WO2008083203A1 - Identification des épitopes de protéines d'enveloppe virale multivalents - Google Patents

Identification des épitopes de protéines d'enveloppe virale multivalents Download PDF

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WO2008083203A1
WO2008083203A1 PCT/US2007/088910 US2007088910W WO2008083203A1 WO 2008083203 A1 WO2008083203 A1 WO 2008083203A1 US 2007088910 W US2007088910 W US 2007088910W WO 2008083203 A1 WO2008083203 A1 WO 2008083203A1
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cell
sequence
sequences
vep
virus
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PCT/US2007/088910
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Xiaofeng Fan
Adrian M. Di Bisceglie
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Saint Louis University
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Priority to CA002674244A priority patent/CA2674244A1/fr
Publication of WO2008083203A1 publication Critical patent/WO2008083203A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B10/00—ICT specially adapted for evolutionary bioinformatics, e.g. phylogenetic tree construction or analysis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention is directed to methods of identifying nucleic acid and polypeptide sequences useful in the development of universal multivalent viral vaccines.
  • RNA viruses such as hepatitis C virus (HCV), human immunodeficiency virus (HIV), dengue virus and influenza virus.
  • HCV hepatitis C virus
  • HAV human immunodeficiency virus
  • dengue virus dengue virus
  • influenza virus A common feature shared by these viruses is the great genetic heterogeneity, shown as multiple serotypes, genotypes, subtypes and quasispecies nature even in single infected individual.
  • the genetic heterogeneity has many physiological and pathogenical results, including immune escape and drug resistance. More importantly, it is a major challenge for vaccine development.
  • a long-term seeking goal is to identify or design a single antigen which, upon vaccination, is able to induce broad neutralizing antibodies against all viral isolates.
  • antigens have not been identified in despite considerable effort.
  • HIV provides an excellent example of this problem.
  • the HIV envelope region is highly diverse and contains multiple potential N-linked glycosylation sites that shield most potential neutralizing epitopes.
  • neutralizing monoclonal antibodies produced from either antibody libraries or transformed B cells, are available against most primary HIV isolates. All recognize conformational epitopes as mapped in 3-D structure of HIV envelope protein, but there is presently no HIV envelope antigen available known to induce such neutralizing antibodies.
  • HIV envelope proteins do induce neutralizing antibodies, but they are very weak for neutralization, and mostly isolate - or genotype-specific.
  • virus neutralization can occur at each step of the virus entry, i.e., attachment, fusion and post-entry.
  • Virus envelope proteins experience conformational alterations at these steps and, as a result, display different neutralizing epitopes.
  • the immune systems especially on the humoral branch, only response well to epitopes displayed before and perhaps during the attachment but not after. In other words, all active immunization with wild-type or wild-type-like viral envelope proteins only elicits neutralizing antibodies to epitopes displayed before and to a less extent during the viral attachment. Neutralizing epitopes displayed after the viral attachment are hardly attacked.
  • DNA shuffling technique is the most attractive.
  • the DNA shuffling technique has been extensively applied by Maxygen (www.maxygen.com) in the search for vaccine antigens of various viral diseases, including dengue and AIDS. Recently, a dengue envelope protein produced by DNA shuffling was shown to elicit broad neutralizing antibodies to all four dengue serotypes, although the titer was found not to be high.
  • a method of identifying a multivalent vaccine epitope comprising (a) providing a data set comprising a plurality of viral envelope protein (VEP) nucleic acid or protein sequences; (b) subjecting said data set to be simulated under evolution models to obtain an ancestral VEP sequence; (c) subjecting said an ancestral VEP sequence to be dedicated for saturated mutatgenesis to obtain a VEP antigen library; (d) constructing a pseudotyped viral particle library, wherein said particles express members of said VEP antigen library; (e) neutralizing said pseudotyped viral particle library with antiserum raised against wild-type VEP sequence; (f) infecting cells with the neutralized viral particle library and selecting variants; (g) selecting survival variants; and (h) obtaining sequence information on said survival variants, wherein these sequences are potential vaccine candidates containing pluriopotent epitopes and deserve for further refinement.
  • VEP viral envelope protein
  • Step (c) of the method may comprise codon optimization.
  • the VEP may be from hepatitis C virus, human immunodeficiency virus, dengue virus, influenza virus, ebola virus, coronavirus, or hantavirus.
  • the plurality of VEP sequences may comprise at least 5, at least 10, at least 15, at least 20 or at least 25 sequences.
  • the simulated evolution may comprise reconstruction of a phylogenetic tree, rooting of the phylogenetic tree, and inference of said ancestral VEP sequence.
