JP2002507393A - Antigen library immunization - Google Patents

Abstract

This invention relates to antigen library immunization which provides a method for obtaining antigens with improved properties for therapeutic and other uses. This method is useful for obtaining improved antigens that can induce an immune response against antigens that are effective in regulating pathogens, cancer and other conditions, and allergies, inflammatory diseases and autoimmune diseases.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to the field of methods for developing immunogens that can induce efficient immune responses to a wide range of antigens.

BACKGROUND Interactions between pathogens and hosts are the result of millions of years of evolution, during which,
The mammalian immune system has evolved sophisticated means of fighting pathogen infestations. However, bacterial and viral pathogens have at the same time gained many mechanisms to improve their virulence and survival in the host, and despite the modern technology of molecular and cell biology, vaccine research and development Offers a major challenge to. Similar to the evolution of pathogen antigens, some cancer antigens appear to have gained a means to down-regulate their immunogenicity as a mechanism to evade the host immune system.

[0003] Efficient vaccine development is also hampered by the antigenic diversity of different strains of pathogens, driven in part by evolutionary forces as a means for pathogens to evade immune defenses. Pathogens can also be expressed, processed, and / or
Alternatively, they reduce their immunogenicity by selecting antigens that are difficult to transport, thereby reducing the availability of immunogenic peptides to molecules that initiate and regulate the immune response. The mechanisms associated with these challenges are complex, polymorphic, and rather poorly characterized. Thus, there is a need for a vaccine that can elicit a protective immune response against bacterial and viral pathogens. The present invention fulfills this and other needs.

SUMMARY OF THE INVENTION The present invention provides a first antigenic determinant from a polypeptide associated with a first disease, and at least one second antigenic determinant from a polypeptide associated with a second disease. A recombinant multivalent antigenic polypeptide comprising a group is provided. Polypeptides associated with this disease include cancer antigens, antigens associated with autoimmune disorders, antigens associated with inflammatory conditions, antigens associated with allergic reactions, antigens associated with infectious agents, and other antigens associated with disease states. May be selected from the group consisting of antigens.

[0005] In another embodiment, the present invention provides a recombinant antigen library comprising a recombinant nucleic acid encoding an antigenic polypeptide. The library is obtained by recombining at least a first form and a second form of a nucleic acid, typically comprising a polynucleotide sequence encoding an antigenic polypeptide associated with a disease, wherein the first form comprises The form and the second form differ from each other by two or more nucleotides, resulting in a library of recombinant nucleic acids.

[0006] Another embodiment of the invention provides a method of obtaining a polynucleotide encoding a recombinant antigen with improved ability to induce an immune response to a disease state. These methods include the following steps: (1) recombining at least the first and second forms of a nucleic acid comprising a polynucleotide sequence encoding an antigenic polypeptide associated with the disease state. Wherein the first and second forms differ from each other by two or more nucleotides, resulting in a library of recombinant nucleic acids; and (2) screening the library to immunize against the disease state. Identifying at least one optimized recombinant nucleic acid encoding an optimized recombinant antigenic polypeptide having improved ability to induce a response.

[0007] These methods optionally further include: (3) converting the at least one optimized recombinant nucleic acid into a further form of the nucleic acid, which is the same as the first and second forms; Recombination to produce a further library of recombinant nucleic acids; (4) screening this further library and encoding a polypeptide having improved ability to induce an immune response against the disease state; Identifying at least one further optimized recombinant nucleic acid; and (5) optionally, the additional optimized recombinant nucleic acid has improved ability to induce an immune response against the disease state. Repeating (3) and (4) until encoding the polypeptide having.

[0008] In some embodiments, the optimized recombinant nucleic acid encodes a multivalent antigenic polypeptide, and the screening is performed using a phage polypeptide in which the recombinant antigen is displayed on the surface of a phage particle. Expressing a library of recombinant nucleic acids in a phage display expression vector so as to be expressed as a fusion protein with a first antibody that is specific for a first serotype of the virulence factor And selecting those phage that bind to the first antibody; and replacing those phage that bind to the first antibody with a second virulence factor.
Contacting with a second antibody that is specific for the serotype of and selecting those phages that bind to the second antibody; wherein those phages that bind to the first and second antibodies are The phage expresses a multivalent antigenic polypeptide.

[0009] The present invention also provides a method for obtaining a recombinant viral vector having enhanced ability to induce an antiviral response in a cell. These methods may include the following steps: (1) recombining at least the first and second forms of the nucleic acid comprising the viral vector, wherein the first and second forms are performed. Morphologically different from each other by two or more nucleotides, resulting in a library of recombinant viral vectors; (2) transfecting the library of recombinant viral vectors into a population of mammalian cells; (3) Staining the cells for the presence of the Mx protein; and (4)
isolating from cells that stain positive for the x protein, wherein the recombinant viral vector from the positively stained cells exhibits enhanced ability to induce an antiviral response.

DETAILED DESCRIPTION Definitions The term “screening” generally describes the process of identifying optimal antigens. Some properties of the antigen can be used in selection and screening, including antigen expression, folding, stability, immunogenicity, and the presence of epitopes from several related antigens. Selection is, in some genetic contexts, identified and physically performed by expression of the selectable marker, which allows cells expressing the marker to survive while other cells die (or vice versa). It is a form of screening in which separation is achieved simultaneously. Screening markers include, for example, luciferase, β-galactosidase, and green fluorescent protein. Selectable markers include drug resistance genes and toxin resistance genes. Due to limitations in studying the primary immune response in vitro, in vivo studies are a particularly useful screening method. In these studies, the antigen was
The immune response is first introduced into the test animal and subsequently the immune response is studied by analyzing the protective immune response or using lymphoid cells from the immunized animal to study the quality or strength of the immune response induced Be studied by Natural selection can and will occur in the course of natural evolution, but in the present method, the selection is performed by a human.

An “exogenous DNA segment,” “heterologous sequence,” or “heterologous nucleic acid” as used herein is derived from a source that is foreign to the particular host cell, or If they are from the same source, they have been modified from their original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to a particular host cell, but has been modified. Heterologous sequence modifications in the applications described herein typically occur through the use of DNA shuffling. Thus, these terms refer to a DNA segment that is foreign or heterologous to the cell, or is homologous to the cell, but in a location within the host cell nucleic acid where the element is not normally found. An exogenous DNA segment is expressed to yield an exogenous polypeptide.

The term “gene” is widely used to refer to any segment of DNA that is associated with a biological function. Thus, a gene comprises a coding sequence and / or regulatory sequences required for expression of the coding sequence. Genes also include, for example, non-expressed DNA segments that form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesis from known or predicted sequence information, and can include sequences designed to have the desired variables.

The term “isolated” when applied to a nucleic acid or protein indicates that the nucleic acid or protein is essentially free of other cellular components that are not related in its natural state. It can be in either a dry or aqueous solution, but is preferably in a homogeneous state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The protein that is the predominant species present in the preparation is substantially purified. In particular, an isolated gene is separated from open reading frames that flank the gene and encode proteins other than the gene of interest. The term "purified" indicates that the nucleic acid or protein gives rise to essentially one band in the electrophoretic gel. In particular, it means that the nucleic acid or protein is at least about 50% pure, more preferably at least about 85% pure, and most preferably at least about 99% pure.

[0014] The term "naturally occurring" is used to describe a substrate that can be found in nature, which is different from an artificially produced one by man. For example, a polymorph present in an organism (including a virus, bacterium, protozoan, insect, plant, or mammalian tissue) that can be isolated from a natural source and that has not been intentionally modified by a human in the laboratory. The peptide or polynucleotide sequence is naturally occurring.

[0015] The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof, in either single-stranded or double-stranded form. Unless specifically limited, the term includes nucleic acids that have similar binding properties as a reference nucleic acid, and include known analogs of naturally occurring nucleotides that are metabolized in a manner similar to the naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes conservatively modified variants thereof (eg, degenerate codon substitutions) and complementary sequences, as well as those sequences explicitly indicated. In particular, degenerate codon substitutions are achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced with mixed base and / or deoxyinosine residues. (B
atzer et al. (1991) Nucleic Acid Res. 19: 5081
Ohtsuka et al., (1985) J .; Biol. Chem. 260: 2605
2608; Cassol et al. (1992); Rossolini et al. (1994).
Mol. Cell. Probes 8: 91-98). The term nucleic acid refers to a gene, c
Used interchangeably with DNA and mRNA encoded by the gene.

“Nucleic acid derived from a gene” refers to a nucleic acid in which a gene for its synthesis or a partial sequence thereof finally acts as a template. Therefore, mRNA
, CDNA reverse transcribed from mRNA, RNA transcribed from cDNA, c
DNA amplified from DNA, RNA transcribed from the amplified DNA, etc., are all derived from the gene, and the detection of such derived products is dependent on the detection of the original gene and / or gene transcript in the sample. Indicates presence and / or abundance.

A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it increases transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. I do. However, enhancers generally function when separated from the promoter by a few kilobases, and because the intron sequences can be of variable length, some polynucleotide elements are operably linked but not contiguous. It may be an untargeted state.

A specific binding affinity between two molecules (eg, a ligand and a receptor) means a preferential binding of one molecule to another in a mixture of molecules. If the binding affinity is from about 1 × 10 ⁴ M ⁻¹ to about 1 × 10 ⁶ M ⁻¹ or more, the binding of the molecule can be considered specific.

The term “recombinant” when used in reference to a cell indicates that the cell replicates the heterologous nucleic acid or expresses a peptide or protein encoded by the heterologous nucleic acid. Recombinant cells can contain genes that are not found in the cell in its native (non-recombinant) form. A recombinant cell can also contain a gene found in the cell in its native form, wherein the gene is modified by artificial means and reintroduced into the cell. The term also includes cells that contain nucleic acid endogenous to the cell that has been modified without removing the nucleic acid from the cell; such modifications may be obtained by gene replacement, site-specific mutation, and related techniques. Modifications included.

A “recombinant expression cassette” or simply “expression cassette” is a nucleic acid element capable of effecting the expression of a structural gene in a host compatible with such sequences, and is a nucleic acid construct produced by recombination or synthesis. . The expression cassette contains at least a promoter, and optionally a transcription termination signal. Typically, a recombinant expression cassette includes a nucleic acid to be transcribed (eg, a nucleic acid encoding a desired polypeptide), and a promoter. Additional factors necessary or useful in effecting expression may also be used as described herein. For example, an expression cassette can also include a nucleotide sequence that encodes a signal sequence that directs secretion of the expressed protein from a host cell. Transcription termination signals, enhancers, and other nucleic acid sequences that affect gene expression, can also be included in the expression cassette.

