Bioinformatics design and experimental validation of influenza A virus multi-epitopes that induce neutralizing antibodies

Abstract

Pandemics caused by influenza A virus (IAV) are responsible for the deaths of millions of humans around the world. One of these pandemics occurred in Mexico in 2009. Despite the impact of IAV on human health, there is no effective vaccine. Gene mutations and translocation of genome segments of different IAV subtypes infecting a single host cell make the development of a universal vaccine difficult. The design of immunogenic peptides using bioinformatics tools could be an interesting strategy to increase the success of vaccines. In this work, we used the predicted amino acid sequences of the neuraminidase (NA) and hemagglutinin (HA) proteins of different IAV subtypes to perform multiple alignments, epitope predictions, molecular dynamics simulations, and experimental validation. Peptide selection was based on the following criteria: promiscuity, protein surface exposure, and the degree of conservation among different medically relevant IAV strains. These peptides were tested using immunological assays to test their ability to induce production of antibodies against IAV. We immunized rabbits and mice and measured the levels of IgG and IgA antibodies in serum samples and nasal washes. Rabbit antibodies against the peptides P11 and P14 (both of which are hybrids of NA and HA) recognized HA from both group 1 (H1, H2, and H5) and group 2 (H3 and H7) IAV and also recognized the purified NA protein from the viral stock (influenza A Puerto Rico/916/34). IgG antibodies from rabbits immunized with P11 and P14 were capable of recognizing viral particles and inhibited virus hemagglutination. Additionally, intranasal immunization of mice with P11 and P14 induced specific IgG and IgA antibodies in serum and nasal mucosa, respectively. Interestingly, the IgG antibodies were found to have neutralizing capability. In conclusion, the peptides designed through in silico studies were validated in experimental assays.

Fig. 1

Sequence alignment of peptides A) P1, B) P2, C) P3 and D) P4 from hemagglutinin and E) P5 from neuraminidase with the corresponding peptides from different influenza virus subtypes (H1N1, H2N2, H3N2, H5N1 and H7N9). These peptides have structural regions that are important for MHCI and MHC II recognition according to epitope predictions

Fig. 2. A

) Root mean square deviation (RMSD), B) root mean square fluctuations (RMSF), and C) radius of gyrations of hybrid peptides P9, P11 and P14

Fig. 3

Peptide-specific IgG antibody titers in the sera of rabbits immunized with unconjugated peptides or peptides conjugated with KLH. Serial dilutions of sera from rabbits inoculated with unconjugated peptides (panel A) or peptides conjugated with KLH (panel B) were added to microplates that had been coated with different peptides. IgG antibodies were detected using a secondary Ab specific for rabbit IgG (1:3000). Sera from rabbits immunized with either the unconjugated peptide 1 or with hybrid peptides P11 and P14 conjugated with KLH showed the highest titer (1:800), whereas sera against the remaining peptides had a lower titer (A₄₉₀ <0.5; 1:800). Samples of serum from a pre-immune rabbit were used as controls. No peptide-specific IgG responses were found in pre-immune sera. Individual samples were run in duplicate, and the data are shown as the mean ± SD

Fig. 4

Peptide-specific IgG or IgA antibody titers in sera and nasal washes of mice immunized with different peptides. Serial dilutions of sera (panels A-B) or nasal washes (panels C-D) from mice inoculated with different peptides were added to microplates that had been coated with specific peptides. Antibodies were detected using a secondary Ab specific for mouse IgG (1:3000) or IgA (1:500). Samples of serum from pre-immune mice were used as controls, and no peptide-specific IgG or IgA responses were found in pre-immune sera or nasal washes, respectively. Individual samples were run in triplicate, and the data are shown as the mean ± SD

Fig. 5

Rabbit IgG antibodies against hybrid peptides P11 and peptide P14 recognized recombinant hemagglutinins (rHA) from several influenza A viruses: A) A/California/07/2009, B) A/Shanghai/2/2013, C), A/Aichi/2/1968 D) A/Cambodia/S1211394/2008 E) A/Japan/305/1957, F) A/Puerto Rico/8/34 and G) A/Brisbane/59/2007 . Serial dilutions of sera from rabbits inoculated with peptides conjugated to KLH were added to microplates that had been coated with purified recombinant hemagglutinins (0.5 μg). Antibodies against P11 and P14 had similar binding activity to the majority of the rHAs, except for A/Brisbane/59/2007 and A/Puerto Rico/8/1934, both of which are H1N1 subtypes. As controls, serum samples from pre-immunized rabbits were tested against the specific peptide

Fig. 6

NA-specific IgG antibody titers in sera from rabbits immunized with different peptides. Serial dilutions of sera from rabbits inoculated with peptide 14 (P14), and peptide 11 (P11) were added to microplates previously coated with neuraminidase protein (0.5 µg). IgG antibodies were detected using a secondary Ab specific for rabbit IgG (1:3000). Individual samples were run in duplicate, and the data are shown as the mean ± SD

Fig. 7

Trapping ELISA. Antibodies from rabbits immunized with P11 or P14 recognized influenza virus immobilized with ConA. Pre-immune serum samples were used as negative controls, and a human serum was used as a positive control (p < 0.0001, pre-immune vs. immunized rabbit)