Isabelle Legastelois

Generation of candidate human influenza vaccine strains in cell culture – rehearsing the European response to an H7N1 pandemic threat

Background  Although H5N1 avian influenza viruses pose the most obvious imminent pandemic threat, there have been several recent zoonotic incidents involving transmission of H7 viruses to humans. Vaccines are the primary public health defense against pandemics, but reliance on embryonated chickens eggs to propagate vaccine and logistic problems posed by the use of new technology may slow our ability to respond rapidly in a pandemic situation.

Suitability of PER.C6 cells to generate epidemic and pandemic influenza vaccine strains by reverse genetics

Reverse genetics, the generation of influenza viruses from cDNA, presents a rapid method for creating vaccine strains. The technique necessitates the use of cultured cells. Due to technical and regulatory requirements, the choice of cell lines for production of human influenza vaccines is limited. PER.C6 cells, among the most extensively characterized and documented cells, support growth of all influenza viruses tested to date, and can be grown to high densities in large bioreactors in the absence of serum or micro carriers. Here, the suitability of these cells for the generation of influenza viruses by reverse genetics was investigated. A range of viruses reflective of vaccine strains was rescued exclusively using PER.C6 cells by various transfection methods, including an animal component-free procedure. Furthermore, a whole inactivated vaccine carrying the HA and NA segments of A/HK/156/97 (H5N1) that was both rescued from and propagated on PER.C6 cells, conferred protection in a mouse model. Thus PER.C6 cells provide an attractive platform for generation of influenza vaccine strains via reverse genetics.

Avian glycan-specific IgM monoclonal antibodies for the detection and quantitation of type A and B haemagglutinins in egg-derived influenza vaccines.

Two IgM monoclonal antibodies (MAbs), Y6F5 and Y13F9, were selected during a screening of clones obtained immunising BALB/c mice with purified envelop proteins of the A/Sydney/5/97 (H3N2) IVR108 influenza strain. These MAbs recognised avian glycans on the haemagglutinin (HA) of the virus. This broad recognition allowed these MAbs to be used as enzyme-labelled secondary antibody reagents in a strain specific enzyme-linked immunosorbent assay (ELISA) in combination with a capture MAb that recognised and allowed the quantitation of the strain specific HA protein present in an egg-produced influenza vaccine. Advantage was taken of these MAbs to develop a universal ELISA in which the MAbs were used both as capture antibody and as enzyme-labelled secondary antibody to detect and quantify the HA protein of any egg-derived influenza vaccine. These avian-glycan specific IgM MAbs may prove to be particularly useful for determining the HA concentration in monovalent egg-derived pandemic influenza vaccines, in which the HA concentration may be lower than 5μg/ml. The HA detection limit in the ELISA assays developed in this study was 1.9μg/ml, as opposed to the 5μg/ml quantitation limit generally accepted for the standard single-radial-immunodiffusion (SRID) assay, the approved technique for quantifying HA content in influenza vaccines. These ELISAs can also be used to quantify influenza HA formulated with emulsion-based or mineral salt adjuvants that could interfere with HA measurement by the SRID assay.

Preparation of genetically engineered A/H5N1 and A/H7N1 pandemic vaccine viruses by reverse genetics in a mixture of Vero and chicken embryo cells

Background  In case of influenza pandemic, a robust, easy and clean technique to prepare reassortants would be necessary.

Characterization and immunogenicity in mice of recombinant influenza haemagglutinins produced in Leishmania tarentolae

The membrane displayed antigen haemagglutinin (HA) from several influenza strains were expressed in the Leishmania tarentolae system. This non-conventional expression system based on a parasite of lizards, can be readily propagated to high cell density (>10(8)cells/mL) in a simple incubator at 26°C. The genes encoding HA proteins were cloned from six influenza strains, among these being a 2009 A/H1N1 pandemic strain from swine origin, namely A/California/07/09(H1N1). Soluble HA proteins were secreted into the cell culture medium and were easily and successfully purified via a His-Tag domain fused to the proteins. The overall process could be conducted in less than 3 months and resulted in a yield of approximately 1.5-5mg of HA per liter of biofermenter culture after purification. The recombinant HA proteins expressed by L. tarentolae were characterized by dynamic light scattering and were observed to be mostly monomeric. The L. tarentolae recombinant HA proteins were immunogenic in mice at a dose of 10μg when administered twice with an oil-in-water emulsion-based adjuvant. These results suggest that the L. tarentolae expression system may be an alternative to the current egg-based vaccine production.

