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

Abstract

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.

Fig. 1

HLA-DR-restricted influenza recall responses from human donors. Human donor 753 panels A and B; donor 6018 panels C and D and donor 709 panels E and F. Panels A, C and E depict recall responses measured in the absence of in vitro peptide expansion while panels B, D and F depict recall responses measured using an in vitro peptide expansion step. CD4⁺ cells from normal human donors were enriched for assay use. The experimental values are expressed as mean IFN-γ net spots/10⁶ CD4⁺ lymphocytes ± SEM for each peptide; tests were performed using triplicate wells. Significant responses from the indicated epitopes were defined as responses > than mean of background responses + (2.0 × Std. Dev.). Background responses were determined using supertype HLA-DR binding peptides from pathogens to which donors were not exposed, i.e., HIV, HBV, HCV and Plasmodium falciparum.

Fig. 2

Schematic diagram of the CD4⁺ 20 epitope DNA insert. The epitope order and amino acid spacer usage are shown. Influenza-derived epitopes were used in addition to the universal helper epitope, PADRE [23].

Fig. 3

Epitope-specific immune responses, animal survival and augmentation of PR8-specific antibody responses. HLA-DR4 transgenic H-2^b mice, (n=14), were immunized with CD4⁺20 epitope DNA construct or empty vector DNA control using Ichor’s in vivo electroporation device with 50 µg per mouse. Additional, a group of mice was also immunized with inactivated PR8. Two weeks post-immunization, two mice per group were sacrificed and splenocytes prepared for an epitope-specific IFN-γ ELISPOT (A). All 20 CD4⁺ epitope-specific responses were evaluated. Only positive responses plus three negative responses as comparators are shown. At this same time, the remaining 12 mice were infected with 100 HAU PR8 (4MLD50). Six days following the PR8 infection, two additional mice were sacrificed from each group for a second epitope-specific ELISPOT evaluation (A). The remaining 10 mice were monitored for survival of the animals (B). HLA-DR4 transgenic mice, (n=10) were immunized with CD4⁺20 epitope DNA vaccine or empty vector DNA control. Two weeks later, mice were immunized with 15 µg inactivated PR8. Seven and 14 days following inactivated PR8 immunization, mice were bled for determination of HA-specific antibody responses (C).

Fig. 3

Epitope-specific immune responses, animal survival and augmentation of PR8-specific antibody responses. HLA-DR4 transgenic H-2^b mice, (n=14), were immunized with CD4⁺20 epitope DNA construct or empty vector DNA control using Ichor’s in vivo electroporation device with 50 µg per mouse. Additional, a group of mice was also immunized with inactivated PR8. Two weeks post-immunization, two mice per group were sacrificed and splenocytes prepared for an epitope-specific IFN-γ ELISPOT (A). All 20 CD4⁺ epitope-specific responses were evaluated. Only positive responses plus three negative responses as comparators are shown. At this same time, the remaining 12 mice were infected with 100 HAU PR8 (4MLD50). Six days following the PR8 infection, two additional mice were sacrificed from each group for a second epitope-specific ELISPOT evaluation (A). The remaining 10 mice were monitored for survival of the animals (B). HLA-DR4 transgenic mice, (n=10) were immunized with CD4⁺20 epitope DNA vaccine or empty vector DNA control. Two weeks later, mice were immunized with 15 µg inactivated PR8. Seven and 14 days following inactivated PR8 immunization, mice were bled for determination of HA-specific antibody responses (C).