  • the phylogenetic tree may comprise a phylogenetic inference method.
  • the phylogenetic inference method may comprise a maximum likelihood method.
  • the rooting of the phylogenetic tree may also comprise the use of molecular clock hypothesis or outgroup criterion.
  • the method may further comprise selecting a nucleotide or amino acid substitution model that fits the data set.
  • the method may also further comprise identifying domains in said ancestral sequence for saturated mutagenesis.
  • Constructing a pseudotyped viral particle library may comprise PCR assembly.
  • the pseudotyped viral particles may be retroviral particles, including lentiviral particles.
  • the antiserum may be human antiserum or from a non-human animal.
  • Selecting survival variants may comprise fluorescence activated cell sorting of infected cells.
  • "a" or "an” may mean one or more.
  • FIG 1 Schematic representation of the relationship between the potential sequence space of a given protein and current methods for library construction. Due to the application of different strategies, each method takes up only a small but distinct part of the space although the possibility for the overlap among methods cannot be totally excluded.
  • RACHITT random chimeragenesis on transient templates (Coco et al, 2001; Coco, 2003); StEP, staggered extension process (Aguinaldo and Arnold, 2003; 1998); ITCHY, iterative truncation for the creation of hybrid enzymes (Ostermeier and Lutz, 2003; Ostermeier et al, 1999); MAX randomization (Hughes et al, 2003); Error-prone PCR (Cirino et al, 2003; Matsumura and Ellington, 2001).
  • FIGS. 2A-B The saturation mutagenesis of an ancestral gene.
  • FIG. 2A The design of mutagenic primers is shown. Each primer consists of three parts (5' to 3'), the overlapped domain for assembly PCR, the middle part for saturation mutagenesis and the priming domain for single PCR.
  • FIG. 2B An example of EGCAL is depicted. Assuming that three domains (green) are identified for saturation mutagenesis, a given ancestral gene should be divided into three overlapped fragments. The saturation mutagenesis is done for each fragment, followed by the assembly PCR. The final product is cloned into appropriate E.coli cells to generate EGCAL.
  • the inventor now presents a novel approach for construction of antigen libraries. Basically, this approach takes into the account of molecular evolution.
  • the first step is the inference of the putative ancestor of a given viral gene by simulating its evolutionary history.
  • the ancestor is then used as the backbone for further saturated mutagenesis at certain regions, which give the higher dN/dS values by scanning entire genetic domain with an appropriate slide window.
  • the ratio of the number of nonsynonymous substitutions per nonsynonymous site (dN) and the number of synonymous substitutions per synonymous site (dS) is generally defined as an indicator of evolutionary pressure. While keeping certain sites stable due to functional constraint, viruses always search sequence space for affordable sites to increase replicate fitness and/or to escape immune/drug attacks.
  • a pseudotyped virus pool will be produced by the transfection of lentiviral vectors containing envelope variants from the antigen libraries.
  • Neutralizing assay based on pseudotyped viral particles is now extensively used for many viruses, including HIV, HCV, SARS, Ebola, etc., because it is a fast and convenient way without the concern for biosafety.
  • This pseudotyped virus pool will be first incubated with antiserum produced with wild-type viral envelope gene, followed by the infection with acceptable cells.
  • the infected pseudotyped viral particles should have viral envelope variants that have not been blocked by antiserum with wild-type viral gene. Therefore, these survival viral envelope variants may contain expected multivalent epitopes that are able to elict broad neutralizing antibodies against all isolates for a given virus.
  • the present invention utilizes a method for developing an ancestral nucleotide sequence through reconstruction of phylogenetic trees.
  • the ancestral nucleotide sequence may be directed to any one of myriad viruses or virus families, with HCV being exemplified herein.
  • the method involves, generally, the steps of retrieving virus nucleic acid sequences from a genetic database (e.g.
  • GenBank GenBank
  • editing and alignment programs include for example Clustal W (Higgins and Sharp, 1988), the BioEdit program available from North Carolina State University (available at www.mbio.ncsu.edu/BioEdit/ bioedit.html), and the SegEd program available in the GCG package (Wisconsin GCG package. Version 10.0. Oxford Molecular Group, Inc.). Any missing information, for example, the genotype for a given sequence, may be determined by phylogenetic analyses, such as for example Molecular Evolutionary Genetics Analysis (MEGA; Kumar et al., 2001). The sequences are then filtered to remove sequences that are below a particular size cut-off.
  • MEGA Molecular Evolutionary Genetics Analysis
  • Model simulation and phylogenetic reconstruction are applied to the now remaining sequences.
