WO2005005614A2

WO2005005614A2 - Combined b-cell and t-cell epitopes on vlp for improved vaccines

Info

Publication number: WO2005005614A2
Application number: PCT/US2004/021381
Authority: WO
Inventors: Vsevolod Katritch; Robert J. Deans; Ole Lund; Søren BRUNAK
Original assignee: Pecos Labs, Inc.
Priority date: 2003-07-03
Filing date: 2004-07-06
Publication date: 2005-01-20
Also published as: WO2005005614A3

Abstract

A composition is described to induce simultaneous humoral (B-cell) and cell-mediated (CTL) immune responses against single or multiple antigens of a pathogenic virus bacteria, or cancer cell. Multiple B-cell and T-cell epitopes are identified in the pathogen proteome by computational and experimental techniques, and genetically inserted into a virus-like particle (VLP). Different insertion sites for B-cell and T-cell epitopes can be chosen at N-terminus, C-terminus or within the sequence of the VLP protein for optimal presentation of the epitopes.

Description

COMBINED B-CELL AND T-CELL EPITOPES ON VLP FOR IMPROVED VACCINES

Field of the Invention This invention relates to methods of vaccine design for prevention and treatment of infectious disease. More specifically it relates to synthetic protein compositions capable of inducing both neutralizing B-lymphocyte and cytotoxic T-lymphocyte (CTL) arms of immunity, specifically directed towards selected epitopes of a virus, bacteria or a pathogenic cell, including cancer cells.

Background Both B-cell (humoral) and CTL ("Cytolytic T Lymphocytes")(cell-mediated) arms of immune system are important components of protective responses against infection, intracellular pathogens and pathogenic molecules, including all viral infections (e.g. common influenza, HIV, HCV, HBV, HPV, JEV etc) and some bacterial infections (e.g. M. tuberculosis). Specific antibodies can neutralize extracellular virus or bacteria load and thus limit or prevent infection of cells in the host (Shibata et al., 1999). Also antibodies can neutralize bacterial toxins, thus reducing pathogenicity of the infection. Complementing these activities, CTL can limit viral production by killing the infected cells, inducing apoptosis, release of antiviral substances, and/or inducing increased intracellular lysis in already infected cells and thus help to prevent or cure the disease. In addition, effective and long lasting response in both arms of immunity usually requires additional support from T-helper (Th1 and Th2) lymphocytes. After binding specific T cell epitopes on the surface of APC, Th1 and Th2 cells supply specific soluble cytokine signals that regulate the balance between antibody and CTL immunity. Thus, effective immunity involves multiple antigen recognition events of specific pathogen immunogenic determinants (epitopes); by T-helper cells followed by molecular recognition by B cells, CTL, or both. Different types of lymphocytes (B-cells, CTL and Th cells) specifically recognize different types of epitopes of the pathogen. Thus, B-cell epitopes are exposed structural features on the surface of viral envelope, bacterial outer membrane or secreted toxins. In contrast, T-cell epitopes are short peptides from any protein of a pathogenic target, which only have to conform to the host antigen- processing and MHC binding mechanisms, most notably class I or class II MHC haplotype restriction mechanisms. Suitable T-cell epitopes occur with an estimated frequency of about one per 200-500 aa sequence^'' , also varying for different hosts populations and pathogens. Therefore, it is likely that a naturally occurring protein antigen does not comprise a suitable T-cell epitope at all, or has only a suboptimal T-cell epitope. Adding selected T-cell epitope or epitopes to such B-cell antigen is beneficial, but requires a special delivery vehicle. Different delivery platforms are usually employed for B-cell and T-cell epitopes, because mechanisms of B-cell and CTL responses are different. For example, a so-called "prime and boost" vaccination regimen has been suggested, when a patient is primed with a T-cell epitopes, administered by a DNA delivery mechanism, and then humoral response is boosted by a recombinant B-cell antigen in a few week interval. At the same time, effective combination of B-cell and T-cell epitopes in one delivery vehicle would be highly beneficial. As a delivery vehicle, VLPs have been shown to be able to induce B-ceil as well as a T-cell response. However, accurate structural representation of B cell epitopes, and the limiting size of an insert has been an obstacle in inserting both T-cell and B-cell epitopes in one VLP formulation.

