Conserved synthetic peptides from the hemagglutinin of influenza viruses induce broad humoral and T-cell responses in a pig model

Abstract

Outbreaks involving either H5N1 or H1N1 influenza viruses (IV) have recently become an increasing threat to cause potential pandemics. Pigs have an important role in this aspect. As reflected in the 2009 human H1N1 pandemia, they may act as a vehicle for mixing and generating new assortments of viruses potentially pathogenic to animals and humans. Lack of universal vaccines against the highly variable influenza virus forces scientists to continuously design vaccines à la carte, which is an expensive and risky practice overall when dealing with virulent strains. Therefore, we focused our efforts on developing a broadly protective influenza vaccine based on the Informational Spectrum Method (ISM). This theoretical prediction allows the selection of highly conserved peptide sequences from within the hemagglutinin subunit 1 protein (HA1) from either H5 or H1 viruses which are located in the flanking region of the HA binding site and with the potential to elicit broader immune responses than conventional vaccines. Confirming the theoretical predictions, immunization of conventional farm pigs with the synthetic peptides induced humoral responses in every single pig. The fact that the induced antibodies were able to recognize in vitro heterologous influenza viruses such as the pandemic H1N1 virus (pH1N1), two swine influenza field isolates (SwH1N1 and SwH3N2) and a H5N1 highly pathogenic avian virus, confirm the broad recognition of the antibodies induced. Unexpectedly, all pigs also showed T-cell responses that not only recognized the specific peptides, but also the pH1N1 virus. Finally, a partial effect on the kinetics of virus clearance was observed after the intranasal infection with the pH1N1 virus, setting forth the groundwork for the design of peptide-based vaccines against influenza viruses. Further insights into the understanding of the mechanisms involved in the protection afforded will be necessary to optimize future vaccine formulations.

Figure 1. VIN1-peptide cocktail acts as a potent immunogen and the elicited sera reacts with different hemagglutinin subtypes and against VIN1-peptides.

(A) Sera from individuals were obtained 15 days after each immunization and were tested for binding to a mixture of the VIN1-peptides (serum dilution 1∶100) by ELISA. (B) Sera from individual pigs were obtained 15 days after the third immunization and were serially diluted and tested for binding to each single peptide by ELISA and (C) Sera described in B) were tested for binding to H5- or H1- recombinant hemagglutinin by ELISA.

Figure 2. VIN1-HA1 derived peptides immunization induces strong T-cell responses in pigs.

(A) Kinetics of the VIN1 peptide-specific T-cell responses induced 15 days after all immunizations measured by IFNγ-ELISPOT. (B) Specific T-cell responses induced 4 weeks after the third immunization were tested for each single peptide by IFNγ-ELISPOT.

Figure 3. Immunization with VIN1-HA1 partially protects pigs in vivo against heterologous challenge with pH1N1.

Influenza viral RNA quantification in BAL was performed by RT-qPCR at 6 dpi, corresponding to necropsy day. Bars indicated positive samples in genome equivalent copies (GEC) per ml of BAL. The detection limit in the assay was 3 log₁₀ GEC/ml.

Figure 4. VIN1-sera recognize pH1N1 virus in vitro.

Indirect immunofluorescence of pH1N1-infected MDCK cells at 16 hpi using as primary antibody: (A) the serum from one pig (representative of the group), immunized three times with VIN1-peptides; (B) the serum from one negative control pig (representative of the group), immunized three times with PBS; and (C) A monoclonal antibody against the NS-1 protein was used as control for the infection (right panel).

Figure 5. Immunization with the VIN1-HA1 peptide induces specific antibodies and T-cells against the heterologous pH1N1 virus.

(A) HI and SNT titers obtained with sera from pigs immunized either with the VIN1-peptides or with saline solution (control), at 6 dpi with the pH1N1 virus. Grey bars represent HI titres and red triangles show SNT. (B) IFNγ-ELISPOT using pH1N1 virus as stimulus and PBMCs from pigs immunized either with the VIN1-peptides or with saline solution (control). The assay was done using PBMCs isolated either before the infection with the pandemic H1N1 virus or at 6 dpi.

Figure 6. VIN1-sera recognize distinct viral subtypes.

Indirect immunofluorescence of either H5N1, SwH3N2 or SwH1N1-infected MDCKs cells at 16 hpi using as primary antibody the serum from one pig (representative of the group), immunized three times with VIN1 peptides.