  • a particular model is selected through a hierarchical likelihood ratio test (hLRT) simulated with the program Modeltest (Posada and Crandall, 1998;
  • a phylogenetic tree is then constructed by heuristic search using a maximum likelihood (ML) approach for all genotypes.
  • ML trees can be constructed using any one or more of known programs (e.g., PAUP, Swofford, Sinauer Associates; PHYML, Guindon and Gascuel, 2003).
  • PAUP PAUP
  • Swofford Sinauer Associates
  • PHYML PHYML
  • Guindon and Gascuel 2003.
  • the rooted tree is then used as a template to simulate an ancestral sequence. Simulation of ancestral sequences at each internal node as well as the most recent common ancestor (MRCA) is conducted using an evolutionary program, such as for example the baseml program of the PAML package (Yang, 1997).
  • the ancestral sequence(s) are reconstructed at the nucleotide level.
  • a frequently observed drawback for ML approach is the intensive computation that is sometime unaffordable, especially when the data set contains a large number of sequences and the complicated evolutionary models are applied.
  • the inventors solved this issue by producing initial ML trees with PHYML program that implants a simple hill-climbing algorithm for heuristic tree search and uses a distance-based tree as a starting point (Guindon and Gascuel, 2003).
  • the trees produced by PHYML are then transferred into PAUP* for further optimization, including the tree rooting under molecular clock hypothesis, which is otherwise impossible for large ML phylogenies.
  • Rooting of the Phylogenetic Tree The root of a phylogenetic tree represents its first and deepest split, and it therefore provides the crucial time point for polarizing the historical sequences of all subsequent evolutionary events. An incorrectly rooted tree can result in profoundly misleading inferences of taxonomic relationships and character evolution. It is mandatory to root the tree to generate a correct topology, which will serve as the template for the inference of ancestral sequences.
  • viral envelope proteins may be subjected to the methods described herein. The following are a possible candidates: hepatitis C virus, human immunodeficiency virus, dengue virus, influenza virus, ebola virus, coronavirus, hepatitis B virus, Japanese encephalitis virus or hantavirus.
  • the term "gene” is used here to refer to the nucleic acid giving rise to a functional protein, polypeptide, or peptide-encoding unit, in this case a viral envelope protein.
  • the polynucleotides of the present invention contemplate shorter lengths that comprise less than all of a complete viral envelope polypeptide, in particular epitopes thereof, including about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330,
  • the invention concerns isolated nucleic acid segments and recombinant vectors incorporating DNA sequences that encode envelope polypeptides or peptides.
  • vectors used in the present invention regardless of the length of the coding sequence itself, may be combined with other vectors used in the present invention.
  • DNA or RNA sequences such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
  • the present invention encompasses the use of vectors that encode all or part of viral envelope polypeptides.
  • vector is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated.
  • a nucleic acid sequence can be "exogenous,” which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found.
  • Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs).
  • gene therapy or immunization vectors are contemplated.
  • One of skill in the art would be well equipped to construct a vector through standard recombinant techniques, which are described in Maniatis et al. (1990) and Ausubel et al. (1996), both incorporated herein by reference.
  • expression vector refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide.
  • Expression vectors can contain a variety of "control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra.
  • a "promoter” is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors.
  • the phrases "operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” means that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence.
  • a promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.
  • a promoter may be one naturally-associated with a gene or sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as "endogenous.”
  • an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence.
  • certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment.
  • a recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment.
  • promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not "naturally-occurring," i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression.
  • sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCRTM, in connection with the compositions disclosed herein (see U.S. Patent 4,683,202 and U.S. Patent 5,928,906, each incorporated herein by reference).
  • control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.
  • promoter and/or enhancer that effectively directs the expression of the nucleic acid segment in the cell type, organelle, and organism chosen for expression.
  • Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al (2001), incorporated herein by reference.
  • the promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides.
  • the promoter may be heterologous or exogenous, i.e., from a different source than the viral envelope sequence.
  • a prokaryotic promoter is employed for use with in vitro transcription of a desired sequence.
  • Prokaryotic promoters for use with many commercially available systems include T7, T3, and Sp6.
  • Table 2 lists several elements/promoters that may be employed, in the context of the present invention, to regulate the expression of a gene. This list is not intended to be exhaustive of all the possible elements involved in the promotion of expression but, merely, to be exemplary thereof.
  • Table 3 provides examples of inducible elements, which are regions of a nucleic acid sequence that can be activated in response to a specific stimulus.
  • a specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be "in- frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.
  • Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector (see Carbonelli et al, 1999; Levenson et ah, 1998; and Cocea, 1997; all incorporated herein by reference).
