Immunization with a Hemagglutinin-Derived Synthetic Peptide Formulated with a CpG-DNA-Liposome Complex Induced Protection against Lethal Influenza Virus Infection in Mice

<div><p>Whole-virus vaccines, including inactivated or live-attenuated influenza vaccines, have been conventionally developed and supported as a prophylaxis. These currently available virus-based influenza vaccines are widely used in the clinic, but the vaccine production takes a long time and a huge number of embryonated chicken eggs. To overcome the imperfection of egg-based influenza vaccines, epitope-based peptide vaccines have been studied as an alternative approach. Here, we formulated an efficacious peptide vaccine without carriers using phosphodiester CpG-DNA and a special liposome complex. Potential epitope peptides predicted from the hemagglutinin (HA) protein of the H5N1 A/Viet Nam/1203/2004 strain (NCBI database, AAW80717) were used to immunize mice along with phosphodiester CpG-DNA co-encapsulated in a phosphatidyl-β-oleoyl-γ-palmitoyl ethanolamine (DOPE):cholesterol hemisuccinate (CHEMS) complex (Lipoplex(O)) without carriers. We identified a B cell epitope peptide (hH5N1 HA233 epitope, 14 amino acids) that can potently induce epitope-specific antibodies. Furthermore, immunization with a complex of the B cell epitope and Lipoplex(O) completely protects mice challenged with a lethal dose of recombinant H5N1 virus. These results suggest that our improved peptide vaccine technology can be promptly applied to vaccine development against pandemic influenza. Furthermore our results suggest that potent epitopes, which cannot be easily found using proteins or a virus as an antigen, can be screened when we use a complex of peptide epitopes and Lipoplex(O).</p> </div

Effect of liposome composition, CG dinucleotide and backbone modification on antibody production.

<p>(<b>A</b>) Effect of the liposome composition. Three BALB/c mice were injected i.p. with a complex of hH5N1 HA233 and MB-ODN 4531(O) co-encapsulated in indicated liposomes on three occasions. The antisera were collected, and then amounts of hH5N1 HA233 epitope-specific total IgG were measured by ELISA. (<b>B</b>) Effect of CG dinucleotide and phosphorothioate backbone modification. Three BALB/c mice were injected i.p. with hH5N1 HA233 and DOPE:CHEMS (1∶1 ratio)-co-encapsulated MB-ODN 4531(O) (Lipoplex(O)+hH5N1 HA233), MB-ODN 4531GC(O) (LipoplexGC(O)+hH5N1 HA233), MB-ODN 4531(S) (Lipoplex(S)+hH5N1 HA233), and complementary MB-ODN 4531(S) (Lipoplex 4531(S)CS+hH5N1 HA233) on three occasions. The antisera were collected, and amounts of hH5N1 HA233-specific total IgG were measured by ELISA.</p

Prophylactic efficacy of a complex of hH5N1 HA233 and Lipoplex(O) against influenza A virus.

<p>BALB/c mice were immunized i.p. twice with a complex of hH5N1 HA233 encapsulated in indicated combination. The immunized mice were challenged intranasally with the rH5N1 virus (PR8/H5Lo) (<b>A–C</b>). After the virus challenge, the survival rate (<b>A</b>) and the body weight (<b>B</b>) were recorded for 20 days (N = 8/group). The lungs were collected at 3 days, 6 days or 30 days after the challenge with the rH5N1 virus (PR8/H5Lo) (<b>C</b>) (N = 3/group). Scale bars in (<b>C</b>), 100 µm. To estimate the viral clearance, the lung viral titers were measured by means of a plaque assay at 3 days or 6 days after the challenge with the rH5N1 virus (<b>D</b>). Lipoplex(O), MB-ODN 4531(O) encapsulated in DOPE:CHEMS (1∶1 ratio) complex; LipoplexGC(O), MB-ODN 4531GC(O) encapsulated in DOPE:CHEMS (1∶1 ratio) complex.</p

Location of B cell epitopes used in this study in the HA structure.