“Polyvalent antigenic polypeptide” or “recombinant polyvalent antigenic polypeptide” comprises 1
A non-naturally occurring polypeptide that includes an amino acid sequence from more than one source polypeptide, which is typically a naturally occurring polypeptide. At least some of the regions of different amino acid sequence constitute epitopes recognized by antibodies found in the mammal injected with the source polypeptide. Source polypeptides from which different epitopes are derived are usually homologous (ie, having the same or similar structure and / or function), and often different isolates, serotypes, strains, From a species or, for example, a different disease state.

The term “identity” or% “identity” as used in the context of two or more nucleic acid or polypeptide sequences, as determined using one of the following sequence comparison algorithms or by visual inspection Two or more sequences or subsequences having the same or specific percentage of amino acid residues or nucleotides when compared and aligned for maximum correspondence.

The phrase “substantially identical” compares in the context of two nucleic acids or polypeptides for the greatest match using one of the following sequence comparison algorithms or as determined by visual inspection: And two or more sequences or subsequences that, when aligned, have at least 60%, preferably 80%, most preferably 90-95% nucleotide or amino acid residue identity. Preferably, the substantial identity exists over a region of the sequence that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequence is substantially over at least about 150 residues. Is the same as In some embodiments, the sequences are substantially identical over the entire length of the coding region.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. If a sequence comparison algorithm is used, the test and reference sequences are entered into a computer, subsequence coordinates are specified, and if necessary, sequence algorithm program variables are specified. Then, the sequence comparison algorithm:
Based on the designated program variables, calculate the% sequence identity for the test sequence compared to the reference sequence.

Optimal alignment of sequences for comparison is described in Smith & Waterm.
an, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. et al. Mol. Bi
ol. 48: 443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sc
i. USA 85: 2444 (1988), performed a computerized implementation of these algorithms (GAP, BESTFIT,
FASTA and TFASTA, Wisconsin Genetics S
software Package, Genetics Computer Gr
up, 575 Science Dr. , Madison, WI)
Or by visual inspection (generally Ausubel et al., See below).

One of the algorithms that is suitable for determining% sequence identity and sequence similarity
One example is the BLAST algorithm, which is described in Altschul et al.
Mol. Biol. 215: 403-410 (1990).
Software for performing BLAST analysis is available from National Cent
er for Biotechnology Information (http
p: // www. ncbi. nlm. nih. It is publicly available via gov /). This algorithm first matches or satisfies some net threshold score T when aligned with words of the same length in the sequence of the database. And identifying high-scoring sequence pairs (HSPs) by identifying short words of length W. T is
It is called the adjacent word score threshold (Altschul et al., Supra). These first adjacent word hits serve as the basis for initiating a search to find longer HSPs containing them. The word hits are then extended in both directions along each sequence, as long as the cumulative alignment score can be increased. Cumulative scores are calculated using the variable M (reward score for matched base pairs; always> 0 for nucleotide sequences).
), And N (penalty score for mismatched residues; always <0). For the amino acid sequence, a cumulative score is calculated using a scoring matrix. Word hit growth in each direction is stopped if: the cumulative alignment score falls off its maximum attainment by an amount X; due to the accumulation of one or more negative scoring residue alignments. And the cumulative score falls below zero; or when the end of any sequence is reached. BLA
The ST algorithm variables W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, and M = 5.
, N = 4, and a comparison of both strands is used. For amino acid sequences, BLAS
As a default, the TP program has a word length (W) of 3, an expected value (E) of 10,
And using the BLOSUM62 scoring matrix (Henikoff
& Henikoff (1989) Proc. Nat'l. Acad. Sci
. ScL USA 89: 10915).

In addition to calculating% sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (eg, Karlin & Al)
tschul (1993) Proc. Nat'l. Acad. Sci. USA
90: 5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (smallest sum pr).
obability (P (N)), which provides an index of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, if the minimum total probability in comparison of a test nucleic acid to a reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001, the nucleic acid is compared to the reference sequence. It is considered similar.

Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase “specifically hybridizes to” refers to the binding of a molecule to only a particular nucleotide sequence under stringent conditions when the sequence is present in complex mixtures (eg, total cell) DNA or RNA. , Double strand formation, or hybridization. "Substantially bind" refers to complementary hybridization between a probe nucleic acid and a target nucleic acid, and by reducing the stringency of the hybridization medium to achieve the desired detection of the target polynucleotide sequence. Includes minor mismatches that can be achieved.

“Stringent hybridization conditions” and “stringent hybridization wash conditions” are sequence-dependent and may be different in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations. Different under variables. Longer sequences hybridize specifically at higher temperatures. An extensive guide to nucleic acid hybridization can be found in Tijssen (1993) Laborat.
ory Techniques in Biochemistry and M
olecular Biology--Hybridization with
Nucleic Acid Probes Part 1, Chapter 2, "Overview
of principals of hybridization and t
he strategy of nucleic acid probe as
says ", Elsevier, New York. Generally, highly stringent hybridization and washing conditions are selected to be about 5 ° C. below the melting point temperature (T _m ) for a particular sequence at a given ionic strength and pH. Typically, under "stringent conditions", a probe will hybridize to its target subsequence, but not to other sequences.

T_mIs the temperature (under a given ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Extremely stringent conditions
Is the T for a particular probe_mIs chosen to be equal to More than 100 complementary on filters in Southern blots or Northern blots
Stringency for the hybridization of complementary nucleic acids with residues
An example of a suitable hybridization condition is 50% form containing 1 mg of heparin.
Amide, 42 ° C., hybridization performed overnight. Highly strike
An example of a gently washing condition is 0.15 M NaCl at 72 ° C. for about 15 minutes.
It is. An example of stringent washing conditions is 0.2 × SSC washing at 65 ° C.
15 minutes (see Sambrook (below) for a description of SSC buffer).
See). Frequently, the background should be high prior to high stringency washes.
Perform low stringency washes to remove probe signals. For example
Moderate stringency wash for duplexes of more than 100 nucleotides
An example of purification is 1 × SSC at 45 ° C. for 15 minutes. For example, more than 100
Examples of low stringency washes for nucleotide duplexes include 4-6 × SSC
At 40 ° C. for 15 minutes. Short probes (e.g., about 10 to 50 nucleotides)
For tide), stringent conditions typically involve less than about 1.0 M Na. ⁺ The salt concentration of the ion, typically about 0.01-1.0 M Na at pH 7.0-8.3.⁺It contains an ionic concentration (or other salt) and the temperature is typically at least about 30 ° C. Stringent conditions can also reduce
This can be achieved with the addition of a stabilizing agent. Generally, certain hybridization assays
Twice that observed for unrelated probes at
A) signal-to-noise ratio indicates the detection of a particular hybridization. Strike
Nucleic acids that do not hybridize to each other under conditions of
If the polypeptides are substantially identical, they are still substantially identical. this thing
Use, for example, the maximum codon degeneracy that a copy of the nucleic acid allows in the genetic code.
Occurs when it is generated.

[0031] A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically identical to the polypeptide encoded by the second nucleic acid. Cross-reactive or specifically binding to it. Thus, for example, where two peptides differ only by conservative substitutions, the polypeptide is typically substantially identical to the second polypeptide.

The phrase “specifically (or selectively) binds to an antibody” or “specifically (or selectively) immunoreactive with” when referring to a protein or peptide,
A binding reaction that is a determinant of the presence of a protein or an epitope derived from the protein in the presence of a heterogeneous population of proteins and other biologics. Thus, under designed immunoassay conditions, certain antibodies bind to certain proteins and do not bind in significant amounts to other proteins present in the sample. Antibodies raised against a multivalent antigenic polypeptide generally bind to the protein from which one or more epitopes were obtained. Specific binding to an antibody under such conditions may require an antibody that is selected for specificity for a particular protein. A variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays, Western blots, or immunohistochemistry are routinely used to select monoclonal antibodies specifically immunoreactive with the protein. For a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity, see Harlow and Lane (1988) Antibodies, A La.
laboratory Manual, Cold Spring Harbor P
publications, New York "Harlow and Lane
"checking. Typically, a specific or selective reaction is at least twice background signal or noise, and more typically between 10 and 10
The background is greater than 0 times.

A “conservatively modified variant” of a particular polynucleotide sequence is a polynucleotide that encodes the same or essentially the same amino acid sequence, or if the polynucleotide does not encode an amino acid sequence, essentially Refers to the same sequence. Due to the degeneracy of the genetic code, the majority of functionally identical nucleic acids encode any given polypeptide. For example, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variants are "silent variants", which are one of the "conservatively modified variants". Every polynucleotide sequence described herein that encodes a polypeptide also describes every possible silent variation, except where otherwise noted. One skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine) is:
It can be modified to produce a functionally identical molecule by standard techniques. Accordingly, each "silent variant" of a nucleic acid which encodes a polypeptide is included in each described sequence.

In addition, one skilled in the art will recognize that individual amino acids that modify, add, or delete a single amino acid or a low percentage of amino acids (typically less than 5%, more typically less than 1%) in a coding sequence. It is understood that a substitution, deletion, or addition is a "conservatively modified variant" if the alteration results in the replacement of the amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Each of the following five groups contains amino acids that are conservative substitutions for one another: Aliphatics: Glycine (G), Alanine (A), Valine (V), Leucine (L),
Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine (R), Lysine (K), Histidine (H); acidic: aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q). For further groupings of amino acids, see Creighton (1984).
Proteins, W.C. H. See also Freeman and Company. In addition, individual substitutions, deletions, or additions that modify, add, or delete a single amino acid or a low percentage of amino acids in a coding sequence are also "conservatively modified variants."

