key: cord-0783595-nno2yjae
authors: Sylvester‐Hvid, C.; Nielsen, M.; Lamberth, K.; Røder, G.; Justesen, S.; Lundegaard, C.; Worning, P.; Thomadsen, H.; Lund, O.; Brunak, S.; Buus, S.
title: SARS CTL vaccine candidates; HLA supertype‐, genome‐wide scanning and biochemical validation
date: 2004-04-23
journal: Tissue Antigens
DOI: 10.1111/j.0001-2815.2004.00221.x
sha: cc42fc360f1a18e72a2db1517715b48cbe4d069d
doc_id: 783595
cord_uid: nno2yjae

Abstract: An effective Severe Acute Respiratory Syndrome (SARS) vaccine is likely to include components that can induce specific cytotoxic T‐lymphocyte (CTL) responses. The specificities of such responses are governed by human leukocyte antigen (HLA)‐restricted presentation of SARS‐derived peptide epitopes. Exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information (Lauemoller et al., Rev Immunogenet 2001: 2: 477–91). The latter was recently established when a causative coronavirus (SARS‐CoV) was isolated and full‐length sequenced (Marra et al., Science 2003: 300: 1399–404). Here, we have combined advanced bioinformatics and high‐throughput immunology to perform an HLA supertype‐, genome‐wide scan for SARS‐specific CTL epitopes. The scan includes all nine human HLA supertypes in total covering >99% of all individuals of all major human populations (Sette & Sidney, Immunogenetics 1999: 50: 201–12). For each HLA supertype, we have selected the 15 top candidates for test in biochemical binding assays. At this time (approximately 6 months after the genome was established), we have tested the majority of the HLA supertypes and identified almost 100 potential vaccine candidates. These should be further validated in SARS survivors and used for vaccine formulation. We suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design.

eradicated. This would require detection assays that can track the disease and intervention measures that can break the chain of transmission. All of these procedures should be simple, yet effective.

Unfortunately, no such diagnostic test is currently available, and controlling transmission by containment solely is complicated and extremely costly. Further complicating any eradication effort, a nonhuman reservoir appears to exist. Thus, a strong case for a SARS vaccine can be made. It would be of significant help in any eradication effort and, should that fail, it could protect infected individuals against the disease.

The SARS-CoV infects epithelial cells in the respiratory tract causing interstitial pneumonia (4) . One would therefore expect that an effective vaccine should induce mucosal immunity such as that effected by secretory immunoglobulin A (IgA), which specifically prevents an infectious agent from penetrating the mucosal epithelium, and by cytotoxic T lymphocytes (CTLs), which specifically eradicate infected cells (5) . IgA responses are generally considered the major protective mechanism; however, there are examples of CTLs, not antibodies, being responsible for early control of mucosal infection (5) . Particularly noteworthy, this is the case for the infectious bronchitis virus of chicks, a prototype of the Coronaviridae family, where primary effector CD8 þ CTLs play a critical role in the elimination of virus during acute infection and subsequent control of the infection (6-10).

Human CTLs are specific for peptides presented in the context of human leukocyte antigen (HLA) molecules [generically known as ''major histocompatibility complex (MHC) molecules'']. Prior to presentation, peptides are generated in the cytosol by limited proteolytic fragmentation of all available protein antigens, translocated to the endoplasmic reticulum, specifically sampled by the MHC molecules and exported to the cell surface, where they await CTL scrutiny. Importantly, the HLA is extremely polymorphic and the peptide binding specificity varies for the different polymorphic HLA molecules (1). It has, however, been suggested that the majority of all major human populations can be covered with three to nine ''HLA supertypes'', where the different members of each supertype bind similar peptides (3). If one knew exactly how peptides were generated and selected, then genomic/proteomic information could be used to predict the outcome of antigen presentation and forecast immunogenicity. Here, we have used advanced immunobioinformatical tools to mimic antigen presentation and, in a highly cost-and timeeffective manner, predicted possible immunogenic epitopes.

