Elsevier

Vaccine

Volume 25, Issue 15, 12 April 2007, Pages 2823-2831
Vaccine

CTL epitopes for influenza A including the H5N1 bird flu; genome-, pathogen-, and HLA-wide screening

https://doi.org/10.1016/j.vaccine.2006.12.038Get rights and content

Abstract

The purpose of the present study is to perform a global screening for new immunogenic HLA class I (HLA-I) restricted cytotoxic T cell (CTL) epitopes of potential utility as candidates of influenza A-virus diagnostics and vaccines. We used predictions of antigen processing and presentation, the latter encompassing 12 different HLA class I supertypes with >99% population coverage, and searched for conserved epitopes from available influenza A viral protein sequences. Peptides corresponding to 167 predicted peptide–HLA-I interactions were synthesized, tested for peptide–HLA-I interactions in a biochemical assay and for influenza-specific, HLA-I-restricted CTL responses in an IFN-γ ELISPOT assay. Eighty-nine peptides could be confirmed as HLA-I binders, and 13 could be confirmed as CTL targets. The 13 epitopes, are highly conserved among human influenza A pathogens, and all of these epitopes are present in the emerging bird flu isolates. Our study demonstrates that present technology enables a fast global screening for T cell immune epitopes of potential diagnostics and vaccine interest. This technology includes immuno-bioinformatics predictors with the capacity to perform fast genome-, pathogen-, and HLA-wide searches for immune targets. To exploit this new potential, a coordinated international effort to analyze the precious source of information represented by rare patients, such as the current victims of bird flu, would be essential.

Introduction

Influenza is a highly contagious, airborne respiratory tract infection associated with a significant disease burden. The annual “mild” influenza epidemics caused by antigenic drift of the virus affects 10–20% of the world's population with up to 5 million cases of serious illness and 500,000 deaths (http://www.who.int/vaccine_research/diseases/ari/en). At various intervals, new influenza subtypes emerge against which no immunity exists in the human population and these may cause global pandemics with an even higher disease toll. The current outbreak of a new influenza subtype A (H5N1), which can be directly, although at this time rarely, transmitted from birds to humans [1], is an example of a potential pandemic flu threat, which has currently reached phase 3 of 6 in the WHO delineation of a flu pandemic (http://www.who.int/csr/disease/avian_influenza/phase/en/index.html). It is highly pathogenic in birds, and when transmitted to humans it carries a mortality of over 50% [2]. So far, all the resulting human cases have been in close contact with infected flocks and there is no known example of a human-to-human transmission of the current H5N1 bird flu. However, it is the largest and most severe influenza epidemic ever registered among birds; since 2003, it has spread rapidly to poultry in many countries in Asia and most recently it seems to have established itself in Turkey. The size of this virus repertoire has caused concerns that re-assortment or mutations of influenza genes occurring in bird populations, or in infected humans, eventually will generate a virus that can be transmitted from person to person causing a highly contagious, and potentially devastating, pandemic [3].

The primary port of entry of the influenza virus is the mucosa of the respiratory tract. The adaptive immune system can provide immune protection against mucosal pathogens through secretory IgA and IgM immunoglobulins, which can effectively prevent the virus from infecting its target cells. Vaccination using inactivated influenza virus preparations remains the primary method of prevention. However, the virus attempts to escape neutralizing antibodies through constantly changing the composition of its surface antigens. This complicates the development of cross-protective immunity, i.e. the ability to cover several different isolates; rather, influenza vaccines must regularly be updated to match existing seasonal epidemic flu isolates. Current vaccine technology is likely to be too slow, and also of too low capacity, to produce enough vaccine against a new emerging flu isolate in time to prevent a true pandemic. Thus, the development of faster and more efficient vaccine technologies capable of delivering new, safe, efficacious and easy-managed protective influenza vaccines is of high priority.