“Subsequence” refers to a nucleic acid or amino acid sequence that includes a portion of a longer sequence of nucleic acids or amino acids (eg, a polypeptide), respectively.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention provides a new approach to vaccine development called "antigen library immunization." No other techniques are available for generating libraries of related antigens, nor for optimizing known protective antigens. Only the most powerful existing methods for identification of vaccine antigens, such as high-throughput sequencing or expression library immunization, test the sequence space provided by the pathogen genome. These approaches are inadequate because they generally only target a single pathogen strain, and because natural evolution directs pathogens to down-regulate their own immunogenicity It seems. In contrast, the immunization protocols of the present invention using shuffled antigen libraries provide a means for identifying novel antigen sequences. The most protective antigens can be selected from these pools by an in vivo challenge model. Antigen library immunization dramatically expands the diversity of available immunogenic sequences, and thus these antigen chimera libraries also provide a means of protection against emerging emerging future pathogen variants . The method of the present invention provides efficient protection against a variety of existing pathogens and allows for the identification of individual chimeric antigens that provide improved vaccines for military and civilian populations.

The method of the present invention provides an evolutionary approach, such as DNA shuffling, specifically an approach that is the optimal approach to improve the immunogenicity of many types of antigens. For example, the method provides a means to obtain optimized cancer antigens useful for preventing and treating malignancies. In addition, an increasing number of self-antigens (causing autoimmune diseases) and allergens (atopy, allergies,
And cause asthma). The immunogenicity and production of these antigens can likewise be improved with respect to the method of the invention.

The antigen library immunization method of the present invention provides a means by which recombinant antigens with improved ability to induce an immune response to a virulence factor can be obtained. "Pathogenic factor"
Refers to an organism or virus that can infect host cells. A virulence factor typically includes and / or encodes a molecule (usually a polypeptide) that is immunogenic in that an immune response is elicited against the immunogenic polypeptide. Frequently, the immune response elicited against an immunogenic polypeptide from one serotype of the virulence factor is incapable of recognizing different serotype virulence factors or other related virulence factors, Therefore we cannot defend against it. In other situations, the polypeptides produced by the virulence factor are not produced in sufficient amounts to cause an infected cell to elicit an effective immune response to the virulence factor, or are not sufficiently immunogenic.

These problems typically involve the recombination of two or more forms of a nucleic acid encoding a polypeptide of a virulence factor or antigen involved in another disease or condition. Solved by These recombination methods, referred to herein as "DNA shuffling," use different forms of nucleic acid at two or more nucleotides as substrates, resulting in a library of recombinant nucleic acids.
The library is then screened and at least one optimized recombinant nucleic acid encoding an optimized recombinant antigen with improved ability to induce an immune response against the virulence factor or other condition is obtained. Identify. The resulting recombinant antigens are frequently chimeric in that they are recognized by antibodies (Abs) reactive against multiple pathogen strains and can generally elicit a broad spectrum of immune responses. Although additional mechanisms appear to be involved, such as cytotoxic T lymphocytes, specific neutralizing antibodies are known to mediate protection against some pathogens of interest.
The concept of inducing the chimeric multivalent antigen to elicit a broad antibody response is further illustrated in FIG.

In a preferred embodiment, the different forms of the nucleic acid encoding the antigenic polypeptide are obtained from members of the family of related virulence factors. This scheme for performing DNA shuffling using nucleic acids from related organisms known as "family shuffling" is described in Crameri et al. ((1988) Nature
e 391: 288-291) and is shown schematically in FIG. Polypeptides of different strains and serotypes of the pathogen generally vary by 60-98%, which allows for efficient family DNA shuffling. Thus, family DNA shuffling provides an effective approach for generating multivalent cross-protective antigens. This method is useful for obtaining individual chimeras that effectively protect against most or all pathogen variants (FIG. 3A).
). In addition, immunization using the entire library or pool of shuffled antigen chimeras also protects against pathogen variants that were not included in the starting population of antigens (eg, chimeras / strains B and A in FIG. 3B). A shuffled library of mutants can result in the identification of a chimeric antigen that protects against the C strain. Thus, this antigen library immunization approach allows the development of immunogenic polypeptides that can elicit an immune response against newly characterized pathogen variants that are less well characterized.

Sequence recombination can be achieved in many different formats and modifications of formats, as described in further detail below. These forms share some general principles. For example, the target for modification will change in different applications where the properties sought to be obtained or improved will change. Examples of candidate targets for gaining or improving properties include a gene that encodes a protein that has immunogenic and / or toxicogenic activity when introduced into a host organism.

The method uses at least two variant forms of the starting target. Variant forms of the candidate substrate may exhibit substantial sequence or secondary structural similarity to each other,
They should also differ in at least one position, and preferably at least two positions. The initial diversity between forms may be the result of natural modifications, for example, different variant forms (homologs) may be obtained from different individuals or strains of an organism, or may have related sequences from the same organism (eg, Allelic variants) or homologs (interspecies variants) from different organisms. Alternatively, initial diversity can be induced. For example, the variant form may be an error-prone transcription of the first variant form (PCR that is prone to error, or the use of a polymerase that lacks proof-reading activity (Liao (1990) Gene 8).
8: 107-111) or by replication of a first form in a mutant strain (mutator host cells are discussed in further detail below and are generally well known). The mutator strain may contain any mutation in any organism that has impaired mismatch repair function. These are mut
S, mutT, mutH, mutL, ovrD, dcm, vsr, umuC, u
Mutant gene products such as muD, sbcB and recJ are included. Impairment can be achieved by genetic mutation, allelic replacement, selective inhibition by added reagents (eg, small compounds or expressed antisense RNA), or other techniques. The impairment can be an impairment of the described gene or of a homologous gene in any organism. Other methods of generating initial diversity include methods well known to those of skill in the art through spontaneous mutagenesis (including, for example, treatment of nucleic acids with chimeric or other mutagens) and errors in cells containing nucleic acids. Repair systems that tend to include (eg, SO
S). The initial diversity between the substrates is generally greatly enhanced in subsequent recombination steps for library generation.

Properties Involved in Immunogenicity The effectiveness of an antigen in inducing an immune response against a pathogen can depend on several factors, many of which are not well understood. The earliest available methods for increasing the effectiveness of antigens are the molecular basis for these factors (molecu
lar basis). However, DNA shuffling and antigen library immunization according to the methods of the present invention are effective even when the molecular basis is not known. The method of the present invention does not rely on a priori assumptions.

[0044] Polynucleotide sequences that can positively or negatively affect the immunogenicity of the antigen encoded by the polynucleotide are frequently scattered throughout the entire antigen gene. Some of these factors are shown schematically in FIG. By recombining different forms of the polynucleotide encoding the antigen using DNA shuffling, and subsequently selecting for a chimeric polynucleotide encoding the antigen that can elicit an improved immune response, a positive response to the immunogenicity of the antigen is obtained. A primary sequence having the effect of Sequences that negatively affect the immunogenicity of the antigen are removed (FIG. 4). It is not required to know the specific sequence involved.

(DNA Shuffling Method) In general, the method of the present invention comprises performing DNA recombination (“shuffling”),
And screening or selection to "evolve" individual genes, whole plasmids or viruses, multigene clusters, or even whole genomes (S
temmer (1995) Bio / Technology 13: 549-55.
3). Repeat cycles of recombination and screening / selection can be performed to further evolve the nucleic acid of interest. Such techniques do not require the extensive analysis and calculations required by conventional methods for polypeptide manipulation. Shuffling allows for the recombination of the majority of mutations with a minimal number of selection cycles, as opposed to natural pairs of recombination events (eg, those that occur during sexual replication). To Thus, the sequence recombination techniques described herein provide a way in which they can provide recombination between mutations in any or all of these, whereby different combinations of mutations can produce the desired result. It offers certain advantages in that it provides a very quick way to test. However, in some instances, structural and / or functional information is available that is not required for sequence recombination, but provides an opportunity for technology modification.

The DNA shuffling methods of the present invention can include at least one of at least four different approaches to improving immunogenic activity and broadening specificity. First, DNA shuffling can be performed on a single gene. Second, some highly homologous genes can be identified by sequence comparison with known homologous genes. These genes can be synthesized and shuffled as a family of homologs to select for recombinants with the desired activity.
The shuffled gene is E. coli. The gene can be cloned into a suitable host cell, such as E. coli, yeast, plants, fungi, animal cells, etc., and the gene encoding the antigen with the desired properties can be identified by the methods described below. Third, whole genome shuffling can be performed to shuffle genes (along with other genomic nucleic acids) encoding antigenic polypeptides. For a whole-genome shuffling approach, it is not necessary to identify which genes are being shuffled. Instead, the bacterial cell or viral genome is combined and shuffled, for example, to obtain a recombinant polypeptide with enhanced ability to elicit an immune response, as measured in any of the assays described below. . Fourth, the gene encoding the antigenic polypeptide can be a codon that has been modified to assess the diversity of mutations that are not present in any naturally occurring gene. Details regarding each of these procedures can be found below.

Sometimes, DNA shuffling, evolution, or molecular hybridization (molecular)
Exemplary formats and examples for sequence recombination, also referred to as breeding, are described by the present inventors and co-workers in the following co-pending applications: US Patent Application No. 08 / 198,431 (1994). Application number PCT / US95 / 02126 (filed February 17, 1995),
Application No. 08 / 425,684 (filed on April 18, 1995), Application No. 08 /
537,874 (filed on October 30, 1995), application number 08 / 564,95.
5 (filed on November 30, 1995), and the application number 08 / 621,859 (1996)
Application number 08 / 621,430 (filed March 25, 1996), application number PCT / US96 / 05480 (filed April 18, 1996),
Application No. 08 / 650,400 (filed May 20, 1996), Application No. 08 /
675,502 (filed on July 3, 1996), application number 08 / 721,824 (
Application No. PCT / US97 / 17300 (filed Sep. 27, 1996)
Application No. PCT / US97 / 24239 (filed Sep. 26, 1995)
Filed on December 17, 1995); Stemmer, Science 270: 1510
(1995); Stemmer et al., Gene 164: 49-53 (1995).
Stemmer, Bio / Technology 13: 549-553 (1
995); Stemmer, Proc. Natl. Acad. Sci. U. S.
A. 91: 10747-10751 (1994); Stemmer, Natu.
re 370: 389-391 (1994); Crameri et al., Nature.
Medicine 2 (1): 1-3 (1996); Crameri et al., Na
cure Biotechnology 14: 315-319 (1996),
Each of which is incorporated by reference in its entirety for all purposes.