  • MCS multiple cloning site
  • "Restriction enzyme digestion” refers to catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in a nucleic acid molecule. Many of these restriction enzymes are commercially available. Use of such enzymes is widely understood by those of skill in the art.
  • a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector.
  • "Ligation” refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology.
  • the vectors or constructs of the present invention will generally comprise at least one termination signal.
  • a “termination signal” or “terminator” is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels.
  • the terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (polyA) to the 3' end of the transcript. RNA molecules modified with this polyA tail appear to more stable and are translated more efficiently.
  • terminator comprises a signal for the cleavage of the RNA, and it is more preferred that the terminator signal promotes polyadenylation of the message.
  • the terminator and/or polyadenylation site elements can serve to enhance message levels and/or to minimize read through from the cassette into other sequences.
  • Terminators contemplated for use in the invention include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not limited to, for example, the termination sequences of genes, such as for example the bovine growth hormone terminator or viral termination sequences, such as for example the SV40 terminator.
  • the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation.
  • a vector in a host cell may contain one or more origins of replication sites (often termed "ori"), which is a specific nucleic acid sequence at which replication is initiated.
  • ori origins of replication sites
  • ARS autonomously replicating sequence
  • the cells containing a nucleic acid construct of the present invention may be identified in vitro or in vivo by including a marker in the expression vector.
  • a marker in the expression vector.
  • Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector.
  • a selectable marker is one that confers a property that allows for selection.
  • a positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection.
  • An example of a positive selectable marker is a drug resistance marker.
  • a drug selection marker aids in the cloning and identification of transformants
  • genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers.
  • markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions other types of markers including screenable markers such as GFP, whose basis is colorimetric analysis, are also contemplated.
  • screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized.
  • the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. All of these terms also include their progeny, which refers to any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations.
  • "host cell” refers to a prokaryotic or eukaryotic cell, and it includes any transformable organisms that are capable of replicating a vector and/or expressing a heterologous gene encoded by a vector.
  • a host cell can, and has been, used as a recipient for vectors.
  • a host cell may be "transfected” or “transformed,” which refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a transformed cell includes the primary subject cell and its progeny.
  • Host cells may be derived from prokaryotes or eukaryotes, depending upon whether the desired result is replication of the vector, expression of part or all of the vector-encoded nucleic acid sequences, or production of infectious viral particles.
  • ATCC American Type Culture Collection
  • An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result.
  • a plasmid or cosmid for example, can be introduced into a prokaryote host cell for replication of many vectors.
  • Bacterial cells used as host cells for vector replication and/or expression include DH5 ⁇ , JM109, and KC8, as well as a number of commercially available bacterial hosts such as SURE ® Competent Cells and SOLOPACKTM Gold Cells (STRATAGENE ® , La Jolla).
  • bacterial cells such as E. coli LE392 could be used as host cells for phage viruses.
  • Prokaryote- and/or eukaryote -based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely available.
  • the insect cell/baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Patents
  • expression systems include STRATAGENE ® 'S COMPLETE CONTROLTM Inducible Mammalian Expression System, which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, an E. coli expression system.
  • an inducible expression system is available from INVITROGEN ® , which carries the T-REXTM (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter.
  • the Tet-OnTM and Tet-OffTM systems from CLONTECH ® can be used to regulate expression in a mammalian host using tetracycline or its derivatives. The implementation of these systems is described in Gossen et al.
  • INVITROGEN ® also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica.
  • a vector such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.
  • a nucleic acid may be introduced into a cell in vitro for production of polypeptides or in vivo for immunization purposes.
  • nucleic acid molecules such as expression vectors may be introduced into cells.
  • the expression vector comprises an HCV infectious particle or engineered vector derived from an HCV genome.
  • an expression vector known to one of skill in the art may be used to express an HCV polypeptide.
  • viral expression vector is meant to include those vectors containing sequences of that virus sufficient to (a) support packaging of the vector and (b) to express a polynucleotide that has been cloned therein. In this context, expression may require that the gene product be synthesized.
  • viral vectors have already been thoroughly researched, including adenovirus, adeno-associated viruses, retroviruses, herpesviruses, and vaccinia viruses.
  • Delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states.
  • One mechanism for delivery is via viral infection where the expression vector is encapsidated in an infectious viral particle.
  • Several non-viral methods for the transfer of expression vectors into cultured mammalian cells also are contemplated by the present invention.
  • the polynucleotide encoding an HCV gene may be stably integrated into the genome of the cell.
  • the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA.
  • Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression vector is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression vector employed.
