Optimized production and purification of Coxsackievirus B1 vaccine and its preclinical evaluation in a mouse model.

Coxsackie B viruses are among the most common enteroviruses, causing a wide range of diseases. Recent studies have also suggested that they may contribute to the development of type 1 diabetes. Vaccination would provide an effective way to prevent CVB infections, and the objective of this study was to develop an efficient vaccine production protocol for the generation of novel CVB vaccines. Various steps in the production of a formalin-inactivated Coxsackievirus B1 (CVB1) vaccine were optimized including the Multiplicity Of Infection (MOI) used for virus amplification, virus cultivation time, type of cell growth medium, virus purification method and formulation of the purified virus. Safety and immunogenicity of the formalin inactivated CVB1 vaccine was characterized in a mouse model. Two of the developed methods were found to be optimal for virus purification: the first employed PEG-precipitation followed by gelatin-chromatography and sucrose cushion pelleting (three-step protocol), yielding 19-fold increase in virus concentration (0.06µg/cm2) as compared to gold standard method. The second method utilized tandem sucrose pelleting without a PEG precipitation step, yielding 83-fold increase in virus concentration (0.24µg/cm2), but it was more labor-intensive and cannot be efficiently scaled up. Both protocols provide radically higher virus yields compared with traditional virus purification protocols involving PEG-precipitation and sucrose gradient ultracentrifugation. Formalin inactivation of CVB1 produced a vaccine that induced a strong, virus-neutralizing antibody response in vaccinated mice, which protected against challenge with CVB1 virus. Altogether, these results provide valuable information for the development of new enterovirus vaccines.

Vaccination strategies against Zika virus.

The epidemic emergence of Zika virus (ZIKV) in 2015-2016 has been associated with congenital malformations and neurological sequela. Current efforts to develop a ZIKV vaccine build on technologies that successfully reduced infection or disease burden against closely related flaviviruses or other RNA viruses. Subunit-based (DNA plasmid and modified mRNA), viral vectored (adeno- and measles viruses) and inactivated viral vaccines are already advancing to clinical trials in humans after successful mouse and non-human primate studies. Among the greatest challenges for the rapid implementation of immunogenic and protective ZIKV vaccines will be addressing the potential for exacerbating Dengue virus infection or causing Guillain-Barré syndrome through production of cross-reactive immunity targeting related viral or host proteins. Here, we review vaccine strategies under development for ZIKV and the issues surrounding their usage.

Pre-clinical development of a hydrogen peroxide-inactivated West Nile virus vaccine.

West Nile virus (WNV) is a mosquito-transmitted pathogen with a wide geographical range that can lead to long-term disability and death in some cases. Despite the public health risk posed by WNV, including an estimated 3 million infections in the United States alone, no vaccine is available for use in humans. Here, we present a scaled manufacturing approach for production of a hydrogen peroxide-inactivated whole virion WNV vaccine, termed HydroVax-001WNV. Vaccination resulted in robust virus-specific neutralizing antibody responses and protection against WNV-associated mortality in mice or viremia in rhesus macaques (RM). A GLP-compliant toxicology study performed in rats demonstrated an excellent safety profile with clinical findings limited to minor and transient irritation at the injection site. An in vitro relative potency (IVRP) assay was developed and shown to correlate with in vivo responses following forced degradation studies. Long-term in vivo potency comparisons between the intended storage condition (2-8°C) and a thermally stressed condition (40±2°C) demonstrated no loss in vaccine efficacy or protective immunity over a 6-month span of time. Together, the positive pre-clinical findings regarding immunogenicity, safety, and stability indicate that HydroVax-001WNV is a promising vaccine candidate.

The Madin-Darby canine kidney cell culture derived influenza A/H5N1 vaccine: a phase I trial in Taiwan.

BACKGROUND: Avian H5N1 influenza has caused human infections globally and has a high mortality rate. Rapid production of effective vaccines is needed. METHODS: A phase 1, randomized, observer-blinded clinical trial was conducted to examine the safety and immunogenicity of an inactivated whole virion vaccine against the influenza A/H5N1 virus produced from the Madin-Darby canine kidney (MDCK) cell line. Participants were randomized to four groups and administered two intramuscular doses of vaccine containing 3 µg hemagglutinin (HA), 3 µg HA with 300 µg aluminum phosphate (AlPO4), 6 µg HA, and 6 µg HA with 300 µg AlPO4, respectively, at two visits, 21 days apart. Serum hemagglutination inhibition (HAI) and neutralizing antibody levels were determined at baseline and on Days 21 and 42. RESULTS: Sixty healthy individuals were enrolled. The neutralization assay showed a significant immune response in the 6 µg with ALPO4 group on Day 42 compared to pre-vaccination levels (11.32±9.77 vs. 4.00±0, p=0.02). The adjuvant effect in neutralization assay was also significant on Day 42 in the 6 µg group (4.52±1.94 without adjuvant vs. 11.32±9.77 with adjuvant, p=0.02). HAI assay also showed an aluminum adjuvant-induced increasing trend in HAI geometric mean titer on Day 42 in the 3 µg and 6 µg groups (6.02 versus 8.20, p=0.05 and 5.74 versus 8.21, p=0.14). The most frequent adverse event was local pain (20% to 60%). There were no vaccine-related severe adverse effects. CONCLUSION: MDCK cell line-derived H5N1 vaccine was well tolerated. It is necessary to investigate further the immunogenicity of higher antigen doses and the role of aluminum adjuvant in augmenting the effect of the vaccine.