Summary of the Invention The present invention provides a composition to induce simultaneous humoral (B-cell) and cell-mediated (CTL) immune responses against single or multiple antigens of a pathogenic virus bacteria, or cancer cell. Multiple B-cell and T-cell epitopes are identified in the pathogen proteome by computational and experimental techniques, and genetically inserted into a virus-like particle (VLP). Different insertion sites for B-cell and T-cell epitopes can be chosen at N-terminus, C-terminus or within the sequence of the VLP protein for optimal presentation of the epitopes. Rationally designed chimeric particles, comprised of B-cell and multiple

T-cell epitopes and a VLP carrier can be developed into more effective and safe prophylactic and therapeutic vaccines against many viral and bacterial infections. Unlike existing vaccine approaches, the novel recombinant vaccines candidates do not contain infectious material or functional toxins, and therefore are potentially safe and side effect-free. Combining well-defined protective epitopes by virtue of self- assembling particles provides a mean to induce effective T cell independent humoral immune response and long lasting immune memory, eliminating the need for multiple vaccinations.

Detailed Description of the Figures Figure 1. An example of HCV B-cell epitope design, (a) A portion of all- beta central domain from E2 antigen model shown as green ribbon, (b) To cut this domain from the rest of the protein, large portions of the E2 protein are replaced by optimally fitting short loops (blue and red balls), selected by ICM-powered conformational search in PDB. The resulting mini-protein includes the selected epitope. Figure 2. T-cell epitope predictions for HCV genome. Advanced neural net algorithms were used to scan the HCV viral genome for high (<50 nM) and moderate (<500 nM) HLA-A2 binding peptides. The epitope atlas places the position of these epitopes relative to sense and reading frames of the viral genome (represented in circular format). As well, a composite of all epitopes placed on the genome is shown on the outer ring.

Figure 3. An example of epitope-VLP chimera design, (a) B-cell epitope is a structurally stable fold of all-beta central domain from E2 antigen, (b) The tip of HBc VLP carrier (green) can be replaced by B-cell epitope miniprotein (c) Two T-cell epitopes (Ep1 >EMGGNITRV in red and Ep2> KLGVPPLRA in blue) from T-cell epitope analysis above, (d) Combination of B-cell epitope, T-cell epitope, and HBc carrier forms structurally feasible protein, capable of assembly into VLP particles (BeTeVax). Figure 4. Fully assembled icosahedral virus-like particles, each comprising

240 copies of a single protein, a) Original HBc protein^'' 1 with B-cell epitopes on the on tips of HBc a-helical spikes, MIR region, colored in green, b) Rationally designed BeTeVaxprotein, with 3D structural epitopes shown in green. Immunogenic loop, E2[522-551] shown in yellow balls, points outwards and is accessible for antibodies on the surface of VLP, while T-cell epitopes, attached to the C-terminus of the protein, point into the interior of the particle.