  • Mutagenesis may be accomplished by a variety of standard, mutagenic procedures. Mutation is the process whereby changes occur in the quantity or structure of an organism. Mutation can involve modification of the nucleotide sequence of a single gene, blocks of genes or whole chromosome. Changes in single genes may be the consequence of point mutations which include the deletion, insertion or substitution of a single nucleotide base within a DNA sequence, or they may be the consequence of changes involving the insertion or deletion of large numbers of nucleotides.
  • Mutations can arise spontaneously as a result of events such as errors in the fidelity of DNA replication or the movement of transposable genetic elements (transposons) within the genome. They also are induced following exposure to chemical or physical mutagens.
  • mutation-inducing agents include ionizing radiations, ultraviolet light and a diverse array of chemical such as alkylating agents and polycyclic aromatic hydrocarbons all of which are capable of interacting either directly or indirectly (generally following some metabolic biotransformations) with nucleic acids.
  • the DNA lesions induced by such environmental agents may lead to modifications of base sequence when the affected DNA is replicated or repaired and thus to a mutation. Mutation also can be site-directed through the use of particular targeting methods.
  • Insertional Mutagenesis is based on the inactivation of a gene via insertion of a known DNA fragment. Because it involves the insertion of some type of DNA fragment, the mutations generated are generally loss-of-function, rather than gain-of-function mutations. However, there are several examples of insertions generating gain-of-function mutations (Oppenheimer et al 1991). Insertion mutagenesis has been very successful in bacteria and Drosophila (Cooley et al 1988) and recently has become a powerful tool in corn (Schmidt et al. 1987); Arabidopsis; (Marks et al, 1991; Koncz et al 1990); and Antirrhinum (Sommer et al 1990).
  • Transposable genetic elements are DNA sequences that can move (transpose) from one place to another in the genome of a cell.
  • the first transposable elements to be recognized were the Activator/Dissociation elements of Zea mays (McClintock,
  • Transposable elements in the genome are characterized by being flanked by direct repeats of a short sequence of DNA that has been duplicated during transposition and is called a target site duplication. Virtually all transposable elements whatever their type, and mechanism of transposition, make such duplications at the site of their insertion. In some cases the number of bases duplicated is constant, in other cases it may vary with each transposition event. Most transposable elements have inverted repeat sequences at their termini. These terminal inverted repeats may be anything from a few bases to a few hundred bases long and in many cases they are known to be necessary for transposition. Prokaryotic transposable elements have been most studied in E. coli and Gram negative bacteria, but also are present in Gram positive bacteria.
  • transposons are generally termed insertion sequences if they are less than about 2 kb long, or transposons if they are longer.
  • Bacteriophages such as mu and D 108, which replicate by transposition, make up a third type of transposable element. Elements of each type encode at least one polypeptide a transposase, required for their own transposition.
  • Transposons often further include genes coding for function unrelated to transposition, for example, antibiotic resistance genes.
  • Transposons can be divided into two classes according to their structure. First, compound or composite transposons have copies of an insertion sequence element at each end, usually in an inverted orientation. These transposons require transposases encoded by one of their terminal IS elements. The second class of transposon have terminal repeats of about 30 base pairs and do not contain sequences from IS elements. Transposition usually is either conservative or replicative, although in some cases it can be both. In replicative transposition, one copy of the transposing element remains at the donor site, and another is inserted at the target site. In conservative transposition, the transposing element is excised from one site and inserted at another.
  • Eukaryotic elements also can be classified according to their structure and mechanism of transportation. The primary distinction is between elements that transpose via an RNA intermediate, and elements that transpose directly from DNA to DNA.
  • Retrotransposons Elements that transpose via an RNA intermediate often are referred to as retrotransposons, and their most characteristic feature is that they encode polypeptides that are believed to have reverse transcriptionase activity.
  • retrotransposon There are two types of retrotransposon. Some resemble the integrated proviral DNA of a retrovirus in that they have long direct repeat sequences, long terminal repeats (LTRs), at each end. The similarity between these retrotransposons and proviruses extends to their coding capacity. They contain sequences related to the gag and pol genes of a retrovirus, suggesting that they transpose by a mechanism related to a retroviral life cycle. Retrotransposons of the second type have no terminal repeats.
  • RNA intermediates RNA intermediates
  • transpose by reverse transcription of RNA intermediates, but do so by a mechanism that differs from that or retrovirus-like elements.
  • Transposition by reverse transcription is a replicative process and does not require excision of an element from a donor site.
  • Transposable elements are an important source of spontaneous mutations, and have influenced the ways in which genes and genomes have evolved. They can inactivate genes by inserting within them, and can cause gross chromosomal rearrangements either directly, through the activity of their transposases, or indirectly, as a result of recombination between copies of an element scattered around the genome. Transposable elements that excise often do so imprecisely and may produce alleles coding for altered gene products if the number of bases added or deleted is a multiple of three.
