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.

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.

Emulsified nanoparticles containing inactivated influenza virus and CpG oligodeoxynucleotides critically influences the host immune responses in mice.

BACKGROUND: Antigen sparing and cross-protective immunity are regarded as crucial in pandemic influenza vaccine development. Both targets can be achieved by adjuvantation strategy to elicit a robust and broadened immune response. We assessed the immunogenicity of an inactivated H5N1 whole-virion vaccine (A/Vietnam/1194/2004 NIBRG-14, clade 1) formulated with emulsified nanoparticles and investigated whether it can induce cross-clade protecting immunity. METHODOLOGY/PRINCIPAL FINDINGS: After formulation with PELC, a proprietary water-in-oil-in-water nanoemulsion comprising of bioresorbable polymer/Span(R)85/squalene, inactivated virus was intramuscularly administered to mice in either one-dose or two-dose schedule. We found that the antigen-specific serum antibody responses elicited after two doses of non-adjuvanted vaccine were lower than those observed after a single dose of adjuvanted vaccine, PELC and the conventional alum adjuvant as well. Moreover, 5 microg HA of PELC-formulated inactivated virus were capable of inducing higher antibodies than those obtained from alum-adjuvanted vaccine. In single-dose study, we found that encapsulating inactivated virus into emulsified PELC nanoparticles could induce better antibody responses than those formulated with PELC-adsorbed vaccine. However, the potency was rather reduced when the inactivated virus and CpG (an immunostimulatory oligodeoxynucleotide containing unmethylated cytosine-guanosine motifs) were co-encapsulated within the emulsion. Finally, the mice who received PELC/CpG(adsorption)-vaccine could easily and quickly reach 100% of seroprotection against a homologous virus strain and effective cross-protection against a heterologous virus strain (A/Whooper swan/Mongolia/244/2005, clade 2.2). CONCLUSIONS/SIGNIFICANCE: Encapsulating inactivated H5N1 influenza virus and CpG into emulsified nanoparticles critically influences the humoral responses against pandemic influenza. These results demonstrated that the use of PELC could be as antigen-sparing in preparation for a potential shortage of prophylactic vaccines against local infectious diseases, in particular pandemic influenza. Moreover, the cross-clade neutralizing antibody responses data verify the potential of such adjuvanted H5N1 candidate vaccine as an effective tool in pre-pandemic preparedness.

Microcarrier-based MDCK cell culture system for the production of influenza H5N1 vaccines.

Current egg-based influenza vaccine production technology, which is labor intensive and slow, would not be able to meet demand during an influenza pandemic. Thus, interest in the emerging technology of using mammalian cells for vaccine production has been great. In this study, Madin-Darby canine kidney (MDCK) cells using microcarrier culture systems were established to produce inactivated whole-virus H5N1 vaccine. The current clade-1 influenza H5N1 vaccine virus (NIBRG-14) was provided by the UK National Institute for Biological Standards and Control. Various process parameters were first optimized in 100-mL scale spinner flasks then scaled up to a 1-L scale bioreactor system. In the 1-L scale bioreactor system, peak virus titer could reach 10(8-9)TCID50/mL using serum-containing medium. After purification and inactivation, hemagglutinin (HA) protein content reached 31.56-43.96 microg/mL in two different runs. In mice immunogenicity studies, two doses of the purified vaccine antigen adjuvanted with aluminum phosphate induced good immune responses in 0.2 and 1.0 microg HA dosages (geometric mean titers of hemagglutination-inhibition antibody: 113 and 242, respectively). This study demonstrates the feasibility of the development of MDCK cell-based inactivated influenza H5 vaccines in microcarrier culture systems and could be valuable to many countries that are planning to establish manufacturing capacity for influenza vaccines.

The conditioned enhancement of neutrophil activity is catecholamine dependent.

Neutrophil activity was elevated in the conditioned mice for the first time through an established conditioned training process. Catecholamines were proved to be important in the regulation of this conditioned innate immunity. In the study, the camphor odor (as the conditioned stimulus, CS) and poly I: C (as the unconditioned stimulus, US) was used to conditionally elevate the activity of the splenic neutrophils. The mechanism(s) responsible for the conditioned enhancement of neutrophil activity was further investigated using the neurochemical blocking assay and immunohistochemical analysis. Results showed that the neutrophil activity was significantly enhanced through the conditioned training process; both reserpine and 6-hydroxydopamine (6-OHDA) significantly blocked this conditioned innate immunity at the conditioned recall stage. Dexamethasone (Dex), however, showed no effect on the conditioned neutrophil response. Tyrosine hydroxylase (TH)-positive cells significantly increased in the locus coeruleus (LC), hypothalamus, and cortex but not in the spleen of the conditioned animals. These results indicate that during the conditioned recall stage, the brain signals the splenic neutrophils via the sympathetic nervous system (SNS) by releasing the peripheral catecholamines in spleen. The activation of the SNS, on the other hand, is also under the influence of catecholamines released in the LC. The hypothalamic pituitary (HP) axis, on the other hand, plays no role in the regulation of the conditioned neutrophil response.

Microglia-derived glial cell line-derived neurotrophic factor could protect Sprague-Dawley rat astrocyte from in vitro ischemia-induced damage.

The interrelationship between microglia and astrocytes in cerebral ischemia was determined in vitro by adding in vitro ischemia-induced supernatant from microglia into astrocytes under the same conditions (glucose-, oxygen- and serum-free). The involvement of glial cell line-derived neurotrophic factor (GDNF) was further investigated by immunoblocking assay and Western blot analysis. Results showed that microglia-derived supernatant protected against in vitro ischemia-induced damage of astrocytes and this protection was pre-blocked by anti-GDNF but not normal rabbit serum. In addition, in vitro ischemia appeared to induce the expression of GDNF in microglia. These results indicate that microglia-derived protection on astrocytes during in vitro ischemia is GDNF-dependent.

Cholinergic and serotonergic activities are required in triggering conditioned NK cell response.

The purpose of the study was to examine the importance of the cholinergic system in triggering the conditioned NK cell response. The fact that serotonergic system can modulate cholinergic functions suggested that it might be involved in conditioned NK cell response. To evaluate the potential pathways, cholinergic and serotonergic antagonists were applied centrally at either the conditioned association or recall stage, to interfere with the conditioned NK cell response. The results showed that both the cholinergic and serotonergic systems were necessary for eliciting the conditioned enhancement of NK cell activity. Involvements of the two systems were found to be critical for establishing the conditioned association and recall of the conditioned response. The blocks are believed to be receptor mediated. The receptors identified to be involved in the regulation of the conditioned NK cell response were: M(1), M(2) and M(3) muscarinic; nicotinic; 5 HT(1) and 5 HT(2) receptors.