Detailed Description of the Invention B-cell epitopes are structural domains of protein, lipid or polysaccharide antigens accessible to B-cell receptor recognition. B-cell epitopes are presented on a surface of a secreted pathogenic protein or on the outer surface of a virus or pathogenic cell. Specific binding of B-cell epitopes to immunoglobulin (Ig) receptors in a B-cell leads to activation of this lymphocyte with subsequent release of secreted lg receptors (antibodies). A B-cell epitope raises a neutralizing humoral immune response if the epitope-induced antibodies specifically cross-react with the whole pathogen or toxin. T-cell epitopes are short linear peptides that can belong to any polypeptide of a pathogenic agent, including proteins expressed internally in an infected or malignant cell. These peptides are produced in the antigen presenting cells (APC) by a multistep processing mechanism, which also inserts epitopes into MHC (major histocompatibility complex) molecules and transports them to the cell surface. Epitopes for cytotoxic T-lymphocytes are 8 to 10 aa (amino acid) long peptides capable of binding MHC class I molecules. T-helper epitopes can be 8 to 15 aa long and must bind MHC class II molecules of a host. Virus-like particles (VLP) are periodic structures with preferably icosahedral symmetry, spontaneously assembled from multiple copies of a viral envelope or capsid protein. Specific modification can be introduced to the protein, e.g. sequence insertions, that does not interfere with protein folding and VLP self- assembly. We document invention of a novel method to rationally design prophylactic and therapeutic vaccines, based on combination of specific B-cell and T-cell epitopes, genetically inserted into a viral structural protein. An important characteristic of the designed chimeric proteins is their ability to self-ensemble into symmetric virus-like particles (VLP), so that B-cell epitopes are presented on the surface of the VLP. Specific selection of most potent B-cell and T-cell epitopes from the whole proteome of the pathogenic virus or bacteria allows for optimal induction of both humoral and cell-mediated arms of immunity. Design of a VLP-based vaccine includes a VLP structural protein as a carrier, a B-cell epitope, preferably represented by a stable structural domain of 50 to 250 aa length, and multiple T-cell epitopes, represented by peptides 8-10 aa each. In the preferred embodiment, B-cell and T-cell epitopes can originate from different antigens and can be inserted into different sites in the VLP protein sequence (e.g. C-term, N-term or internal site). The design method assures correct folding of the structural epitope domain and the whole chimeric protein, as well as self-assembly of the chimeric proteins into a VLP. Specific immune response to the selected epitopes is achieved by optimal presentation of the B-cell epitope on the surface of the assembled VLP. Usual size of acceptable insertions in VLP is rather limited. For example, 250 aa is a longest documented insertion into HBcAg, compatible with the VLP assembly. In some cases insertions can accommodate a whole antigen. In most other cases, a structural domain of the antigen can be identified, so that it is conformationally stable, smaller than 250 aa, and optimally presents a selected B- cell epitope. Thus, a VLP can be designed to represent any selected B-cell epitope. At the same time, it is likely that optimal T-cell epitopes will not be present in such insert, and CTL immunity will not be induced. Therefore, we suggest using secondary insertion sites in VLP protein sequence to accommodate several selected T cell (MHC class I and class II) epitopes, identified by one of the known algorithms.

Methodology Design of B-cell epitopes Identification Knowledge of 3D structure of an antigen is inferred either from available X- ray structure or from a model by homology. All atom 3D models built with ICM homology modeling procedures can be used for accurate analysis of fold domain structure. Also, the model is used for mapping of known functional and immunogenic sites of the polypeptide sequence to targets on the surface of the protein. Stable fold domain structures, representing a important surface targets are selected as candidate B-cell epitope. Evaluation and optimization Relative fold stabilities of the candidate polypeptides are assessed with an ICM procedure based on fast and accurate evaluation of free energy of the polypeptide model. This free energy function, which includes conformational strain, implicit entropy and implicit solvation terms, was specifically developed and normalized to study stability of polypeptide chains^. A similar free energy function has also been recently used in successful ab-initio folding of a 23-residue ββα- peptide^.

Design of T-cell epitopes Method of CTL epitopes identification, described in patent application WO2001 DK0000059 relates to the by the combination of biochemical assays, statistical matrix calculations, and artificial neural networks^"⁷. A set of peptide libraries are used to generate complete unbiased matrices representing peptide- MHC interactions used to generate a primary prediction of MHC binding for all possible non-redundant peptides. The best binders are subject to a quantitative biochemical binding assay and subsequently a computerised artificial neural network prediction program built from these in vitro experimental MHC-I binding data. The method further comprises improving the identified epitope by replacing amino acids, and testing the identified CTL epitopes in vitro and in vivo models.