Non-conventional expression systems for the production of vaccine proteins and immunotherapeutic molecules

ABSTRACT The increasing demand for recombinant vaccine antigens or immunotherapeutic molecules calls into question the universality of current protein expression systems. Vaccine production can require relatively low amounts of expressed materials, but represents an extremely diverse category consisting of different target antigens with marked structural differences. In contrast, monoclonal antibodies, by definition share key molecular characteristics and require a production system capable of very large outputs, which drives the quest for highly efficient and cost-effective systems. In discussing expression systems, the primary assumption is that a universal production platform for vaccines and immunotherapeutics will unlikely exist. This review provides an overview of the evolution of traditional expression systems, including mammalian cells, yeast and E.coli, but also alternative systems such as other bacteria than E. coli, transgenic animals, insect cells, plants and microalgae, Tetrahymena thermophila, Leishmania tarentolae, filamentous fungi, cell free systems, and the incorporation of non-natural amino acids.

Vero/CHOK1, a novel mixture of cell lines that is optimal for the rescue of influenza A vaccine seeds

Seasonal and pandemic influenza vaccine manufacturing is challenged with a tight production schedule. Reverse genetics constitutes a rapid method for creating viruses. Vero and CHOK1 cells were found to be an appropriate cell mixture for the generation of influenza reassortants by reverse genetics under the constraints of vaccine production, such as the use of regulatory-compliant cells and culture media devoid of components of animal origin. In addition, no further amplification in cell or egg substrates was required, thus reducing the time needed to obtain reassortant seed virus. In parallel, the cloning step was shown to be dramatically improved, permitting the rapid vRNA expression of influenza viruses. In addition, nucleoporation of the cells was conducted to more efficiently target the nucleus and avoid the use of chemical reagents containing proteins of animal origin. In conclusion, the reverse genetics system for influenza A viruses reported in this study was shown to be rapid, simple to perform and totally animal component-free to best comply with the requirements of health authorities for the production of a vaccine seed.

Optimization of influenza A vaccine virus by reverse genetic using chimeric HA and NA genes with an extended PR8 backbone

The yield of influenza antigen production may significantly vary between vaccine strains; for example the A/California/07/09 (H1N1)-X179A vaccine virus, prepared during 2009 influenza pandemic, presented a low antigen yield in eggs compared to other seasonal H1N1 reassortants. In this study a bi-chimeric virus expressing HA and NA genes with A/Puerto Rico/8/34 (H1N1) (PR8) and X179A domains was rescued by reverse genetics using a mixture of Vero/CHOK1 cell lines (Medina et al. [7]). The bi-chimeric virus obtained demonstrated to yield much larger amounts of HA than X179A in eggs as measured by single-radial-immunodiffusion (SRID), the reference method to quantify HA protein in influenza vaccine. Such kind of optimized virus using PR8 backbone derived chimeric glycoproteins could be used as improved seed viruses for vaccine production.

A latex agglutination assay to quantify the amount of hemagglutinin protein in adjuvanted low-dose influenza monovalent vaccines

To formulate inactivated influenza vaccines, the concentration of hemagglutinin (HA) must be accurately determined. The standard test currently used to measure HA in influenza vaccines is the Single Radial Immunodiffusion (SRID) assay. We developed a very rapid, simple and sensitive alternative quantitative HA assay, namely the Latex Agglutination Assay (LAA). The LAA uses the Spherotest® technology, which is based on the agglutination of HA-specific immunoglobulin-coated latex beads. The amount of HA in a sample is calculated from the level of bead agglutination by a simple absorbance measurement at 405nm against a standard curve generated using a monovalent vaccine standard. In less than 2hours, tens of samples could be quantified using the LAA as opposed to 2days for the SRID assay. Ten steps are required to complete an SRID assay as compared to 6 steps for the LAA, from sample preparation through spectrophotometric analysis. Furthermore, the limit of detection of the LAA was found to be approximately 15ng HA/mL, similar to an ELISA, with the quantification of less than 1.8μg HA/mL. The quantification limit of the SRID is usually considered to be approximately 5μg HA/mL. The development of the assay and a comparison of the titers obtained by SRID and LAA for several monovalent vaccines corresponding to various strains were performed. For A/H5N1 and A/H1N1 monovalent vaccines, the LAA was found to be linear and accurate as compared to the SRID. The precision of the LAA was close to that of the standard test, and good reproducibility from one laboratory to another was observed. Moreover, the LAA enabled HA quantification in AlOOH-adjuvanted and in emulsion-adjuvanted low-dose vaccines as well as unadjuvanted vaccines. In conclusion, LAA may be useful to rapidly and accurately measure influenza HA protein in monovalent vaccines, especially in those containing less than 5μg/mL of HA in the presence of an adjuvant.