We have developed a protocol for rapid production of the hemagglutinin (HA) of H5N1 avian pandemic influenza viruses. This procedure does not require access to the pandemic influenza virus strains. The ability to work with these pathogens is restricted due to the select agent regulations, USDA control measures, and biosafety level constraints. Using only DNA sequences from the CDC/WHO pandemic influenza virus database, we synthesized chemically genes, de novo, in a procedure optimized for protein expression in E. coli. This procedure can reduce the response time required to produce a vaccine to less than one month compared to 6-8 months needed for the traditional egg-based preparation of influenza vaccines. In addition to this new de novo gene protocol, we have developed cloning protocols utilizing influenza viruses as templates for reverse transcription-polymerase chain reaction gene amplification. Using these procedures and protocols, bacterial seed clones/cultures have been constructed and used to produce pilot plant quantities of recombinant HA proteins. With this recombinant material, we have now developed standardized downstream processing protocols for protein extraction, purification, refolding, and vaccine formulation. We have produced recombinant HA immunogens from four representative influenza A viruses:? ? H3N2 A/California/7/2004 (standard control from human 2006 influenza vaccine)? H5N1 A/Vietnam/1203/2004 Clade 1? H5N1 A/Indonesia/5/2005 Clade 2, subclade 1? H5N1 A/Bar-headed Goose/Qinghai/1A/2005 Clade 2, subclade 2? ? The recombinant protein constructs were designed to represent the mature configuration of HA with the amino acid domain spanning the viral membrane deleted from the carboxyl terminal. This domain was replaced with a Gly3X-His6X tag to facilitate purification of the expressed protein using Ni-ion chelating chromatography.? ? Recombinant HA (rHA) from four representative virus strains was produced in either E. coli BL21(DE3) or Rosetta 2(DE3) bacteria and purified from isolated inclusion bodies by urea solubilization of the inclusion body protein and by Ni-ion column chromatography. The purified rHA protein derived from the A/Vietnam/1203/2004 and A/Indonesia/5/2005 H5N1 influenza virus was further processed for use in vaccine formulation and immunization studies.? ? Solubilized rHA was processed by rapid dilution into refolding buffer, extensive dialysis and spin-filter concentration. Various vaccine candidates were formulated by either adsorbing the rHA onto alum or treating the protein with formalin, or both.? ? Injected into young mice three times (2.5-5ug/mouse), rHA induced antibodies with hemagglutination inhibition titers of 40 and 80 or higher, suggesting that rHA could induce protective immune responses against influenza virus infection (FDA guidelines require a minimum titer of 40). Our preliminary data suggest that the alum-absorbed rHA vaccine, produced in just four weeks, can fulfill the FDA requirements. Antibody neutralization experiments based on plaque reduction analysis and inhibition of viral replication within a unique tissue culture cell line are planned.? ? Recent literature reports suggest that antibodies to the exposed N-terminal of 23 amino acids (M2e) of the mature matrix 2 protein (M2) may ameliorate disease symptoms. The M2 protein provides an ion-channel through the viral membrane and is recognized as a target for prophylaxis and treatment with the antiviral drug, Amantadine. Unlike the virions surface proteins, HA and NA, which are subject to constant genetic drift and shift, the M2 protein is highly conserved. This is likely due to its protected location within the viral membrane, preventing a strong host immune response. However, recent studies show that when the exposed 23-amino acid M2e peptide was genetically fused to the N-terminus of hepatitis B virus core particles as a carrier, the chimeric protein conferred complete protection against a lethal heterologous influenza virus challenge in a mouse model.? ? We bound a synthetic M2e peptide to a genetically detoxified diphtheria toxin (DT-H21G) via thioether linkages. MADLI-MS analyses showed an average of 7 chains of M2e per DT molecule rendering the ratio of DT : M2e as 1 : 0.3. This conjugate, in aqueous form or alum adsorbed, was injected into mice sc, at 2.5 mcg peptide/0.1 mL PBS, 2 or 3 times, 2 weeks apart. Sera were collected 1 week after the last injection. ELISA showed high anti-M2e levels after the third injection, with or without alum (GM=0.04 mg/ml after the 2nd injection; GM=1.4 mg/ml after the 3rd; and GM=1.4 mg/ml after the 3rd injection of alum adsorbed conjugate (0.4 to 1.4 p<0.0005). An antibody response was also observed against the DT(H21G) carrier. Our preliminary results suggest that this candidate vaccine may induce immunity against heterologous strains of influenza A virus.