Genetic insertion of the B-cell and T-cell epitopes into VLP The structure-based design of epitope-VLP chimeric protein focuses on optimization of folding of the protein, assembly of icosahedral VLP, and display of the surface target antigen. Structural models of VLPs. All atom 3D models of VLP protein and the assembled VLP particle are built either from available X-ray structure or by homology modeling. The models are verified with available biochemical or immunological data to insure accurate prediction of surface accessible residues. Identification of insertion sites Possible insertion regions are selected in the protein sequence, those exposed on surface of the assembled VLP. While insertion sites for T-cell epitopes can be exposed on both internal and external surfaces of a hollow VLP particle, B- cell epitopes must be inserted on the outer surface of VLP to be accessible to antibodies. Insertion sites are verified individually by expressing a chimeric VLP with a "probe" peptide or fold domain insertion and testing assembly of VLP. Subsequently, double insertions are verified by expressing VLP with different combinations of individual insertions. - Optimization of insertion sites and flexible linkers The discovered insertion sites can be optimized by combination of computational and experimental techniques. Given relatively long and flexible linkers connecting VLP carrier with the epitope, the choice of insertion residues is relatively degenerate, so that the same optimal insertion points can be used for different epitopes. For example, the tip of the spike of the HBc protein, MIR, is an insertion site, which accepts foreign epitopes^. The protein residues between G73 and G94 are disposable, providing a variety of possibilities for design. We take all of the combinations of insertion points in this range and perform a simple check by fast minimization of (Gly)₃ peptides attached to these residues. The polypeptide with the least conformational strain and distance between points less than 10 A is chosen for the next step of design. If this simple check does not differentiate between candidate models, we take one of the most extensively used insertion sites, i.e. residues 76 and 80. Compile a panel of candidate linkers We consider two different sequence contents for linkers: the traditional poly-

Gly linkers and a Gly-Ser-Ala repeat, another widely used linker^. We consider all linker lengths between 3 and 10 amino acids and limit the difference between the lengths of the two linkers to 2 amino acids, thus having -50 polypeptides to test. Perform conformational modeling of chimeric polypeptides We perform modeling of the candidate chimeric protein considering large- scale rearrangements between the foreign epitope domain and HBc domain and conformational changes in two flexible linkers connecting domains. This type of calculation is highly facilitated in ICM internal coordinate representation where domains of the protein are considered as rigid bodies, and only small portion of the polypeptide, corresponding to the flexible linkers, is optimized^'' 0. Energy function is calculated as in loop modeling (see D.1.2), and global energy optimization is performed until convergence. The best 100 alternative conformations for each polypeptide are collected and saved in the conformational stack. Evaluate fold stability and self-assembly of chimeric VLP Successful design should satisfy VLP self-assembly and fold stability requirements, and both are checked in the following procedure: Using icosahedral symmetry pattern of the HBc virion particle, we build a full VLP for the chimeric protein and check the VLP for steric clashes between protein subunits. For these tests, a 23-mer uniformly representing all the contacts within VLP is used instead of the full VLP to simplify calculations. Free energy of the 23-mer is calculated for all candidate conformations including both inter- and intramolecular terms. A few candidate polypeptides with lowest energy conformations are subsequently selected for experimental testing.

Example Clearance from Hepatitis C (HCV) chronic infection has been shown to correlate with activation of both B-cell and T-cell arms of immunity. While most important B-cell epitopes belong to the HCV surface glycoprotein E2, most effective T-cell epitopes with isotype-conserved sequences belong to internal proteins. Therefore our approach to combination of B-cell and T-cell epitope in a VLP can be highly beneficial for a successful HCV vaccine. We have designed a BeTeVaxvaccine candidate with multiple HCV epitopes combined with a VLP carrier from another virus. In this example VLP carrier the VLP carrier is derived from Hep B virus core particle (HBc). Multiple HCV T-cell epitope have been selected as described in patent application WO0155177A3. Multiple B-cell epitopes have been selected and chimeric VLP particles have been designed as described previously (grant application NIH SBIR R43 AI054038-01 , April 31 , 2002). Modeling, performed to generate BeTeVax particles is illustrated in Figures 1-4.

Claims

1. A composition comprising a VLP structural protein as a carrier, a B-cell epitope, and multiple T-cell epitopes.

2. A composition according to claim 1 , wherein the B-cell epitope is represented by a stable structural domain of 50 to 250 aa length.

3. A composition according to claim 1 , wherein the T-cell epitopes are represented by peptides of 8-10 aa each.

4. A composition according to claim 1 , wherein B-cell and T-cell epitopes originate from different antigens.

5. A composition according to claim 1 , wherein B-cell and T-cell epitopes are inserted into different sites in the VLP protein sequence.

6. A composition according to claim 1 , wherein the epitopes are insertions into the VLP of 250 aa or less.

7. A composition according to claim 1 , wherein the T-cell epitopes are inserted into the VLP at one or more secondary insertion sites in the VLP protein sequence to accommodate several selected T cell (MHC class I and class II) epitopes.