A novel lamprey antibody sequence to multimerize and increase the immunogenicity of recombinant viral and bacterial vaccine antigens.

Hemagglutinin, the major surface protein of influenza viruses, was recombinantly expressed in eukaryotic cells as a monomer instead of its native trimer, and was only immunogenic when administered with an adjuvant [Pion et al. 2014]. In order to multimerize this antigen to increase its immunogenicity, a cysteine-rich peptide sequence found at the extreme C-terminus of lamprey variable lymphocyte receptor (VLR)-B antibodies was fused to various recombinant hemagglutinin (rHA) proteins from A and B influenza virus strains. The rHA-Lamp fusion (rHA fused to the lamprey sequence) protein was expressed in Leishmania tarentolae and Chinese hamster ovary (CHO) cells and shown to produce several multimeric forms. The multimers produced were very stable and more immunogenic in mice than monomeric rHA. The lamprey VLR-B sequence was also used to multimerize the neuraminidase (NA) of influenza viruses expressed in CHO cells. For some viral strains, the NA was expressed as a tetramer like the native viral NA form. In addition, the lamprey VLR-B sequence was fused with two surface antigens of Shigella flexneri 2a, the invasion plasmid antigen D and a double mutated soluble form of the membrane expression of the invasion plasmid antigen H namely MxiH. The fusion proteins were expressed in Escherichia coli to produce the respective multimer protein forms. The resulting proteins had similar multimeric forms as rHA-Lamp protein and were more immunogenic in mice than the monomer forms. In conclusion, the VLR-B sequence can be used to increase the immunogenicity of recombinant viral and bacterial antigens, thus negating the need for adjuvants.

Influenza A and B virus-like particles produced in mammalian cells are highly immunogenic and induce functional antibodies.

Influenza virus-like particles (VLPs) represent an attractive alternative to traditional influenza vaccine formulations. Influenza VLPs mimic the natural virus while lacking the genetic material, are easily recognized by the immune system, and are considered safe. The use of a mammalian cell platform offers many advantages for VLP production, such as flexibility and the same glycosylation patterns as a human virus. In this study, the influenza VLPs containing hemagglutinin (HA), neuraminidase (NA) and matrix M1 proteins were expressed in CHO-K1, Vero or 293 T cell lines using transient transfection. After production in 3L bioreactor and purification, extensive characterization was performed on two batches of VLPs produced in 293 T, the best cell line for VLP expression; one batch expressed the HA and NA genes from A/Hong Kong/4801/2014 (H3N2) strain and the other, HA and NA genes from B/Phuket/3073/2013. Characterizations provided evidence that mammalian VLPs closely emulate the exterior of authentic virus particles in terms of both antigen presentation and biological properties. The two VLPs produced contained more NA proteins on their surface with a HA:NA ratio around 1:1 than influenza viruses which present a HA:NA ratio of around 4:1. Immunogenicity studies in BALB/c mice demonstrated that the VLPs, administered intra-muscularly, were highly immunogenic at low doses, with the induction of functional antibodies against HA and NA. Immunogenicity was also shown in a human in vitro model (MIMIC® system). In conclusion, we believe that influenza vaccines made of VLPs produced in mammalian cell lines, constitute a potential alternative to the classical influenza vaccines.

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.

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

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.

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.

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.

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.

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.

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. OBJECTIVES: We sought to generate an H7 candidate vaccine virus suitable for administration to humans whose generation and amplification avoided the use of eggs. METHODS: We generated a suitable H7 vaccine virus by reverse genetics. This virus, known as RD3, comprises the internal genes of A/Puerto Rico/8/34 with surface antigens of the highly pathogenic avian strain A/Chicken/Italy/13474/99 (H7N1). The multi-basic amino acid site in the HA gene, associated with high pathogenicity in chickens, was removed. RESULTS: The HA modification did not alter the antigenicity of the virus and the resultant single basic motif was stably retained following several passages in Vero and PER.C6 cells. RD3 was attenuated for growth in embryonated eggs, chickens, and ferrets. RD3 induced an antibody response in infected animals reactive against both the homologous virus and other H7 influenza viruses associated with recent infection by H7 viruses in humans. CONCLUSIONS: This is the first report of a candidate H7 vaccine virus for use in humans generated by reverse genetics and propagated entirely in mammalian tissue culture. The vaccine has potential use against a wide range of H7 strains.

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. OBJECTIVES: Using reverse genetics, we prepared two vaccine reassortants (A/H5N1 x PR8 and A/H7N1 x PR8) exhibiting the envelope glycoproteins from non-pathogenic avian viruses, A/Turkey/Wisconsin/68 (A/H5N9) and A/Rhea/New Caledonia/39482/93 (A/H7N1) and the internal proteins of the attenuated human virus A/Puerto Rico/8/34 (H1N1). METHODS: The transfection was accomplished using a mixture of Vero and chicken embryo cells both of which are currently being used for vaccine manufacturing. RESULTS: This process was reproducible, resulting in consistent recovery of influenza viruses in 6 days. Because it is mainly the A/H5N1 strain that has recently crossed the human barrier, it is the A/PR8 x A/H5N1 reassortant (RG5) that was further amplified, either in embryonated hen eggs or Vero cells, to produce vaccine pre-master seed stocks that met quality control specifications. Safety testing in chickens and ferrets was performed to assess the non-virulence of the reassortant, and finally analysis using chicken and ferret sera immunized with the RG5 virus showed that the vaccine candidate elicited an antibody response cross-reactive with the Hong Kong 1997 and 2003 H5N1 strains but not the Vietnam/2004 viruses. CONCLUSIONS: The seeds obtained could be used as part of a pandemic vaccine strain 'library' available in case of propagation in humans of a new highly pathogenic avian strain.