Other methods for obtaining recombinant polynucleotides and / or obtaining diversity in nucleic acids used as substrates for shuffling include, for example, homologous recombination (PCT / US98 / 05223; publication no. WO 98/42727);
Oligonucleotide-directed mutagenesis (for review, see Smith, Ann. Re.
v. Genet. 19: 423-462 (1985); Botstein and Shortle, Science 229: 1193-1201 (1985);
Carter, Biochem. J. 237: 1-7 (1986); Kunke
1, "The efficiency of oligonucleotide
directed mutagenesis "Nucleic acid
& Molecular Biology, Eckstein and Lille
y, Ed., Springer Verlag, Berlin (1987). The following are included in these methods: oligonucleotide-directed mutagenesis (
Zoller and Smith, Nucl. Acad. Res. 10: 6487
-6500 (1982), Methods in Enzymol. 100: 4
68-500 (1983), and Methods in Enzymol. 1
54: 329-350 (1987)), phosphorothioate (phosphothioate).
Hioate) Modified DNA Mutagenesis (Taylor et al., Nucl. Acids)
Res. 13: 8749-8864 (1985); Taylor et al., Nucl.
Acids Res. 13: 8765-8787 (1985); Nakamay
e and Eckstein, Nucl. Acids Res. 14: 9679-
9698 (1986); Sayers et al., Nucl. Acids Res. 16
: 791-802 (1988); Sayers et al., Nucl. Acids Re
s. 16: 803-814 (1988)), mutagenesis using uracil-containing templates (Kunkel, Proc. Nat'l. Acad. Sci. USA 82:
488-492 (1985) and Kunkel et al., Methods in E.
nzymol. 154: 367-382)); Gapped double-stranded DNA
(Kramer et al., Nucl. Acids Res. 12:
9441-9456 (1984); Kramer and Fritz, Metho.
ds in Enzymol. 154: 350-367 (1987); Kram.
er et al, Nucl. Acids Res. 16: 7207 (1988)); and Fritz et al., Nucl. Acids Res. 16: 6987-6999 (
1988)). Further suitable methods include point mismatch repair (Kramer et al.,
Cell 38: 879-887 (1984)), Mutagenesis using a repair-deficient host strain (Carter et al., Nucl. Acids Res. 13: 4431-4).
443 (1985); Carter, Methods in Enzymol.
154: 382-403 (1987)), deletion mutagenesis (Eghtedarza).
deh and Henikoff, Nucl. Acids Res. 14: 511
5 (1986)), restriction selection and restriction purification (Wells et al., Phil. Tra.
ns. R. Soc. London. A317: 415-423 (1986)), Mutagenesis by total gene synthesis (Nambiar et al., Science 223: 129).
9-1301 (1984); Sakamar and Khorana, Nucl.
Acids Res. 14: 6361-6372 (1988); Wells et al.
Gene 34: 315-323 (1985); and Grundstrom.
Et al., Nucl. Acids Res. 13: 3305-3316 (1985).
Kits for mutagenesis are commercially available (eg, Bio-Rad, Amer).
sham International, Anglian Biotechno
logic).

The hybridization procedure generally exhibits some degree of sequence identity to each other (ie, at least about 30%, 50%, 70%, 80%, or 90% sequence identity),
Begin with at least two substrates that differ from each other at a particular site. The difference can be any type of mutation (eg, substitution, insertion, and deletion). Frequently, the different segments differ from each other at about 5-20 positions. For recombination resulting in increased diversity compared to the starting material, the starting materials must differ from each other at at least two nucleotide positions. That is, if only two substitutions are present, they should be present in at least two different positions. For example, if three substitutions are present, one substrate may differ from the second at a single site, and the second may differ from the third at a different single position. The starting DNA segments can be natural variants of each other (eg, allelic variants or species variants). The segments can also be from non-alleles (eg, different genes within a superfamily, such as the family of Yersinia V antigens) that exhibit some structural and usually functional relationships. The starting DNA segments can also be variants that can be derived from each other. For example, one DNA segment can be produced by PCR replication, which tends to contain errors in the other, and the nucleic acid can be processed by chemical or other mutagens, or by replacement of a mutagenesis cassette. Induced mutations can also be prepared by amplifying one (or both) of the segments in the mutagenized strain or by inducing a repair system that tends to be error-prone in the cell. In these situations, strictly speaking, the second
DNA segments are not a single segment but a large family of related segments. The different segments forming the starting material are often the same length or substantially the same length. However, this need not be true; for example, one segment may be another subsequence. A segment can be present as part of a larger molecule, such as a vector, or can be in isolated form.

[0050] The starting DNA segment is recombined by any of the sequence recombination formats provided herein to generate a diverse library of recombinant DNA segments. Such libraries, from less than 10, 10 ^5, 10 ^9, 10 ¹² may vary widely to a size having a more or larger members. In some embodiments, the starting segment and the resulting recombinant library include the full-length coding sequence, and any essential regulatory sequences required for expression (eg, promoter and polyadenylation sequences). . In other embodiments, the recombinant DNA segments in the library can be inserted into a common vector that provides the necessary sequences for expression before performing the screening / selection.

Substrates for the Evolution of Optimized Recombinant Antigens The present invention provides methods for obtaining recombinant polynucleotides encoding antigens that exhibit improved ability to elicit an immune response to a virulence factor. I do. The method is applicable to a wide range of virulence factors, including potential biological fighting factors, and other organisms and polypeptides that can cause disease and toxicity in humans and other animals. . The following examples are merely illustrative and not limiting.

1. Bacterial Pathogens and Toxins In some embodiments, the methods of the invention are applied to bacterial pathogens, and toxins produced by bacteria and other organisms. One of skill in the art can use this method to obtain a recombinant polypeptide that can elicit an immune response to a pathogen, as well as a recombinant toxin that is less toxic than the native toxin polypeptide. Often, the polynucleotide of interest encodes a polypeptide that is present on the surface of the pathogenic organism.

The pathogens for which the methods of the present invention are useful for producing protective immunogenic recombinant polypeptides are, inter alia, Yersinia species. Yersinia vest
is is the causative agent of the plague and is one of the most pathogenic bacteria known to be bacteria with an LD ₅₀ value of less than 10 in mice. Pneumonic forms of the disease are easily spread among humans by aerosol or infectious droplets and can be lethal within a few days. A particularly preferred target for obtaining recombinant polypeptides capable of protecting against Yersinia infection is the V antigen, which is a 37 kDa virulence factor, induces a protective immune response, and is currently a subunit vaccine. (Brubaker (1991) Current Inves
tigations of the Microbiology of Yer
sinae, 12: 127). V antigen alone is not toxic, but Y. The pestis isolate is non-pathogenic. Yersinia V antigen is E
. E. coli has been successfully produced by several groups (Lea
ry et al., (1995) Infect. Immun. 3: 2854). Antibodies that recognize the V antigen may provide passive protection against homologous strains but not against heterologous strains. Similarly, immunization with purified V antigen only protects against homologous strains. To obtain cross-protective recombinant V antigens, in a preferred embodiment, various Yersin
V antigen genes from ia species are subjected to family shuffling. For example,
Y. pestis, Y .; enterocolitica, and Y. pseudo
The genes encoding V antigens from O. tuberculosis are 92-99% identical at the DNA level, making them ideal for optimization using family shuffling according to the methods of the present invention. After shuffling, the library of recombinant nucleic acids can induce an improved response and / or screen and / or for recombinant nucleic acids encoding recombinant V antigen polypeptides that can have greater cross-protection. Selected.

Bacillus anthracis, a causative agent of anthrax, is another example of a bacterial target for which the methods of the present invention are useful. Anthrax protective antigen (PA) provides a protective immune response in test animals, and antibodies to PA also provide some protection. However, the immunogenicity of PA is relatively poor, so typically multiple injections are required when wild-type PA is used. Lethal factor (
Simultaneous vaccination with LF) can improve the efficacy of wild-type PA vaccine, but toxicity is the limiting factor. Thus, the DNA shuffling and antigen library immunization methods of the present invention can be used to obtain non-toxic LF. Various B. anthr
The polynucleotide encoding LF from the Acis strain is subjected to family shuffling. The resulting library of recombinant LF nucleic acids can then be screened to identify nucleic acids encoding recombinant LF polypeptides that exhibit reduced toxicity. For example, those skilled in the art will appreciate that tissue culture cells can be transformed with recombinant L in the presence of PA.
Clones that can be inoculated with the F polypeptide and that survive the cells can be selected. If desired, one skilled in the art can then backcross the non-toxic LF polypeptide to maintain the immunogenic epitope of LF. Those selected by the first screen may then be subjected to a second screen. For example, one of skill in the art can test the ability of a recombinant non-toxic LF polypeptide to induce an immune response (eg, a CTL or antibody response) in a test animal, such as a mouse. In a preferred embodiment, the recombinant non-toxic LF polypeptide is then anthrac
The ability to induce protective immunity in test animals against challenge by different strains of is is tested.

B. Anthracis protective antigen (PA) is also a suitable target for the methods of the invention. B. Nucleic acids encoding PA from various strains of anthracis are subjected to DNA shuffling. Those skilled in the art will then appreciate, for example, E.I. c
Oli can be screened for proper folding using polyclonal antibodies. Screening for the ability to induce a broad spectrum of antibodies in test animals is also typically used, either alone or in addition to preliminary screening methods. In a presently preferred embodiment, recombinant polynucleotides exhibiting desired properties can be backcrossed such that the immunogenic epitope is maintained. Eventually, the selected recombinant will be transformed into B. in the test animal. Anthracis is tested for its ability to induce protective immunity against different strains.