  • Transposable elements themselves may evolve in unusual ways. If they were inherited like other DNA sequences, then copies of an element in one species would be more like copies in closely related species than copies in more distant species. This is not always the case, suggesting that transposable elements are occasionally transmitted horizontally from one species to another.
  • Chemical mutagenesis offers certain advantages, such as the ability to find a full range of mutant alleles with degrees of phenotypic severity, and is facile and inexpensive to perform.
  • the majority of chemical carcinogens produce mutations in DNA.
  • Benzo[a]pyrene, N-acetoxy-2-acetyl amino fluorene and aflotoxin Bl cause GC to TA transversions in bacteria and mammalian cells.
  • Benzo[a]pyrene also can produce base substitutions such as AT to TA.
  • N-nitroso compounds produce GC to AT transitions. Alkylation of the 04 position of thymine induced by exposure to n-nitrosoureas results in TA to CG transitions.
  • a high correlation between mutagenicity and carcinogenity is the underlying assumption behind the Ames test (McCann et ah, 1975) which speedily assays for mutants in a bacterial system, together with an added rat liver homogenate, which contains the microsomal cytochrome P450, to provide the metabolic activation of the mutagens where needed.
  • Ames test McCann et ah, 1975
  • several carcinogens have been found to produce mutation in the ras proto-oncogene.
  • N-nitroso-N-methyl urea induces mammary, prostate and other carcinomas in rats with the majority of the tumors showing a G to A transition at the second position in codon 12 of the Ha-ras oncogene.
  • Benzo[a]pyrene-induced skin tumors contain A to T transformation in the second codon of the Ha-ras gene.
  • Ionizing radiation causes DNA damage and cell killing, generally proportional to the dose rate. Ionizing radiation has been postulated to induce multiple biological effects by direct interaction with DNA, or through the formation of free radical species leading to DNA damage (Hall, 1988). These effects include gene mutations, malignant transformation, and cell killing. Although ionizing radiation has been demonstrated to induce expression of certain DNA repair genes in some prokaryotic and lower eukaryotic cells, little is known about the effects of ionizing radiation on the regulation of mammalian gene expression (Borek, 1985). Several studies have described changes in the pattern of protein synthesis observed after irradiation of mammalian cells.
  • ionizing radiation treatment of human malignant melanoma cells is associated with induction of several unidentified proteins (Boothman et al, 1989).
  • Synthesis of cyclin and co-regulated polypeptides is suppressed by ionizing radiation in rat REF52 cells, but not in oncogene-transformed REF52 cell lines (Lambert and Borek, 1988).
  • Other studies have demonstrated that certain growth factors or cytokines may be involved in x-ray-induced DNA damage.
  • platelet-derived growth factor is released from endothelial cells after irradiation (Witte, et al, 1989).
  • the term "ionizing radiation” means radiation comprising particles or photons that have sufficient energy or can produce sufficient energy via nuclear interactions to produce ionization (gain or loss of electrons).
  • An exemplary and preferred ionizing radiation is an x-radiation.
  • the amount of ionizing radiation needed in a given cell generally depends upon the nature of that cell.
  • an effective expression-inducing dose is less than a dose of ionizing radiation that causes cell damage or death directly.
  • Means for determining an effective amount of radiation are well known in the art.
  • an effective expressioninducing amount is from about 2 to about 30 Gray (Gy) administered at a rate of from about 0.5 to about 2 Gy/minute.
  • an effective expression inducing amount of ionizing radiation is from about 5 to about 15 Gy. In other embodiments, doses of 2-9 Gy are used in single doses. An effective dose of ionizing radiation may be from 10 to 100 Gy, with 15 to 75 Gy being preferred, and 20 to 50 Gy being more preferred.
  • radiation may be delivered by first providing a radiolabeled antibody that immunoreacts with an antigen of the tumor, followed by delivering an effective amount of the radiolabeled antibody to the tumor.
  • radioisotopes may be used to deliver ionizing radiation to a tissue or cell.
  • Random mutagenesis also may be introduced using error prone PCR (Cadwell and Joyce, 1992). The rate of mutagenesis may be increased by performing PCR in multiple tubes with dilutions of templates.
  • One particularly useful mutagenesis technique is alanine scanning mutagenesis in which a number of residues are substituted individually with the amino acid alanine so that the effects of losing side-chain interactions can be determined, while minimizing the risk of large-scale perturbations in protein conformation (Cunningham et al, 1989).
  • techniques for estimating the equilibrium constant for ligand binding using minuscule amounts of protein have been developed (Blackburn et al. , 1991; U.S.
