Universal influenza DNA vaccine encoding conserved CD4+ T cell epitopes protects against lethal viral challenge in HLA-DR transgenic mice.

The goal of the present study was to design a vaccine that would provide universal protection against infection of humans with diverse influenza A viruses. Accordingly, protein sequences from influenza A virus strains currently in circulation (H1N1, H3N2), agents of past pandemics (H1N1, H2N2, H3N2) and zoonotic infections of man (H1N1, H5N1, H7N2, H7N3, H7N7, H9N2) were evaluated for the presence of amino acid sequences, motifs, that are predicted to mediate peptide epitope binding with high affinity to the most frequent HLA-DR allelic products. Peptides conserved among diverse influenza strains were then synthesized, evaluated for binding to purified HLA-DR molecules and for their capacity to induce influenza-specific immune recall responses using human donor peripheral blood mononuclear cells (PBMC). Accordingly, 20 epitopes were selected for further investigation based on their conservancy among diverse influenza strains, predicted population coverage in diverse ethnic groups and capacity to recall influenza-specific responses. A DNA plasmid encoding the epitopes was constructed using amino acid spacers between epitopes to promote optimum processing and presentation. Immunogenicity of the DNA vaccine was measured using HLA-DR4 transgenic mice and the TriGrid in vivo electroporation device. Vaccination resulted in peptide-specific immune responses, augmented HA-specific antibody responses and protection of HLA-DR4 transgenic mice from lethal PR8 influenza virus challenge. These studies demonstrate the utility of this vaccine format and the contribution of CD4(+) T cell responses to protection against influenza infection.

Formulation and characterization of a ten-peptide single-vial vaccine, EP-2101, designed to induce cytotoxic T-lymphocyte responses for cancer immunotherapy.

Effective vaccines that mediate clinical responses in cancer patients may require generation of broadly specific cytotoxic T lymphocytes (CTL) directed against multiple epitopes and tumor-associated antigens (TAA). Pursuant to this goal we developed a synthetic peptide vaccine, EP-2101, composed of 10 synthetic peptide epitopes and formulated in Montanide ISA 51 adjuvant. Nine of the HLA-A*0201-restricted CTL epitopes were derived from five well-characterized TAA. The universal HLA-DR binding epitope PADRE was also included for T-cell help. Herein we describe studies on the formulation and characterization of the EP-2101 vaccine which supports generation of a sterile single-vial emulsion using standardized processes. The physicochemical properties of the peptides were highly disparate and as such, solubilization studies were required to identify a process which supported sterile filtration of the EP-2101 peptide mix. A homogenization-based formulation process with Montanide ISA 51 and 0.5 mg/ml of each peptide was developed to generate a water-in-oil emulsion. Physical studies indicated the vaccine emulsion to be stable, with little change in visual appearance, viscosity and water droplet size for at least three months. The physical stability of individual peptides in the vaccine emulsion was demonstrated using HPLC and immunogenicity of the vaccine formulation was confirmed in HLA-A*0201/K(b) transgenic mice where T-cell responses could be induced to all epitopes in EP-2101 following vaccination. Our study process is scalable for production of approximately 1.5 liters of potent experimental vaccine for preclinical animal toxicity and phase 1 clinical testing in patients with breast, colon or lung cancer.