The complete SARS genome/proteome was obtained from Gen-Bank (NC004718) and virtually digested into all 9862 unique nonamer peptides (2) . Thus, close to 10,000 binding predictions were made for each of the nine HLA supertypes. Artificial neural networks (ANNs) were used to predict the binding affinity quantitatively when the corresponding data were available [e.g. for A*0201 (11, 12) ]. The performance of the ANNs is high, as the correlation coefficient between predicted and measured binding is 0.85. The remaining HLA bindings were predicted using weight matrices derived from Gibbs sampling sequence-weighting methods with pseudocount correction for low counts as well as differential position-specific anchor weighting (Nielsen et al. manuscript in preparation). These weight matrices were calculated from available nonamer data from the SYFPEITHI and MHCPEP databases with the peptides clustered into the nine supertypes (A1, A2, A3, A24, B7, B27, B44, B58, and B62). The positive predictive value of the matrix-driven prediction has been found to be around 66%, whereas the negative predictive value has been found to be around 97% (Lamberth et al. unpublished observation). Proteasomal processing was predicted using NETCHOP 2.0 (13). NETCHOP 2.0 has been found to be superior to other proteasomal prediction algorithms (14) . Peptides with a NETCHOP 2.0 score below 0.5 (i.e. poorly predicted proteasomal processing) were excluded from further analysis. Finally, we excluded all peptides that did not represent epitopes conserved in all SARS isolates. Figure 1 shows a representative example for a member of the HLA-A3 supertype, the HLA-A*1101 (this haplotype is particularly common in Southeast Asia).

For each HLA supertype, the 15 top-ranking nonamer peptides were synthesized by standard 9-fluorenylmethyloxycarbonyl (FMOC) chemistry, purified by reversed-phase high-performance liquid chromatography (at least 80%, usually >95% purity) and validated by mass spectrometry. The interaction of these epitope candidates with the appropriate HLA was subsequently validated in a biochemical binding assay (15) . Briefly, denatured and purified recombinant HLA heavy chains were diluted into a renaturation buffer containing HLA light chain, b 2 -microglobulin, and graded concentrations of the peptide to be tested, and incubated at 18 C for 48 h allowing equilibrium to be reached. We have previously demonstrated that denatured HLA molecules can de novo fold efficiently, however, only in the presence of appropriate peptide (16) . The concentration of peptide-HLA complexes generated was measured in a quantitative enzyme-linked immunosorbent assay and plotted against the concentration of peptide offered (15) (Fig. 2) . Because the effective concentration of HLA (3-5 nM) used in these assays is below the equilibrium dissociation constant (K D ) of most high-affinity peptide-HLA interactions, the peptide concentration leading to half-saturation of the HLA is a reasonable approximation of the affinity of the interaction. An initial screening procedure was employed whereby a single high concentration (20,000 nM) of peptide was incubated with one or more HLA molecules. If no complex formation was found, the peptide was assigned as a non-binder to the HLA molecule(s) in question, conversely, if complex formation was found in the initial screening, a full titration of the peptide was performed to determine the affinity of binding.