[0056] Staphylococcus aureus and Streptococcus
The us toxin is another example of a target polypeptide that can be altered using the methods of the present invention. Staphylococcus aureus and group A Strept
The strains of ococci are included in a range of diseases including food poisoning, toxic shock syndrome, scarlet fever, and various autoimmune disorders. They secrete various toxins, including at least five cytotoxic toxins, including clotting enzymes, and various enterotoxins. Enterotoxins are identified as MHC class II molecules and T
Classified as superantigen in crossing cell receptors and causing constitutive T cell activation (Fields et al. (1996) Nature 384: 1
88). This results in accumulation of pathogenic levels of cytokines that can lead to multiple organ defects and death. At least 30 related but still different enterotoxins can be sequenced and grouped pathologically into families. Crystal structures have been obtained for some members alone and in combination with MHC class II molecules. Particular mutations in the MHC class II binding site of the toxin are:
They can strongly reduce their toxicity and form the basis of an attenuated vaccine (Woody et al. (1997) Vaccine 15: 133). However, while a successful immune response to one type of toxin may provide protection against closely related family members, little protection against toxins from other families is observed. Family shuffling of enterotoxin genes from members of the various families can be used to obtain recombinant toxin molecules that have reduced toxicity and can induce a cross-protective immune response. Shuffling antigens can also be screened in suitable animal models, such as mice or monkeys, to identify antigens that elicit neutralizing antibodies. An example of such an assay is an ELISA in which raised antibodies inhibit the binding of enterotoxin to MHC complexes and / or T cell receptors on cells or in purified form.
Formats may be included. These assays may also include formats in which the added antibody inhibits T cells from cross-linking to appropriate antigen presenting cells.

Cholera is an ancient, potentially lethal disease caused by the bacterium Vibrio cholerae, and effective vaccines for its prevention are not yet available. Much of the pathogenicity of the disease is caused by cholera enterotoxin. Ingestion of microgram quantities of cholera toxin can cause severe diarrhea causing loss of tens of liters of water. Cholera toxin is a complex of a single catalytic A subunit with a pentameric ring of identical B subunits. Each subunit is itself inactive. The B subunit binds to specific ganglioside receptors on the surface of intestinal epithelial cells and triggers entry of the A subunit into cells. Regulatory G-proteins, A-subunit ADP-ribosylate, initiate a cascade of events that cause massive sustained flow of electrolytes and water into the intestinal lumen, resulting in extreme diarrhea.

The B subunit of cholera toxin is an attractive vaccine target for a number of reasons. It is a major target for protective antibodies created during cholera infection and contains epitopes for antitoxins that neutralize the antibody. The B subunit is non-toxic without the A subunit, is orally efficacious, and stimulates the production of a strong IgA dominant gastric mucosal immune response, which is essential for protection against cholera and cholera toxin. It is. The B subunit has also been investigated for use as an adjuvant in other vaccine preparations, and thus evolved toxins may provide a general improvement for a variety of different vaccines. Enterotoxinogenic E
. The thermolabile enterotoxin (LT) from the E. coli strain is structurally related to cholera toxin and is 75% identical at the DNA sequence level. Reduced toxicity and V. cholerae and E.C. In order to obtain an optimized recombinant toxin molecule exhibiting increased ability to induce a protective immune response against E. coli,
The gene encoding the relevant toxin is subjected to DNA shuffling.

The recombinant toxin is then tested for one or more several desired traits. For example, one of skill in the art would recognize improved cross-reactivity of antibodies raised against recombinant toxin polypeptides, lack of toxicity in cell culture assays, and protective immune responses against pathogens and / or against the toxin itself. One can screen for the ability to induce. The shuffled clones
Can be screened by phage display and / or
LISA and ELISA assays can be screened for the presence of epitopes from different serotypes. The variant protein having multiple epitopes can then be purified and used to immunize a mouse or other test animal. Animal sera are then assayed for antibodies to different B chain subtypes, and variants that elicit a broad cross-reactive response are further evaluated in a pathogenic challenge model. E. FIG. coli and V. coli. cholera
The e-toxin can also act as an adjuvant that can enhance mucosal immunity and oral delivery of vaccines and proteins. Thus, one of skill in the art can test a library of recombinant toxins to enhance adjuvant activity.

[0060] Shuffled antigens can also be screened for improved expression levels and stability of the B chain pentamer, which may be less stable in the presence of the A chain in the hexamer complex. The heat treatment step and the addition of denaturing agents (eg, salts, urea, and / or guanidine hydrochloride) can be included prior to the ELISA assay to determine the yield of correctly folded molecules by the appropriate antibody.
It is desirable to screen for stable monomeric B chain molecules in an ELISA format, for example, using an antibody that binds to the monomeric B chain but does not bind to the pentameric B chain. Further, the ability of the shuffled antigen to elicit neutralizing antibodies in a suitable animal model, such as a mouse or monkey, can be screened. For example, antibodies that bind to the B chain and inhibit binding to its specific ganglioside receptor on the surface of intestinal epithelial cells can prevent disease. Similarly, antibodies that bind to the B chain and block its pentamerization or block A chain binding may be useful in preventing disease.

Bacterial antigens that can be improved by DNA shuffling for use as vaccines also include, but are not limited to, Helicobacter pylori antigens CagA and VacA (Blaser (1996) Alim
ent. Pharmacol. Ther. 1: 73-7; Blaser and C
rabtree (1996) Am. J. Clin. Pathol. 106: 56
5-7; Censini et al. (1996) Proc. Nat'l. Acad. Sc
i. USA 93: 14648-14643). Other suitable H. pylori antigens are described, for example, in Chatha et al. (1997) Indian J. Biol. Med. Res
. 105: 170-175, four immunoreactive proteins from 45 kDa to 65 kDa; pylori GroES homolog (HspA
(Kansau et al. (1996) Mol. Microbiol. 22: 1013).
-1023). Other suitable bacterial antigens include, but are not limited to: gingivalis 43 kDa and fimbulin (41 kDa) proteins (Boutsl et al. (1996) Oral Mi.
crobiol. Immunol. 11: 236-241); Streptococcus pneumoniae surface protein A (Billes et al. (1996) Ann. NY Acad. Sci. 79).
7: 118-126); Chlamydia psittaci antigen, 80-9.
0 kDa protein and 110 kDa protein (Buendia et al. (199)
7) FEMS Microbiol. Lett. 150: 113-9); Chlamydia exoglycolipid antigen (GLXA) (Whittum-Hudson et al. (
1996) Nature Med. 2: 1116-1121); molecular weight range 92
-98, 51-55, 43-46, and 31.5-33 kDa Chlamy
dia pneumoniae species-specific antigens, and 12, 26 and 65-
A genus-specific antigen in the range of 70 kDa (Halme et al. (1997) Scand.
Immunol. 45: 378-84); Neisseria gonoreh.
orae (GC) or Escherichia coli phase variable milkiness (O
pa) Proteins (Chen and Gotschlich (1996) Proc
. Nat'l. Acad. Sci. USA 93: 14851-14856),
Cutts and Wilson (1997) parasitology 114.
: Any of the twelve immunodominant proteins of Schistosoma mansoni described in 245-55 (molecular weight ranges from 14-208 kDa)
Brucella abortus (De Mot et al. (1996) Curr.
Microbiol. 33: 26-30) 17 kDa protein antigen; Nocardia actinomycetes Rhodococcus sp. Gene homolog of the 17 kDa protein antigen of Gram-negative pathogenic Brucella abortus identified in NI86 / 21 (DeMot et al. (1996) Curr. Microb
iol. 33: 26-30); Staphylococcus enterotoxin (SE) (Wood)
(1997) FEMS Immunol. Med. Microbiol. 17
: 1-10), 42 kDa M.P. hyopneumoniae NrdF ribonucleotide reductase R2 protein or M. hyopneumoniae
15 kDa subunit protein (Fagan et al. (1997) Infect
. Immun. 65: 2502-2507), meningococcal antigen PorA protein (Feavers et al. (1997) Clin. Diagn. Lab. Immun.
ol. 3: 444-50); S. pneumoniae surface protein A (PsaA) (McDa
niel et al. (1997) Gene Ther. 4: 375-377); tu
Larensis outer membrane protein FopA (Fulop et al. (1996) FEMS
Immunol. Med. Microbiol. 13: 245-247); A
Major outer membrane proteins (Hartmann) in strains of the genus ctinobacillus
(1996) Zentralbl. Bakteriol. 284: 255-2
62); p60 or Listeria monocytogenes listeriolysin (Hly) antigen (Hess et al. (19)
96) Proc. Nat'l. Acad. Sci. USA 93: 1458-1
463); Salmonella enteritidis and S.M. pull
flagellum (G) antigens (Holt and Chaubal (1
997). Clin. Microbiol. 35: 1016-1020); B
acillus anthracis protective antigen (PA) (Ivins et al. (19)
95) Vaccine 13: 1779-1784); Echinococcu
s granulosus antigen 5 (Jones et al. (1996) Parasito
logy 113: 213-222); Shigella dysenteri
ae1 and the rol gene of Escherichia coli K-12 (
Klee et al. (1997) J. Am. Bacteriol. 179: 2421-2425
); Cell surface protein Rib and α of group B streptococci (Larsson et al. (1)
996) Infect. Immun. 64: 3518-3523); Pathogenic Ye
rsinia spp. 37 encoded in a 70 kb virulence plasmid
kDa secreted polypeptide (Leary et al. (1995) Contrib. Micr
obiol. Immunol. 13: 216-217 and Roggenkam
p, et al. (1997) Infect. Immun. 65: 446-51); OspA (outer membrane surface protein A) of Lyme disease spirocheta Borrelia burgdorferi (Li et al. (1997) Proc. Nat'l. Acad. Sci.
. USA 94: 3584-3589, Padilla et al. In
fact. Dis. 174: 739-746, and Wallich et al. (199
6) Infection 24: 396-397); B encoding Omp28
rucella melitensis group 3 antigen gene (Lindler et al. (1
996) Infect. Immun. 64: 2490-2499); Strep.
tococcus mutans PAc antigen (Murakami et al. (1997)
) Infect. Immun. 65: 794-797); Pneumolysin (pn
eumolysin), Pneumococcal neurominidas
e, autolysin, hyaluronidase, and 37 kDa pneumococcal surface adhesin A (Paton et al. (1997) Microb. Drug Resist. 3: 1).
-10); Salmonella typhi 29-32, 41-45, 63
-71 x 10 (3) MW antigen (Perez et al. (1996) Immunology
89: 262-267); K antigen as a marker for Klebsiella pneumoniae (Priamukhina and Morozova (19)
96) Klin. Lab. Diagn. 47-9); molecular mass about 60, 40, 2
Nocardia antigens of 0 and 15-10 kDa (Prokesova et al. (1996)
) Int. J. Immunopharmacol. 18: 661-668); S
taphylococcus aureus antigen ORF-2 (Rieneck et al. (1997) Biochim Biophys Acta 1350: 128-
132); GlpQ antigen of Borrelia hermsii (Schwan et al. (1996) J. Clin. Microbiol. 34: 2483-2492).
Cholera protective antigen (CPA) (Sciorino (1996) J. Diar;
rheal Dis. Res. 14: 16-26); Streptococ
us mutant 190 kDa protein antigen (Senpuku et al. (1996) Or
al Microbiol. Immunol. 11: 121-128); Ant
hrax toxin protective antigen (PA) (Sharma et al. (1996) Prote)
in Expr. Purif. 7: 33-38); Clostridium p.
erfringens antigens and toxoids (Strom et al. (1995) Br.
J. Rheumatol. 34: 1095-1096); Salmonella.
enteritidis SEF14 hippocampus antigen (Thorns et al. (1996)
) Microb. Pathog. 20: 235-246); Yersinia
pestis capsular antigen (F1 antigen) (Titball et al. (1997) Infec)
t. Immun. 65: 1926-1930); Mycobacterium.
Laplae 35 kilodalton protein (Tricas et al. (1996) I
nfect. Immun. 64: 5171-5177); Moraxella (
CD (Yang), a major outer membrane protein from Branhamella catarrh
(1997) FEMS Immunol. Med. Microbiol. 17
187-199); pH 6 antigen of Yersinia pestis (PsaA
Protein) (Zav'yalov et al. (1996) FEMS Immunol.
Med. Microbiol. 14: 53-57); Leishmaniam.
ajor major surface glycoprotein gp63 (Xu and Liew (1994) V
accine 12: 1534-1536; Xu and New (1995) I.
Munology 84: 173-176); mycobacterial heat shock protein 65, mycobacterium antigen (Mycobacterium leprae)
hsp65) (Lowrie et al. (1994) Vaccine 12: 1537.
-1540; Ragno et al. (1997) Arthritis Rheum. 40
: 277-283; Silva (1995) Braz. J. Med. Biol.
Res. 28: 843-851); Mycobacterium tuberc.
ulosis antigen 85 (Ag85) (Huygen et al. (1996) Nat. Me.
d. 2: 893-898); Mycobacterium tuberculo.
sis, M.S. bovis, and the 45/47 kDa antigen complex of BCG (APA
(Horn et al. (1996) J. Immunol. Methods 197: 1).
51-159); 65 kDa heat shock protein, hsp65 (Tascon
Et al. (1996) Nat. Med. 2: 888-892); mycobacterial antigens MPB64, MPB70, MPB57, and the alpha antigen (Yamada et al. (199)
5) Kekaku 70: 639-644); tuberculosis
38 kDa protein (Vordermeier et al. (1995) Vaccin
e 13: 1576-1582); Mycobacterium tuberc.
MPT63, MPT64, and MPT59 antigens from ulosis (Manc
a et al. (1997) Infect. Immun. 65: 16-23; Oettin
ger et al. (1997) Scand. J. Immunol. 45: 499-503
Wilcke et al. (1996) Tuber. Lung Dis. 77: 250-
256); 35 kDa protein of Mycobacterium leprae (Tricas et al. (1996) Infect. Immun. 64: 5171-
5177); ESAT-6 antigen of pathogenic mycobacteria (Brandt et al. (19)
96) J. Amer. Immunol. 157: 3527-3533; Pollock and Andersen (1997) J. Mol. Infect. Dis. 175: 1251
-1254); Mycobacterium tuberculosis 16
kDa antigen (Hsp16.3) (Chang et al. (1996) J. Biol. Ch.
em. 271: 7218-7223); and Mycobacterium.
leprae 18 kDa protein (Baumart et al. (1996) Inf
ect. Immun. 64: 2274-2281).