<p>(<b>A</b>) Schematic representation of predicted B cell epitopes used in this study. Red, hH5N1 HA58; Green, hH5N1 HA113; Yellow, hH5N1 HA175; Orange, hH5N1 HA233. (<b>B</b>) Location of receptor-binding site. Red, 130-loop; Green, 190-helix; Yellow, 220-loop. (<b>C</b>) Schematic representation of previously reported B cell epitopes. Red, LAH; Green, H5-F10 and CR6261; Yellow, H5-1C9; Cyan, H5-1B12. The image of HA structure of the A/Duck/Singapore/3/1997 strain was created with the use of PyMOL (<a href="http://www.pymol.org" target="_blank">www.pymol.org</a>) and the HA structure was obtained from the Protein Data Bank (PDB: 1JSM).</p

Prophylactic efficacy of a complex of hH1N1-WSN HA233 and Lipoplex(O) against influenza A virus.

<p>BALB/c mice were immunized i.p. two times with a complex of hH1N1-WSN HA233 and Lipoplex(O) (Lipoplex(O)+hH1N1-WSN HA233). The immunized mice were challenged intranasally with the rH5N1 virus (PR8/H5Lo) or the maA/WSN/1933 virus. After the virus challenge, the survival rate (<b>A</b>) and the body weight (<b>B</b>) were recorded for 22 days (N = 8/group). Lungs were collected at 3 days or 6 days after the challenge with the rH5N1 virus or the maA/WSN/1933 virus (N = 3/group) (<b>C</b>). Scale bars in (<b>C</b>), 100 µm. The lung viral titers were measured by means of a plaque assay to estimate the viral clearance at 3 days or 6 days after the challenge with the maA/WSN/1933 virus or rH5N1 virus (<b>D</b>).</p

Effects of epitope-specific antibodies on influenza A viruses.

<p>Virus neutralization assays were performed with antisera collected from mice immunized with a complex of hH5N1 HA233 and Lipoplex(O). The plaque number was counted and the neutralization percentage was calculated.</p

Reported monoclonal antibodies against B cell epitopes of HA2 polypeptide derived from influenza A virus strains.

a<p>Previously reported monoclonal antibodies against each highly conserved B cell epitope in the HA2 polypeptide.</p>b<p>We synthesized previously reported B cell epitope sequences from the HA2 polypeptide to prepare a complex containing each epitope and Lipoplex(O) in this study.</p>c<p>Abbreviations of each B cell epitope used in this study.</p

Alignment of the sequences corresponding to H5N1 HA233 epitope in influenza A virus subtypes.

<p>Alignment of the sequences corresponding to H5N1 HA233 epitope in influenza A virus subtypes.</p

Lack of production of hH5N1 HA233-specific antibodies by an inactivated rH5N1 virus.

<p>BALB/c mice were injected i.p. with UV-inactivated rH5N1 virus, a complex of the UV-inactivated rH5N1 virus encapsulated in DOPE:CHEMS, or a complex of the UV-inactivated rH5N1 virus and Lipoplex(O) on three occasions. The sera were collected, and the titers of rH5N1 virus-specific total IgG (<b>A</b>) and hH5N1 HA233-specific total IgG (<b>B</b>) were measured by ELISA.</p

IgG production by a complex of Lipoplex(O) and conserved sequences corresponding to hH5N1 HA233 epitope.

<p>Fourteen amino acid long conserved sequences were synthesized; the sequences correspond to hH5N1 HA233 epitope of the A/Vietnam/1203/2004 strain; they were obtained from influenza A virus H5N1 strains and influenza A virus subtypes. Three BALB/c mice were immunized i.p. three times with a complex of each peptide and Lipoplex(O). The antisera were collected 10 days after the final immunization, and then amounts of each peptide-specific total IgG (<b>A,D</b>), IgG1 (<b>B</b>) and IgG2a (<b>C</b>) were measured by ELISA. The ELISA plates were coated with hH5N1-HA 233 peptide.</p