The resulting binding isotherms were analyzed by one-site hyperbola regression (Prism 1 GraphPad) determining the concentration of HLA employed (3-5 nM, data not shown), the K D of the interaction (Table 1 ) and the goodness of the curve fit (R 2 was always >0.95 and in the majority of cases it was >0.98, data not shown) (15) . In general, intermediate and high-affinity binders have K D s better (i.e. lower) than 500 nM and 50 nM, respectively, and the higher the affinity, the more likely the peptide is going to be a T-cell epitope (17) . Table 1 summarizes the data for HLA-A*0301 and HLA-A*1101. The pep- for the top-ranking peptides, only 22 (or 2-3%) of the 952 combinations involves cross-responses, where a peptide predicted to be a top-ranking binder to one HLA molecule turns out to be a binder to a member of another HLA supertype, i.e. it supports the contention that HLA supertypes effect significant diversification of anti-SARS CTL responses. Conversely, the overlap between different members of the same HLA supertype appears to be extensive. Thus, 13 of the 15 peptides predicted to be good binders to A*0301 were found to bind to another member of the A3 supertype, HLA-A*1101. Similarly, nine of the 15 peptides predicted to be good binders to A*1101 were found to bind to HLA-A*0301 (Table 1) Once all nine supertypes have been tested, we would project to have found well over 100 different vaccine candidates. These would all have been predicted to be successfully processed by the proteasome and biochemically validated for HLA binding. Therefore, there should be a high probability that these peptides are indeed presented to CTLs. Once that occurs, there is an approximately 50% chance of being able to raise a CTL response (18) . Thus, our data do in all likelihood include some 50 CTL epitopes. To identify these from the >100 binding peptides, one could search for the corresponding CTL reactivities in peripheral blood of SARS survivors using robust and reasonably simple technologies such as interferon-g secretion from stimulated whole blood T cells (19) Peptide binders to HLA-A*0301 (top frame) and HLA-A*1101 (bottom frame), sorted according to predicted binding strength, were synthesized and the affinities of binding to A*0301 and A*1101 were determined. The peptide sequence is given in single-letter code and the measured binding affinity is given as the KD. In the near future, the genome of any pathogen can be fully sequenced in a matter of days. The bioinformatics tools currently being developed and perfected will be able to use such genomic information to predict immune epitopes computationally, and the corresponding immunological tools will currently be able to validate these predictions in a matter of weeks to months. We predict that epitope identification in the near future will be as fast as a DNA sequencing in handling whole organisms. With the dissemination of these tools, one could envision that clinicians and scientists anywhere would be able to analyze pathogens of their interest (or agent of bioterrorism, or tumor cell) for the purpose of fast identification of immunogenic epitopes (1) . The timeline of the present SARS epidemic has demonstrated how fast modern science can identify a pathogen and decipher its genome. Using this information in a fast and rational design of vaccines, immunobioinformatics promises to take this development one step further.

Identifying cytotoxic T cell epitopes from genomic and proteomic information: ''The human MHC project

The genome sequence of the SARS-associated coronavirus

Nine major HLA class I supertypes account for the vast preponderance of HLA-A and -B polymorphism

SARS coronavirus: a new challenge for prevention and therapy

Recent advances in mucosal vaccines and adjuvants

Specific cytotoxic T lymphocytes are involved in in vivo clearance of infectious bronchitis virus

Cytotoxic T lymphocyte responses to infectious bronchitis virus infection

Cytotoxic T lymphocytes are critical in the control of infectious bronchitis virus in poultry

Adoptive transfer of infectious bronchitis virus primed alphabeta T cells bearing CD8 antigen protects chicks from acute infection

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Sensitive quantitative predictions of peptide-MHC binding by a ''Query by Committee'' artificial neural network approach

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Prediction of proteasome cleavage motifs by neural networks

Predicting proteasomal cleavage sites: a comparison of available methods

Establishment of a quantitative ELISA-based assay capable of determining peptide-MHC class I interaction

Efficient assembly of recombinant major histocompatibility complex class I molecules with preformed disulfide bonds

The relationship between class I binding affinity and immunogenicity of potential cytotoxic T cell epitopes

Immunodominance in MHC class I restricted T lymphocyte responses

Cytokine-based human whole blood assay for the detection of antigen-reactive T cells

Recombinant polyepitope vaccines for the delivery of multiple CD8 cytotoxic T cell epitopes

Delivery of multiple CD8 cytotoxic T cell epitopes by DNA vaccination

Immunogenicity of a human immunodeficiency virus (HIV) polytope vaccine containing multiple HLA A2 HIV CD8(þ) cytotoxic T-cell epitopes

An HLA-A2 polyepitope vaccine for melanoma immunotherapy

Papillomavirus virus-like particles for the delivery of multiple cytotoxic T cell epitopes

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The development of multi-epitope vaccines: epitope identification, vaccine design and clinical evaluation

Optimization of epitope processing enhances immunogenicity of multiepitope DNA vaccines

The Danish MRC (grant 22-01-0272), the 5th Framework Programme of the European Commission (grant QLGT-1999-00173), the NIH (grant AI49213-02), and the Danish National Research Foundation supported this work. Support for this work has been obtained from SIGA Technologies.