2. Viral Pathogens The methods of the present invention are also useful for obtaining recombinant nucleic acids and polypeptides with enhanced ability to induce an immune response against viral pathogens. The bacterial recombinants described above are typically administered in the form of a polypeptide, and the protective conferring recombinant is preferably administered in the form of a nucleic acid as a genetic vaccine.

One illustrative example is Hunter virus. The glycoprotein of the virus typically accumulates in the Golgi membrane of infected cells. This poor expression of glycoproteins prevents the development of effective genetic vaccines for these vaccines. The method of the present invention is directed to recombinants that exhibit enhanced expression for improved immunogenicity by performing DNA shuffling on nucleic acids encoding glycoproteins and when administered in host cells and / or as genetic vaccines. Solve this problem by identifying A convenient screening method for these methods is to express the recombinant polynucleotide as a fusion protein to PIG, which results in the display of the polypeptide on the surface of the host cell (Whitehorn et al. (1995) Biotechnology
y (NY) 13: 1215-9). The cells that express the increased amount of the antigenic polypeptide on the cell surface are then sorted and recovered using fluorescence activated cell sorting. This preliminary screening can be followed by an immunogenicity test in a mammal such as a mouse. Finally, in a preferred embodiment, the recombinant nucleic acids can be tested as genetic vaccines for their ability to protect test animals against challenge by the virus.

Flaviviruses are another example of a viral pathogen for which the methods of the invention are useful for obtaining recombinant polypeptides or genetic vaccines that are effective against the viral pathogen. Flaviviruses are three clusters of antigenically related viruses: dengue fever 1-4 (62-77% identity), Japanese encephalitis virus, St. Louis encephalitis virus, and Mary Valley encephalitis virus (75-82% identity), And tick-borne encephalitis virus (77-96% identity). Dengue virus can induce protective antibodies against SLE and yellow fever (40-50% identity), but few effective vaccines are available. In order to obtain genetic vaccines and recombinant polypeptides that exhibit enhanced cross-reactivity and immunogenicity, the polynucleotide encoding the envelope protein of the relevant virus should have D
Used for NA shuffling. The resulting recombinant polynucleotides can be tested for their ability to induce a broadly reactive neutralizing antibody response, either as a genetic vaccine or using the expressed polypeptide. Finally, preferred clones in preliminary screening can be tested for their ability to protect test animals against viral challenge.

Viral antigens that can be evolved by DNA shuffling for improved activity as vaccines include, but are not limited to: influenza A virus N2 neuraminidase (Kilbourne et al. (1995)
Vaccine 13: 1799-1803); Dengue virus envelope (E) and premembrane (prM) antigens (Feighny et al. (1994) A
m. J. Trop. Med. Hyg. 50: 322-328; Putnak et al. (
1996) Am. J. Trop. Med. Hyg. 55: 504-10); HI
V antigens Gag, Pol, Vif, and Nef (Vogt et al. (1995) Vac
cine 13: 202-208); HIV antigens gp120 and gp160 (
Achour et al. (1995) Cell. Mol. Biol. 41: 395-40
0; Hone et al. (1994) Dev. Biol. Stand. 82: 159-1
62); gp41 epitope of human immunodeficiency virus (Eckhart et al. (19)
96) J. Amer. Gen. Virol. 77: 2001-2008); rotavirus antigen VP4 (Mattion et al. (1995) J. Virol. 69: 5132-5).
137); rotavirus protein VP7 or VP7sc (Emslie et al.,
(1995) J. Mol. Virol. 69 1747-1754; Xu et al. (1995)
J. Gen. Virol. 76: 1971-1980); herpes simplex virus (HSV) glycoprotein gB, gC, gD, gE, gG, gH, and gI (F
leck et al. (1994) Med. Microbiol. Immunol. (Be
rl) 183: 87-94 [Mattion, 1995]; Ghiasi et al.
995) Invest. Ophthalmol. Vis. Sci. 36: 135
2-1360; McLean et al. (1994) J. Am. Infect. Dis. 170
1100-1109); the immediate early protein ICP47 of herpes simplex virus type 1 (HSV-1) (Banks et al. (1994) Virology 200:
236-245); immediate early (IE) protein ICP of herpes simplex virus
27, ICP0, and ICP4 (Manikan et al. (1995) J. Vir.
ol. 69: 4711-4716); influenza virus nucleoprotein and hemagglutinin (Deck et al. (1997) Vaccine 15: 71-78).
Fu et al. (1997) J .; Virol. 71: 2715-2721); B19 parvovirus capsid protein VP1 (Kawase et al. (1995) Viro.
logic 211: 359-366) or VP2 (Brown et al. (1994) V
irology 198: 477-488); hepatitis B virus core and e-antigen (Schödel et al. (1996) Intervirology 39: 104).
-106); B-type surface antigen (Shiau and Murray (1997) J. M.
ed. Virol. 51: 159-166); hepatitis B surface antigen fused to the core antigen of the virus (supra); hepatitis B virus core pre-S2 particles (Neme
ckova et al. (1996) Acta Virol. 40: 273-279); H
BV pre S2-S protein (Kutinova et al. (1996) Vaccine
14: 1045-1052); VZV glycoprotein I (Kutinova et al. (
1996) Vaccine 14: 1045-1052); rabies virus glycoprotein (Xiang et al. (1994) virology 199: 132-14).
0; Xuan et al. (1995) Virus Res. 36: 151-161) or ribonucleocapsid (Hooper et al. (1994) Proc. Nat'l.
Acad. Sci. USA 91: 10908-10912); human cytomegalovirus (HCMV) glycoprotein B (UL55) (Britt et al. (1995)
J.). Infect. Dis. 171: 18-25); secreted or non-secreted forms of the hepatitis C virus (HCV) nucleocapsid protein or the middle (pre-S2 and S) or major (S) surface of the hepatitis B virus (HBV) Fusion protein with antigen (Inchauspe et al. (1997) DNA
Cell Biol. 16: 185-195; Major et al. (1995) J.
. Virol. 69: 5798-5805); hepatitis C virus antigen; core protein (pC); E1 (pE1) and E2 (pE2) alone or as fusion proteins (Saito et al. (1997) Gastroenterology 11).
2: 1321-1330); a gene encoding an RS virus fusion protein (
PFP-2) (Falsey and Walsh (1996) Vaccine 1
4: 1214-1218; Piedra et al. (1996) Pediatr. Inf
ect. Dis. J. 15: 23-31); VP6 and VP7 of rotavirus
Gene (Choi et al. (1997) Virology 232: 129-138;
Jin et al. (1996) Arch. Virol. 141: 2057-2076);
Human papillomavirus E1, E2, E3, E4, E5, E6, and E7 proteins (Brown et al. (1994) Virology 201: 46-54;
Dillner et al. (1995) Cancer Detect. Prev. 19:
381-393; Krul et al. (1996) Cancer Immunol. Im
munother. 43: 44-48; Nakagawa et al. (1997) J. Am. I
nfect. Dis. 175: 927-931); human T lymphotropic virus type I gag protein (Porter et al. (1995) J. Med. Virol.
45: 469-474); Epstein-Barr virus (EBV) gp340 (M
ackett et al. (1996) J. Am. Med. Virol. 50: 263-271).
Epstein-Barr virus (EBV) latent membrane protein LMP2 (Lee et al. (1996) Eur. J. Immunol. 26: 1875-1883); Epstein-Barr virus nuclear antigens 1 and 2 (Chen and Cooper (199);
6) J.I. Virol. 70: 4849-4853; Khanna et al. (1995)
Virology 214: 633-637); measles virus nucleoprotein (
N) (Fooks et al. (1995) Virology 210: 456-465).
And cytomegalovirus glycoprotein gB (Marshall et al. (199)
4) J.I. Med. Virol. 43: 77-83) or glycoprotein gH (R
asmussen et al. (1994) J. Am. Infect. Dis. 170: 673-
677).

