Using influenza viruses as templates for RT-PCR gene amplification or genomic sequencing data, we have produced unique recombinant HA proteins (rHA) from the four 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? ? These protein constructs were designed to represent the mature configuration of the HA protein 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 rHA using Ni-ion chelating chromatography.? ? rHA from the four constructs were produced in E. coli BL21 (DE3) or Rosetta 2 (DE3) bacteria and purified from isolated inclusion bodies by solubilization of the inclusion body protein in urea and Ni-ion column chromatography. The construct derived from the A/Vietnam/1203/2004 H5N1 influenza virus was subsequently used for further studies described below.? ? Solubilized rHA was refolded by rapid dilution into refolding buffer and extended dialysis. The rHA was concentrated using a spin-filter method. Various vaccine candidates were prepared by treating the purified rHA with formalin, its adsorption onto alum or by both.? ? Groups of 10 mice were injected at 0, 2, and 4 weeks, sc, with 5ug or 15ug/0.1mL PBS, of the vaccine candidates. Sera were collected one week after the last injection and analyzed by ELISA, Western blot, and hemagglutinin inhibition assays using horse red blood cells and H5N1 virus. All sera reacted in ELISA. The group of mice injected three times with 15ug rHA-alum produced the highest HA antibody levels with a geometric mean of 449 EU. The second highest levels were from mice injected three times with either 5 ug rHA-formalin/alum or 5ug rHA-alum with GMs of 212 and 177 EU, respectively. The lowest levels of anti-HA (10 to 26 EU) were in groups immunized twice with 5ug of any of the HA vaccine formulations.? ? Sera from mice injected three times with rHA showed hemagglutinin inhibition titers of 40 and 80 or higher suggesting that rHA could produce a protective immune response against influenza virus infection (FDA requires a titer of 40). Our preliminary data suggest that our rHA vaccine can be produced in three to four weeks and formulated with alum, can induce IgG anti-HA levels consistent with those recommended by the FDA for new vaccines against pandemic influenza.? ? Recent literature reports suggest that antibodies to the exposed N-terminal 23 amino acids (M2e) portion 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, likely due to its protected location within the viral membrane preventing a strong host immune response, also explaining why it has not been considered as a vaccine target. 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. (1).? ? Our approach to utilizing the M2e peptide as a vaccine was to chemically synthesize the 23-amino acid peptide and bind it to a genetically detoxified diphtheria toxin (DT-H21G) via thioether linkages. MADLI-MS analyses showed an average of 7 chains M2e per DT molecule rendering the ratio of DT : M2e as 1 : 0.3. This conjugates was injected into mice sc, 2.5 ug peptide/0.1mL PBS, 2 or 3 times, 2 weeks apart, or 3 times adsorbed onto alum. 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 immune response was also observed against the DT(H21G) carrier. Our preliminary results suggest that this vaccine candidate may induce immunity against heterologous strains of influenza A virus.
