Development of live cultural pandemic influenza vaccine Vector-Flu

Background The threat of pandemic A/H1N1 influenza remains a matter of considerable public concern. Recent influenza outbreaks underline the importance of rapid production of a reserve of vaccine sufficient for pandemic and interpandemic periods. Traditional vaccines intended for seasonal influenza do not satisfy this demand, because they are unable to induce cross-reactive antibodies to pandemic flu. Live influenza vaccines made in the cell culture offer potential advantages because it is possible to produce a considerable amount of these vaccines over a short period. Their use eliminate concerns about allergic reactions to egg proteins, and they allow the antigenic composition of the vaccine to be closer to the circulating influenza virus strains. The goal of this work was to develop a live attenuated vaccine against pandemic influenza, Vector-Flu, derived from cold-adapted strain A/17/California/2009/38 (H1N1) and to study the immunogenic and protective properties of the vaccine using ferrets and mice.


Background
The threat of pandemic A/H1N1 influenza remains a matter of considerable public concern. Recent influenza outbreaks underline the importance of rapid production of a reserve of vaccine sufficient for pandemic and interpandemic periods. Traditional vaccines intended for seasonal influenza do not satisfy this demand, because they are unable to induce cross-reactive antibodies to pandemic flu.
Live influenza vaccines made in the cell culture offer potential advantages because it is possible to produce a considerable amount of these vaccines over a short period. Their use eliminate concerns about allergic reactions to egg proteins, and they allow the antigenic composition of the vaccine to be closer to the circulating influenza virus strains.
The goal of this work was to develop a live attenuated vaccine against pandemic influenza, Vector-Flu, derived from cold-adapted strain A/17/California/2009/38 (H1N1) and to study the immunogenic and protective properties of the vaccine using ferrets and mice.

Animals
Female ferrets were 4-5 month old and were obtained from the Animal Farm Rodniki (Pushkino, Moscow region, Russia).

Determination of influenza virus infectious activity
The infectious activity of influenza virus was determined by titration in 10-12-day-old chick embryos. 10-fold dilutions (0.2 ml) of virus-containing fluid were inoculated into the allantoic cavity of chick embryos. The embryos were incubated for 48 hours at a temperature of 35°C. After the incubation, the allantoic fluid was harvested from the embryos to determine the virus infectious activity by agglutination reaction with 1% chicken red blood cells. The virus titer was calculated according to the Reed-Muench method and expressed as log EID 50 /0.2 ml [2].

Control of immunogenicity of the Vector-Flu vaccine
18 ferrets were anesthetized to take blood samples (5 ml) from the caudal vein; they were then immunized with 200 µl of the Vector-Flu vaccine at 6.6-6.8 log EID 50 dose delivered into each nostril. One ferret subgroup was immunized with a single dose and another subgroup received two doses of vaccine with a 10-day interval between vaccinations. At day 21 after the last immunization, the sedated animals were bled from the caudal vein to obtain sera and the antibody titers were Hemagglutination Inhibition (HAI) test. HAI was performed by a routine technique [3] with some modifications. The assayed sera were pre-treated with the receptor destroying enzyme (RDE). The hemagglutination reaction was performed with 1% chicken red blood cells (RBC). The HAI titer was determined as the reciprocal dilution of the last row which contained nonagglutinated RBC.
Microneutralization assay. The assay was performed in compliance with the WHO guidelines [3] with some modifications. MDCK cells supplemented with equal volumes of serum and influenza virus were mixed and incubated in 5% CO 2 at 37°C. The presence of the virus was detected by enzyme immunoassay using the monoclonal antibodies to type A influenza virus NP protein (CDC, Atlanta). Neutralizing antibody titer was defined as reciprocal of the highest serum dilution that provided 50% inhibition of the virus growth in cell culture.

Results
The live MDCK-derived Vector-Flu vaccine was produced in accordance with the established production specifications. Ever in Russia, based on MDCK cell line, we have developed seed and working banks of cells; cell banks are certified in accordance with WHO recommendations [1].
The vaccine production technology involved the following steps: • microcarrier cultivation of MDCK cells in fermenters in serum-free medium; • A/17/California/2009/38 (H1N1) virus growth in the cell culture; • virus-containing fluid collection and purification; • introduction of stabilizers and biodegradable polymer; • freeze-drying; ready-to-use vaccine formulation production.
7 laboratory batches of vaccine Vector-Flu have been produced and certified; they have specific activity index as 7.33-7.5 lg EID 50 /ml. Ferret immune response data to administration of the live MDCK-derived Vector-Flu vaccine are listed in the

Conclusions
We have developed live cultural influenza vaccine production technology. The technology used for pandemic influenza vaccine Vector-Flu production has a versatile use mode, and it could be used for growing live cultural vaccines based on the other influenza virus subtypes. We have obtained the patent of Russia #2330885 for the developed technology. Table 1 The titers of ferret blood serum to A/H1N1 influenza virus strains before and after immunization with the Vector-Flu vaccine determined by HAI and microneutralization assay.