Preparation, characterization, and immunogenicity in mice of a recombinant influenza H5 hemagglutinin vaccine against the avian H5N1 A/Vietnam/1203/2004 influenza virus

Abstract

Production of influenza vaccines requires a minimum of six months after the circulating strain is isolated and the use of infectious viruses. The hemagglutinin (protective antigen) of circulating influenza viruses mutates rapidly requiring reformulation of the vaccines. Our goal is to eliminate the risk of working with infectious virus and reduce significantly the production time. A cDNA fragment encoding the influenza virus A/Vietnam/1203/2004 (H5N1) HA gene was prepared using RT-PCR with viral RNA as a template. Recombinant HA (rHA) protein was produced in Escherichia coli and purified from isolated inclusion bodies by urea solubilization and Ni-ion column chromatography. Vaccine candidates were prepared by treating the rHA with formalin, adsorption onto alum or with both. Mice were injected subcutaneously with candidate vaccines 2 or 3 times 2 weeks apart. Sera were collected one week after the last injection and antibody measured by ELISA and hemagglutination inhibition (HI). The highest antibody response (449 EU) was elicited by three injections of 15 μg alum-adsorbed rHA. Dosages of 5 μg of rHA formulated with formalin and alum, and 5 μg alum adsorbed rHA elicited IgG anti-HA of 212 EU and 177 EU, respectively. HI titers, ≥ 40 were obtained in ≥ 80% of mice with three doses of all formulations. We developed a method to produce rHA in a time-frame suitable for annual and pandemic influenza vaccination. Using this method, rHA vaccine can be produced in three to four weeks and when formulated with alum, induces HA antibody levels in young outbred mice consistent with the FDA guidelines for vaccines against epidemic and pandemic influenza.

Dendrimer-RNA nanoparticles generate protective immunity against lethal Ebola, H1N1 influenza, and Toxoplasma gondii challenges with a single dose

“…Thus, this platform is capable of generating protection against representative diseases from all three categories of the National Institute of Allergy and Infectious Diseases' Priority Pathogen List for emerging and rapidly increasing threats (52). Importantly, generation of a new MDNP vaccine system composed of these dendrimers and replicon RNA takes only about 1 wk, in contrast to the cell culture and fertilized egg systems that can take 6 mo or more to develop (53)(54)(55)(56)(57)(58). In addition, postproduction purification required for this MDNP system is minimal, as is the risk of contaminating allergens relative to existing vaccine systems (59)(60)(61).…”

Chimpanzees as an animal model for human norovirus infection and vaccine development

“…The vaccine (50 μg per dose) was prepared by mixing 300 μg of VLP solution in 5.308 mL of Tris buffer [0.05M Tris (pH 7.8), 0.14 M NaCl] for the vaccine, or 5.308 mL of Tris alone for the placebo, with 0.692 mL of Alhydrogel (Brenntag Biosector) added to both vaccine and placebo for a total of 6 mL. The mixture was incubated at 4°C overnight, and centrifuged at 1,500 rpm for 5 min (49). The pellet was resuspended in PBS for a final volume of 6 mL and stored at 4°C in 1-mL aliquots.…”

Production and stabilization of the trimeric influenza hemagglutinin stem domain for potentially broadly protective influenza vaccines

“…Because of these complications, in vivo Escherichia coli expression produced low yields of only 2 mg/L, and the HA stem polypeptides accumulated as inclusion bodies (1). Although some refolding methods have been attempted (14)(15)(16), the recovery yields of soluble products have been low, and properly folded stable trimeric assembly has not been confirmed.…”