Evolutionarily conserved protein sequences of influenza a viruses, avian and human, as vaccine targets

Abstract

Background: Influenza A viruses generate an extreme genetic diversity through point mutation and gene segment exchange, resulting in many new strains that emerge from the animal reservoirs, among which was the recent highly pathogenic H5N1 virus. This genetic diversity also endows these viruses with a dynamic adaptability to their habitats, one result being the rapid selection of genomic variants that resist the immune responses of infected hosts. With the possibility of an influenza A pandemic, a critical need is a vaccine that will recognize and protect against any influenza A pathogen. One feasible approach is a vaccine containing conserved immunogenic protein sequences that represent the genotypic diversity of all current and future avian and human influenza viruses as an alternative to current vaccines that address only the known circulating virus strains.

Methodology/principal findings: Methodologies for large-scale analysis of the evolutionary variability of the influenza A virus proteins recorded in public databases were developed and used to elucidate the amino acid sequence diversity and conservation of 36,343 sequences of the 11 viral proteins of the recorded virus isolates of the past 30 years. Technologies were also applied to identify the conserved amino acid sequences from isolates of the past decade, and to evaluate the predicted human lymphocyte antigen (HLA) supertype-restricted class I and II T-cell epitopes of the conserved sequences. Fifty-five (55) sequences of 9 or more amino acids of the polymerases (PB2, PB1, and PA), nucleoprotein (NP), and matrix 1 (M1) proteins were completely conserved in at least 80%, many in 95 to 100%, of the avian and human influenza A virus isolates despite the marked evolutionary variability of the viruses. Almost all (50) of these conserved sequences contained putative supertype HLA class I or class II epitopes as predicted by 4 peptide-HLA binding algorithms. Additionally, data of the Immune Epitope Database (IEDB) include 29 experimentally identified HLA class I and II T-cell epitopes present in 14 of the conserved sequences.

Conclusions/significance: This study of all reported influenza A virus protein sequences, avian and human, has identified 55 highly conserved sequences, most of which are predicted to have immune relevance as T-cell epitopes. This is a necessary first step in the design and analysis of a polyepitope, pan-influenza A vaccine. In addition to the application described herein, these technologies can be applied to other pathogens and to other therapeutic modalities designed to attack DNA, RNA, or protein sequences critical to pathogen function.

Figure 1. An overview of the methodology of this study.

Figure 2. Entropy plots of avian influenza A viruses, excluding H5N1 subtype, for each of three decades: 1977–1986, 1987–1996, 1997–2006 (data as of September 30, 2006).

Figure 3. Entropy plots of the sequence alignments of recorded H5N1 viruses isolated from avian and human hosts (data as of September 30, 2006).

Figure 4. Entropy plots of recorded human influenza A subtypes H1N1, H3N2, and H1N2 from 1918–2006 (data as of September 30, 2006).

Figure 5. Entropy-sequence conservation relationship, plotted from data in this study (see Figure 2– 4).

The boxed region indicates area whereby conservation of ≥90% correlates to entropy of 0.8 or less.

Figure 6. Highly conserved sequences of influenza A viruses in human H1N1, H3N2, H1N2, H5N1, avian H5N1, and other avian subtypes circulating between 1997 and 2006.

A region in the viral proteome is considered as highly conserved when it has identical sequence conservation of at least 9 contiguous amino acids in 80% or more of the protein sequences of the analyzed dataset. The index of virus colored symbol is as shown at the top of the figure.

Figure 7. Highly conserved sequences of influenza A viruses and their predicted HLA class I and II supertype-restricted T-cell epitopes by NetCTL, ARB, TEPITOPE, and MULTIPRED systems.

The color symbols corresponding to the prediction systems are as shown at the top of the figure. Only conserved sequences containing predicted alleles are shown. NetCTL predicts all of the listed class I supertypes; MULTIPRED predictions cover A2 and A3; and ARB predicts each of the class I except B8, B27, B39, B58, and B62. Predictions of HLA class II supertypes by MULTIPRED AND TEPITOPE is described in Materials and Methods.

Figure 8. Highly conserved sequences of influenza A viruses and their associated HLA-restricted T-cell epitope based on data obtained from IEDB (www.immuneepitope.org/).

Only sequences with identified sites are included. The first amino acid of each identified allele is shown in bold.