Pilot scale production of highly efficacious and stable enterovirus 71 vaccine candidates.

BACKGROUND: Enterovirus 71 (EV71) has caused several epidemics of hand, foot and mouth diseases (HFMD) in Asia and now is being recognized as an important neurotropic virus. Effective medications and prophylactic vaccine against EV71 infection are urgently needed. Based on the success of inactivated poliovirus vaccine, a prototype chemically inactivated EV71 vaccine candidate has been developed and currently in human phase 1 clinical trial. PRINCIPAL FINDING: In this report, we present the development of a serum-free cell-based EV71 vaccine. The optimization at each step of the manufacturing process was investigated, characterized and quantified. In the up-stream process development, different commercially available cell culture media either containing serum or serum-free was screened for cell growth and virus yield using the roller-bottle technology. VP-SFM serum-free medium was selected based on the Vero cell growth profile and EV71 virus production. After the up-stream processes (virus harvest, diafiltration and concentration), a combination of gel-filtration liquid chromatography and/or sucrose-gradient ultracentrifugation down-stream purification processes were investigated at a pilot scale of 40 liters each. Although the combination of chromatography and sucrose-gradient ultracentrifugation produced extremely pure EV71 infectious virus particles, the overall yield of vaccine was 7-10% as determined by a VP2-based quantitative ELISA. Using chromatography as the downstream purification, the virus yield was 30-43%. To retain the integrity of virus neutralization epitopes and the stability of the vaccine product, the best virus inactivation was found to be 0.025% formalin-treatment at 37 °C for 3 to 6 days. Furthermore, the formalin-inactivated virion vaccine candidate was found to be stable for >18 months at 4 °C and a microgram of viral proteins formulated with alum adjuvant could induce strong virus-neutralizing antibody responses in mice, rats, rabbits, and non-human primates. CONCLUSION: These results provide valuable information supporting the current cell-based serum-free EV71 vaccine candidate going into human Phase I clinical trials.

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.

Evaluation of the purity of a purified, inactivated hepatitis A vaccine (VAQTA).

Manufacture of VAQTA, an inactivated hepatitis A virus vaccine, includes extensive purification of the intact virus particle to remove endogenous components from the host cell culture lysate as well as compounds introduced in the upstream purification process. Analysis of the final purified hepatitis A virus product by SDS-PAGE prior to inactivation shows that greater than 95% of the protein in the preparation is found in four protein bands, which have been confirmed to be hepatitis A virus capsid proteins VP0, VP1, VP2 and VP3 based on Western blot and mass spectrometry analyses. Validation of the manufacturing process and direct analysis of the final product were used to demonstrate that no other specific host cell-derived components are detected and that process residuals are all below the limits of detection of the assays used. Establishment of a rigorous standard of high purity for this product was pursued to minimize the impact of impurities during clinical development of this product and will facilitate the incorporation of this product into combination vaccines.

[A trial to produce an inactivated Lassa fever vaccine]. / Opyt polucheniia inaktivirovannoi vaktsiny likhoradki Lassa.

A single and double immunization of Papio hamadryas monkeys with an inactivated concentrated purified adsorbed preparation of Lassa virus in doses of 0.5-1.0 and 1.1-2.1 mg by protein, respectively, protected the animals against the development of the disease and occurrence of viremia after an intramuscular challenge with 0.4 PFU dose. Upon aerogenic infection of monkeys protection of 50% of the animals was achieved by a single immunization with the inactivated preparation in a dose 1.1 mg by protein.

Preparation and immunogenicity of an inactivated hepatitis A vaccine.

A hepatitis A vaccine was prepared by formaldehyde inactivation of purified hepatitis A virus (HAV) LSH/S strain grown on human diploid MRC-5 cells. The vaccine was devoid of residual infectivity in vitro and failed to induce in marmoset monkeys any pathological features or variations of haematological and clinical chemistry values. Infectious HAV particles were not detected in faeces and sera of the vaccinated primates by ELISA or after passages in MRC-5 cells. The immunogenicity of the vaccine was evaluated by injecting guinea-pigs with 0.8, 0.2 or 0.05 micrograms of HAV antigen adsorbed onto 0.5 and 1 mg of Al (OH)3 or 0.3 mg of AlPO4. The antibody response, measured by a competitive radioimmunoassay, was dose- and adjuvant-dependent. One injection of 0.2 micrograms of AlPO4-adsorbed HAV antigen induced seroconversion in 100% of animals and high levels of specific and neutralizing serum antibodies. A further increase of antibody titres was observed after the second and third inoculations. These results show that this vaccine formulation is safe and immunogenic in animal models, and suggest that it should be evaluated further by human clinical studies.

[A comparison of the protective properties of preparations of the virion and nonvirion ("soluble") antigens of the tick-borne encephalitis virus]. / Sopostavlenie protektivnykh svoistv preparatov virionnogo i nevirionnogo ("rastvorimogo") antigenov virusa kleshchevogo éntsefalita.

A comparative assessment of the protective properties of virion (VA) and nonvirion ("soluble") (NA) antigens of tick-borne encephalitis virus prepared as inactivated samples close in their parameters to vaccine preparations was carried out. The NA in the preparations free from VA or containing only trace, nonprotective amounts of it, was shown to have significantly lower protective properties than VA and exerted no booster effect on the protective activity when added to VA preparations.