It is known that CD8+ T cell responses also play a major role in the control of primary influenza virus infection [4], [5]. In mice, CTLs against conserved epitopes contribute to protective immunity against influenza viruses of various subtypes [6], [7]. Identification of CTL epitopes, especially conserved epitopes shared by multiple viral strains, might therefore be a robust vaccine strategy against emerging influenza epidemics. One could argue that CTLs, being specific for short immunogenic peptides and being diversified by the highly polymorphic HLA system, are easier to target against conserved, and thereby potentially cross-protective, epitopes. Obviously, it is easier to find conservation in a short primary peptide sequence, a CTL target, than in a longer tertiary protein structure, such as a characteristic antibody target. Also, the HLA-restricted CTL immune system targets different epitopes in different individuals thereby reducing the risk of a population-wide virus escape through removal of highly conserved epitope targets. The flipside of trying to exploit the potential advantages of HLA-restricted CTL immunity is that one would have to identify several different CTL targets to encompass the diversity of the HLA system, and obtain sufficient coverage of the human population. This problem, however, appears somewhat alleviated by the recent discovery that HLA molecules largely can be grouped into 12 different “supertypes” of overlapping specificities [8].

In a recent study, a limited search for novel flu CTL epitopes was performed; only one epitope was found with a search restricted to only a part of the genome, one flu isolate and one HLA-I molecule [9]. Here, we have performed a genome-, pathogen- and HLA-wide search for new CTL epitopes directed against influenza A virus. The genome-wide aspect assures that all influenza virus proteins are considered, the pathogen-wide assures that maximum conservation is achieved, whereas the HLA-wide aspect assures maximum coverage of human populations. Others and we have developed immuno-bioinformatics methods to identify CTL epitopes. Here, we have used recently improved methods based upon combined HLA binding, TAP binding, and proteasome processing predictions [10], [11], [12], [13]. Note, that these tools have been developed using large databases such as SYFPEITHI and Los Alamos database as biological benchmarks, i.e. they have been designed with optimal immune epitope identification in mind. The predicted peptides were synthesized and tested by biochemical methods for binding to the appropriate recombinant HLA-I protein, and by IFN-γ ELISPOT analysis for CTL immune responses using PBMCs from healthy, elderly and HLA-typed Danish subjects, which are assumed to have been exposed to multiple influenza infections in their past. Here – even at the limited scale of this study – we suggest several HLA-I-restricted, influenza-specific 9mer epitopes, many of which are conserved among several different influenza virus isolates even including the H5N1 bird flu virus. Combining their HLA-restriction specificity, they cover virtually the entire human population.

Section snippets

Collection of blood samples

Buffy coats of 500 ml whole blood from healthy Danish donors (age range: 35–65 years; donors have given written informed consent) were obtained from The Blood Bank at Rigshospitalet (Copenhagen, Denmark) and used within 24 h to isolate peripheral blood mononuclear cells (PBMC). The donors were selected, according to serological typing of their HLA-A and -B haplotype, to maximize coverage of the 12 HLA class I supertypes. A high-resolution sequence-based typing (SBT) of the HLA-A and HLA-B loci

Prediction of HLA-I binding, proteasome cleavage site, and TAP binding

An example of the proteins used as input for combined predictions of HLA-I binding, TAP binding and proteasome cleavage site, here exemplified by the proteins from the influenza A/Puerto Rico/8/34/Mount Sinai (H1N1) virus isolate, is shown in Table 2. Note that the search for conserved CTL epitopes skewed the selection towards the polymerases and the nucleoprotein, whereas it strongly deselected for the classical antibody targets, the Hemagglutinin and the Neuraminidase. For each of the 12

Discussion

One of the major drawbacks of a peptide-based CTL vaccine strategy is that the restricting HLA genes are extremely polymorphic resulting in a vast diversity of peptide-binding HLA specificities and a low population coverage for any given peptide–HLA specificity. To increase population coverage, one might include defined epitopes for each HLA-I allele, however, this would lead to a vaccine comprising hundreds of peptides. One way to reduce this complexity is to group HLA-I molecules into

Acknowledgements

This work was supported by NIAID Contracts no. HHSN266200400083C, HHSN266200400025C and EU 6FP 503231. We thank Ms. Trine Devantier for her excellent technical assistance.

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