroe, NY.