  • Patents 5,221,605 and 5,238,808) The ability to perform functional assays with small amounts of material can be exploited to develop highly efficient, in vitro methodologies for the saturation mutagenesis of antibodies.
  • the inventors bypassed cloning steps by combining PCR mutagenesis with coupled in vitro transcription/translation for the high throughput generation of protein mutants.
  • the PCR products are used directly as the template for the in vitro transcription/translation of the mutant single chain antibodies. Because of the high efficiency with which all 19 amino acid substitutions can be generated and analyzed in this way, it is now possible to perform saturation mutagenesis on numerous residues of interest, a process that can be described as in vitro scanning saturation mutagenesis (Burks et al., 1997).
  • In vitro scanning saturation mutagenesis provides a rapid method for obtaining a large amount of structure-function information including: (i) identification of residues that modulate ligand binding specificity, (ii) a better understanding of ligand binding based on the identification of those amino acids that retain activity and those that abolish activity at a given location, (iii) an evaluation of the overall plasticity of an active site or protein subdomain, (iv) identification of amino acid substitutions that result in increased binding.
  • a method for generating libraries of displayed polypeptides is described in U.S. Patent 5,380,721.
  • the method comprises obtaining polynucleotide library members, pooling and fragmenting the polynucleotides, and reforming fragments therefrom, performing PCR amplification, thereby homologously recombining the fragments to form a shuffled pool of recombined polynucleotides.
  • Site-specific mutagenesis represents a powerful tool for the dissection and engineering of protein- ligand interactions (Wells, 1996; Braisted et al., 1996).
  • the technique provides for the preparattion and testing of sequence variants by introducing one or more nucleotide sequence changes into a selected DNA.
  • Site-specific mutagenesis uses specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent, unmodified nucleotides. In this way, a primer sequence is provided with sufficient size and complexity to form a stable duplex on both sides of the deletion junction being traversed.
  • a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
  • the technique typically employs a bacteriophage vector that exists in both a single-stranded and double-stranded form.
  • Vectors useful in site-directed mutagenesis include vectors such as the M 13 phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art.
  • Double- stranded plasmids are also routinely employed in site-directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid.
  • An oligonucleotide primer bearing the desired mutated sequence, synthetically prepared, is then annealed with the single-stranded DNA preparation, taking into account the degree of mismatch when selecting hybridization conditions.
  • the hybridized product is subjected to DNA polymerizing enzymes such as E. coli polymerase I (Klenow fragment) in order to complete the synthesis of the mutation-bearing strand.
  • E. coli polymerase I Klenow fragment
  • codon bias Most of amino acids are encoded by more than one codon. For a given amino acid, different species may favor different codons, which creates possible codon bias.
  • CAI codon adaption index
  • Retroviral vectors are some of the most commonly pseudotyped viruses.
  • Murine leukemia virus (MLV) a retrovirus that is able to infect many different cell types, can be manipulated to infect a wide range of cells by pseudotyping.
  • Pseudotyped retroviruses can be engineered in the laboratory using packaging cell lines that produce gag and pol proteins from one virus and env proteins from a second virus.
  • non-targeted, pseudotyped retroviruses based upon MLV and carrying the envelope protein of highly promiscuous vesicular stomatis virus (VSV) have been produced (Yee et. al, 1994).
  • These vectors give titers higher than 10 9 (cf 10 6 for MLV based RVs) and are more stable, facilitating their concentration.
  • These MLV/VSV pseudotyped RVs show a very wide infection spectrum and are able to infect even fish cells.
  • GaLV SEATO-MOMULV hybrid particles were generated at titers approximately equivalent to those obtained with the MoMuLV particles, and the infection spectrum correlates exactly with the previously reported in vitro host range of wild-type GaLV SEATO, i.e., bat, mink, bovine and human cells.
  • the apparent titers of HTLV-I MoMuLV (1-10 CFU/ml) were substantially lower than the titers achieved with either the MoMuLV or GaLV- MoMuLV recombinant virions.
  • the HTLV-I hybrid virions were able to infect human and mink cells (Wilson et al, 1989).
  • the inventors will introduce putative vaccine envelope sequences into expression vectors that will be introduced into host cells and sequently infected with viral vectors lacking envelope sequences.
  • the pseudotyped particles will contain the vaccine envelope sequence(s) to be tested by neutralization assays, as described below.
  • the virus population will be mixed, i.e., it will contain a large number of distinct virus envelope sequences.
  • the population will be homogenous, i.e., it will contain a single envelope sequence, thereby permitting more precise assessment of the degree of virus neutralization.