3. Parasites Parasite-derived antigens can also be optimized by the methods of the present invention. These include Schistosoma mansoni, S.A. haematobium,
Or S. schistosome intestinal associated antigen CAA (circulating anodic antigen) and CCA (
Circulating cathodic antigen
(Deeler et al. (1996) Parasitology 112: 21-35.
); A multiple antigen peptide consisting of two different protective antigens from the parasite Schistosoma mansoni (multiple antigen pep)
ide) (MAP) (Ferru et al. (1997) Parasite Immu)
nol. 19: 1-11); Leishmania parasite surface molecule (Leza)
ma-Davila (1997) Arch. Med. Res. 28: 47-53
); loa third stage larva (L3) antigen (Akue et al. (1997) J. In.
fact. Dis. 175: 158-63); Theileria annul
The genes Tams1-1 and Tams1-2 encoding the 30 kDa and 32 kDa major merozoite surface antigens of ata (Ta) (d'Oliveira et al. (1)
996) Gene 172: 33-39); Plasmodium falci.
param merozoite surface antigen 1 or 2 (al-Yaman et al. (1995) T
rans. R. Soc. Trop. Med. Hyg. 89: 555-559; B
Eck et al. (1997) J. Am. Infect. Dis. 175: 921-926; R
Zepczyk et al. (1997) Infect. Immun. 65: 1098-1
100); a B epitope based on the circumsporozoite (CS) protein from Plasmodium berghei, (PPPPNPND) 2 and Plasmodium yoelii,
(QGPGAP) 3QG; berghei T helper epitope KQIRDSITEWS (Reed et al. (1997) Vaccine 15: 4.
82-488); Plasmo encoded by NYVAC-Pf7 derived from sporozoites (circum sporozoite protein and sporozoite surface protein 2)
and hepatic (liver stage antigen 1), blood (merozoite surface protein 1, serine repeat antigen, and apical membrane antigen 1), including, but not limited to, sexual (25 kDa) The sexual stage antigen) stage is inserted into a single NYVAC genome and NYVAC-Pf7 (Tine et al. (
1996) Infect. Immun. 64: 3833-3844); Plas.
medium falciparum antigen Pfs230 (Williamson
(1996) Mol. Biochem. Parasitol. 78: 161-
169); Plasmodium falciparum apical membrane antigen (AMA-
1) (Lal et al., (1996) Infect. Immun. 64: 1054-1).
059); Plasmodium falciparum protein Pfs28
And Pfs25 (Duffy and Kaslow (1997) Infect.
Immun. 65: 1109-1113); Plasmodium falci
param merozoite surface protein, MSP1 (Hui et al. (1996) Inf
ect. Immun. 64: 1502-1509); malaria antigen Pf332 (
(Ahlborg et al. (1996) Immunology 88: 630-635).
Plasmodium falciparum erythrocyte membrane protein 1 (Bar
uch et al. (1995) Proc. Nat'l. Acad. Sci. USA 93
: 3497-3502; Baruch et al. (1995) Cell 82: 77-8.
7); Plasmodium falciparum merozoite surface antigen, Pf
MSP-1 (Egan et al. (1996) J. Infect. Dis. 173: 76).
5-769); Plasmodium falciparum antigen SERA, E
BA-175, RAP1 and RAP2 (Riley (1997) J. Phar
m; Pharmacol. 49: 21-27); Schistosoma ja
ponicum paramyosin (Sj97) or a fragment thereof (Yang et al. (1995) Biochem. Biophys. Res. Commun. 212)
: 1029-1039); and Hsp70 (Maresc) in parasites.
a and Kobayashi (1994) Experientia 50:10.
67-1074).

(4. Allergy) The present invention also provides a method for obtaining a reagent useful for treating allergy. 1
In one embodiment, the method comprises the steps of creating a library of recombinant polynucleotides encoding the allergen, and a set thereof that when used as an immunotherapeutic reagent for treating allergy, exhibit improved properties. Screening the library to identify the replacement polynucleotide. For example, specific immunotherapy of allergies with natural antigens can be performed with high affinity Ig on mast cells.
It carries the risk of anaphylaxis induction that can be initiated by cross-linking of the E receptor. Therefore, allergens that are not recognized by existing IgE are desired.
The method of the present invention provides a method by which such an allergen variant can be obtained. Another improved property of interest is the induction of a broader immune response with increased safety and efficacy.

The synthesis of polyclonal IgE and allergen-specific IgE requires multiple interactions between B cells, T cells and specialized antigen presenting cells (APCs). Untreated, immature B cells have specific B cells whose cell surface immunoglobulin (s
It is triggered when the allergen is recognized by Ig). However, costimulatory molecules expressed by activated T cells in both soluble and membrane-bound forms are essential for the differentiation of B cells into IgE-secreting plasma cells. Activation of T helper cells
In the context of MHC class II molecules on the plasma membrane of (eg, monocytes, dendritic cells, Langerhans cells or immature B cells), recognition of antigenic peptides is required. Professional APCs can efficiently capture antigens and peptide-MHC class II complexes are formed in the post-Golgi, a proteolytic intracellular component, which is then transported to the plasma membrane where they Recognized by the T cell receptor (TCR) (Whitton (1998) Curr. Top. Microbio
l. Immunol. 232: 1 to 13). In addition, activated B cells have CD80
(B7-1) and CD86 (B7-2, B70). This is CD28
And provides a costimulatory signal for T cell activation resulting in T cell proliferation and cytokine synthesis. Atopic individuals from allergen-specific T cells generally belong to the T _H 2 cell subsets, these activated cells also, IL-4 and IL-13 (expressed by activated helper T cells Along with the associated membrane-bound costimulatory molecule, which differentiates B cells into IgE-secreting plasma cells.

[0069] Mast cells and eosinophils are important cells in inducing allergic symptoms in target organs. High affinity Ig on mast cells, basophils or eosinophils
Recognition of specific antigens by IgE binding to the E receptor results in receptor cross-linking, which leads to degranulation of cells and rapid release of mediator molecules such as histamine, prostaglandins and leukotrienes that cause allergic symptoms. . Currently, immunotherapy for allergic diseases includes desensitization treatment with escalating doses of the allergen injected into the patient. These treatments result in skewing of the immune response towards T _H 1 phenotype and specific IgG against allergens /
Increase the ratio of IgE antibodies. Because these patients have circulating IgE antibodies specific for the allergen, these treatments involve a significant risk of an anaphylactic reaction. In these reactions, free circulating allergens are recognized by IgE molecules that bind to high affinity IgE receptors on mast cells and eosinophils. Allergen recognition results in cross-linking of receptors leading to the release of mediators such as histamine, prostaglandins and leukotrienes (which cause allergic symptoms and sometimes anaphylactic reactions). Other problems associated with desensitization include the low effectiveness and difficulty of producing reproducibly allergen extracts.

The method of the present invention provides a means for obtaining an allergen, which when used in a genetic vaccine, provides a means of circumventing the problems that limit the usefulness of previously known desensitization treatments. For example, by expressing the antigen on the surface of a cell (eg, a muscle cell), the risk of an anaphylactic reaction is significantly reduced. This can be conveniently obtained by using a gene vaccine vector encoding the transmembrane form of the allergen. The allergens can also be modified in such a way that they are efficiently expressed in a transmembrane form, further reducing the risk of anaphylactic reactions. Another advantage provided by the use of genetic vaccines for desensitization is that genetic vaccines can include cytokines and accessory molecules. These are further allowed direct the immune response to the T _H 1 phenotype, thus reducing the amount of IgE antibodies produced, and increases the effectiveness of the treatment. To further reduce IgE production, shuffled allergens can be administered using vectors evolved to induce mainly IgG and IgM responses with little or no IgE response (eg, Filed February 11, 1998, US patent application Ser. No. 09 /
021,769).

In these methods, the polynucleotide encoding a known allergen or a homolog or fragment thereof (eg, an immunogenic peptide) is a DN
A vaccine vector and used to immunize allergic and asthmatic individuals. Alternatively, the shuffled allergen is E. coli. co
expressed in a production cell, such as a li or yeast cell, and subsequently purified,
It is used to treat patients or prevent allergic diseases. DNA shuffling or other methods of recombination can be used to acquire allergens that activate T cells but cannot elicit an anaphylactic reaction. For example, a library of recombinant polynucleotides encoding variants of an allergen can be expressed in a cell, such as an antigen presenting cell, which is then contacted with a PBMC or T cell clone from an atopic patient. Those library members that efficiently activate _TH cells from atopic patients can be assayed for T cell proliferation or by cytokine synthesis (eg, I
L-2, IL-4, IFN-γ synthesis). These recombinant allergen variants that are positive in the in vitro test may then be subjected to an in vivo test.