  • viruses and hence virus envelope sequences
  • the antibodies may come from a variety of sources, including natural (serum) and synthetic (polyclonal immune sera; monoclonal antibodies) sources.
  • the antibody preparation will be diluted to appropriate concentrations and, alternatively, multiple concentrations of antibody may be used in replicate experiments.
  • the antibody preparation is then mixed with the pseudotyped viral particles and incubated for an appropriate period of time. Virus infectivity is then assessed by plating of the neutralized virus onto permissive cells, followed by incubation under conditions permitting uptake and replication of the virus in the cells.
  • Assessing of virus replication can be undertaken by several different means.
  • cells may be solubilized and Western blot or radioimmune precipitation may be performed.
  • virus replication can be assessed by incorporated of a radiolabel, such as tritiated thymidine, into the produced genomes and assessing by scintillation counting.
  • plaque assays may be performed where virus is plated onto confluent cell monolayers followed by observations of areas of cell death, called "plaques.”
  • HCV E1E2 sesquences were retrieved from the GenBank (www.ncbi.nlm.nih.gov/Genbank/index.html) and Los Alamos HCV database (hcv.lanl.gov) (Kuiken et al, 2005). Each sequence was manually examined to determine its genotype and exact length. Sequences were edited and aligned with Clustal W (Higgins and Sharp, 1988), BioEdit
  • the final data set consists of 119 full-length HCV E1E2 sequences, designated number 1 to 119, respectively matching with their GenBank accession numbers AFOl 1753, AF271632, AF290978, AF511948, AF511949, AF511950, AF529293, AJ278830, AJ557444, AY388455, AY615798, AY695436, AY695437, AY885238, AY958064, D10749, DQ061303, DQ061307, DQ061312, DQ061318, DQ061322, DQ061326, DQ061327, M62321, AB049087, AB049090, AB049093, AB049096, AB154186, AB154188, AB154192, AB154194, AB154198, AB154200, AB154202, AB154206, AF165056, AF176573, AF207758,
  • Model selection The inventor then evaluated the most appreciate nucleotide substitution model for these 119 HCV sequences. This was done by using hierarchical likelihood ratio tests (hLRTs) that were simulated with the program Modeltest for total of 56 evolutionary models (Goldman, 1993; Posada and Crandall, 1998; Posada and Crandall, 2001).
  • GTR General time Reversible model
  • I invariable sites
  • G gamma distribution shape parameter
  • codon usages of HCVAl and HCVA2 were optimized based on mammal species with program JCat (Grote et al., 2005) that implanted an algorithm for the calculation of the codon adaption index (CAI), a prevailing empirical measure of expressivity (Sharp and Li, 1987).
  • CAI codon adaption index
  • the nucleotide sequences after codon optimization are designated as HCVAI l for HCVAl and HCVA22 for HCVA2, respectively. All sequences are shown below.
  • Ruhl et al Cell, 50:1057, 1987. Ruiken et al, Bioinformatics, 21(3):379-384, 2005. Rumar et al, Bioinformatics, 17:1244-1245, 2001. Kunz et al., Nucl. Acids Res., 17:1121, 1989.

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Abstract

La présente invention concerne des procédés pour identifier les antigènes de vaccin multivalents dérivés de protéines d'enveloppe.
PCT/US2007/088910 2006-12-28 2007-12-27 Identification des épitopes de protéines d'enveloppe virale multivalents WO2008083203A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030185806A1 (en) * 1995-04-24 2003-10-02 John Wayne Cancer Institute Pluripotent vaccine against enveloped viruses
US6984488B1 (en) * 1996-11-07 2006-01-10 Ramot At Tel-Aviv University Ltd. Determination and control of bimolecular interactions

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030185806A1 (en) * 1995-04-24 2003-10-02 John Wayne Cancer Institute Pluripotent vaccine against enveloped viruses
US6984488B1 (en) * 1996-11-07 2006-01-10 Ramot At Tel-Aviv University Ltd. Determination and control of bimolecular interactions

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DORIA-ROSE N.A. ET AL.: "Human Immunodeficiency Virus Type 1 subtype B Ancestral Envelope Protein Is Functional and Elicits Neutralizing Antibodies in Rabbits Similar to Those Elicited by a Circulating Subtype B Envelope", J. OF VIROLOGY, vol. 79, no. 17, September 2005 (2005-09-01), pages 11214 - 11224, XP008112620 *
FLOREA L. ET AL.: "Epitope prediction algorithms peptide-based vaccine design", PROC. IEEE COMPUT. SOC. BIOINFORM. CONF., vol. 2, 2003, pages 17 - 26, XP008112593 *

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