Examples of allergies that can be treated include house dust mites, weed pollen, birch pollen, ragweed pollen, hazel pollen, cockroaches, rice, olive tree pollen, fungi, mustard, bee venom But are not limited to these. The antigen of interest is the allergen der p1 (Scobie et al., (19
94) Biochem. Soc. Trans. 22: 448S; Yssel et al. (
1992). Immunol. 148: 738-745), der p2 (C
hua et al. (1996) Clin. Exp. Allergy 26: 829-83
7), der p3 (Smith and Thomas (1996) Clin. E)
xp. Allergy 26: 571-579), der p5, der pV
(Lin et al. (1994) J. Allergy Clin. Immunol. 9).
4: 989-996), der p6 (Bennett and Thomas (1
996) Clin. Exp. Allergy 26: 1150-1154), d
er p7 (Shen et al., (1995) Clin. Exp. Allergy 2
5: 416-422), der f2 (Yuki et al., (1997) Int. A.
rch. Allergy Immunol. 112: 44-48), der f
3 (Nishiyama et al., (1995) FEBS Lett. 377: 62-
66), der f7 (Shen et al., (1995) Clin. Exp. Alle
rgy 25: 1000-1006); Mag 3 (Fujikawa et al. (19)
96) Mol. Immunol. 33: 311-319), mites (eg, Dermatophagoides pteronyssinus, Der).
matophagoides farinae, Blomia tropicala
lis). Targets as antigens also include: house dust mite allergen Tyr p2 (Eriksson et al. (1998)
) Eur. J. Biochem. 251: 443-447), Lep d1 (S
chmidt et al. (1995) FEBS Lett. 370: 11-14), and glutathione S-transferase (O'Neill et al. (1995) Imm
unol Lett. 48: 103-107); a 25,589 Da 219 amino acid polypeptide homologous to glutathione S-transferase (O'Neil).
1 (1994) Biochim. Biophys. Acta. 1219: 52
Blot5 (Arruda et al. (1995) Int. Arch.
Allergy Immunol. 107: 456-457); bee venom phospholipase A2 (Carballido et al. (1994) J. Allergy Cli).
n. Immunol. 93: 758-767; Jutel et al. (1995) J. Am. I
mmunol. 154: 4187-4194); Bovine dermis / dandruff antigen BDA11.
(Rautiainen et al. (1995) J. Invest. Dermatol.
105: 660-663) and BDA20 (Mantyjarvi et al. (199)
6) J.I. Allergy Clin. Immunol. 97: 1297-130
3); Major equine allergen Ecucl (Gregoire et al.
Biol. Chem. 271: 32951-29959); Jumper an
tM. pirosula allergen Myr p I and its homolog allergenic polypeptide Myr p2 (Donovan et al. (1996) Bioche
m. Mol. Biol. Int. 39: 877-885); Tick Blomia
tropicalis 1-13, 14, 16 kD allergen (Caraba
Ilo et al. (1996) J. Mol. Allergy Clin. Immunol. 98
: 573-579); cockroach allergen Blag Bd90K (Helm et al. (1996) J. Allergy Clin. Immunol. 98: 172-).
180) and Blag 2 (Arruda et al., (1995) J. Biol.
Chem. 270: 19563-19568); cockroaches Cr-PI allergen (Wu et al., (1996) J. Biol. Chem. 271: 17937-179).
43); fire ant venom allergen, Sol i 2 (Schmidt et al. (1996) J. Allergy Clin. Immunol. 98: 82-8).
8); insect Chironomus thummi major allergen Chit
1-9 (Kipp et al. (1996) Int. Arch. Allergy Imm.
unol. 110: 348-353); canine allergen Can f1 or feline allergen Fel d1 (Ingram et al. (1995) J. Allergy C).
lin. Immunol. 96: 449-456); for example, albumin from horses, dogs or cats (Goubran Botros et al. (1996) Immu).
No. 88: 340-347); molecular weight 22 kD, 25 kD or 60
Deer allergen that is kD (Spitzauer et al. (1997) Clin. Ex.
p. Allergy 27: 196-200); as well as the major 20 kd allergen of cows (Ylonen et al. (1994) J. Allergy Clin. Im.
munol. 93: 851-858).

Pollen and grass allergens are also useful in vaccines, especially after antigen optimization according to the method of the invention. Such allergens include, for example: Hor v9 (Astwood and Hill (1996) Gen.
e 182: 53-62, Lig v1 (Batanero et al., (1996) C
lin. Exp. Allergy 26: 1401-1410); Lol p1
(Muller et al. (1996) Int. Arch. Allergy Immun.
ol. 109: 352-355), Lol pII (Tamborini et al. (1
995) Mol. Immunol. 32: 505-513), Lol pVA,
Lol pVB (Ong et al. (1995) Mol. Immunol. 32: 295).
-302), Lol p9 (Blaher et al., (1996) J. Allergy.
Clin. Immunol. 98: 124-132); Par JI (Cos
ta et al. (1994) FEBS Lett. 341: 182 to 186; Sallu
sto et al. Allergy Clin. Immunol. 97:
627-637), Par j 2.0101 (Duro et al. (1996) FEBS).
Lett. 399: 295-298); Bet v1 (Faver et al. (199)
6) J.I. Biol. Chem. 271: 19243-19250), Bet v.
2 (Rihs et al. (1994) Int. Arch. Allergy Immuno.
l. 105: 190-194); Dac g3 (Guerin-Marchan).
d et al. (1996) Mol. Immunol. 33: 797-806); Phl
p1 (Petersen et al. (1995) J. Allergy Clin. Imm
unol. 95: 987-994), Phl p5 (Muller et al. (1996)
) Int. Arch. Allergy Immunol. 109: 352-35
5), Ph1 p6 (Petersen et al. (1995) Int. Arch. Al)
large Immunol. 108: 55-59); Cry j I (Son
e et al. (1994) Biocehm. Biophys. Res. Commun. 1
99: 619-625), Cryj II (Namba et al. (1994) FEB).
S Lett. 353: 124-128); Cor a 1 (Schenk et al. (
1994) Eur. J. Biochem. 224: 717-722); cyn
d1 (Smith et al. (1996) J. Allergy Clin. Immuno.
l. 98: 331-343), cyn d7 (Suphiglu et al. (199)
7) FEBS Lett. 402: 167-172); Pha a1 and Ph
a Isoforms of a5 (Suphioglu and Singh (1995)
Clin. Exp. Allergy 25: 853-865); Chao 1
(Suzuki et al. (1996) Mol. Immunol. 33: 451-460).
); For example, profilin (Val) derived from pollen of timothy grass or birch
enta et al. (1994) Biochem. Biophys. Res. Commu
n. 199: 106-118); P0149 (Wu et al. (1996) Plant.
Mol. Biol. 32: 1037-1042); Ory s1 (Xu et al. (1
995) Gene 164: 255-259); and Amb a V and Amb t 5 (Kim et al. (1996) Mol. Immunol. 33: 873).
８880; Zhu et al. (1995) J. Mol. Immunol. 155: 5064-50
73).

Vaccines against food allergens can also be developed using the methods of the present invention. Suitable antigens for shuffling include, for example: profilin (Rihs et al. (1994) Int. Arch. Allergy Immu).
nol. 105: 190-194); a rice allergenic cDNA belonging to the α-amylase / trypsin inhibitor gene family (Alvarez et al. (199)
5) Biochim Biophys Acta 1251: 201-204)
The major olive allergen, Ole e I (Lombardero et al. (199)
4) Clin Exp Allergy 24: 765-770); Sina
1. Major allergens from mustard (Gonzalez De La Pena)
(1996) Eur J. et al. Biochem. 237: 827-832); Parvalbumin, a major salmon allergen (Lindstrom et al. (1996)
Scand. J. Immunol. 44: 335-344); Major Allergen M
apple allergens such as al d1 (Vanek-Krebitz et al. (199)
5) Biochem. Biophys. Res. Commun. 214: 538
551); and peanut allergens such as Ara hI (Burks et al. (1995) J. Clin. Invest. 96: 1715-1721).

The methods of the present invention can also be used to develop a recombinant antigen that is effective against allergy to fungi. Fungal allergens useful in these vaccines include, but are not limited to, the following allergens: Clado
Cla h III of sporium herbarum (Zhang et al. (19)
95) J. Amer. Immunol. 154: 710-717); Basidiomycete Psilology
allergen Psic2 from be cubensis, fungal cyclophilin (
Horner et al. (1995) Int. Arch. Allergy Immuno
l. 107: 298-300); Cladosporium herbarum
Hsp70 (Zhang et al. (19) cloned from a cDNA library of
96) Clin Exp Allergy 26: 88-95); Penici
lium notatum 68 kD allergen (Shen et al. (1995) C
lin. Exp. Allergy 26: 350-356); Aldehyde dehydrogenase (ALDH) (Achatz et al. (1995) Mol. Immunol).
. 32: 213-227); enolase (Achatz et al. (1995) Mol.
Immunol. 32: 213-227); YCP4 (Id.); Acidic ribosome protein P2 (Id.).

Other allergens that can be used in the method of the present invention include latex allergens such as the major allergen from natural rubber latex (Hev b 5) (Akasawa et al. (1996) J. Biol. Chem. 271). :
25389-25393; Slater et al. Biol. Chem
. 271: 25394-25399).

The present invention also provides a solution to another shortcoming of vaccination as a treatment for allergy and asthma. Genetic vaccination mainly induces a CD8 ⁺ T cell response, whereas induction of an allergen-specific IgE response depends on CD4 ⁺ T cells and their help to B cells. T _H 2 type cells is particularly effective have your induction of IgE synthesis. Because they secrete high levels of IL-4, IL-5 and IL-13 (switching to IgE synthesis and directed to the Ig isotype). IL-5 also induces eosinophilia. The method of the present invention comprises:
CD4 ⁺ T cells responses effectively induced, and can be used to generate a recombinant antigens directed differentiation into T _H 1 phenotype of these cells.

5. Inflammatory and Autoimmune Diseases Autoimmune diseases are immune responses that attack tissues or cells of one's own body, or pathogen-specific immune responses that are also harmful to one's own tissues or cells. Alternatively, it is characterized by non-specific immune activation that is detrimental to its own tissues or cells.
Examples of autoimmune diseases include, but are not limited to, rheumatoid arthritis, SLE, diabetes mellitus, myasthenia gravis, reactive arthritis, ankylosing spondylitis and multiple sclerosis. These and other inflammatory conditions, including IBD, psoriasis, pancreatitis and various immunodeficiencies, can be treated with antigens that are optimized using the methods of the invention.

[0079] These conditions are often characterized by accumulation of inflammatory cells, such as lymphocytes, macrophages, and neutrophils, at the site of inflammation. Changes in cytokine production levels are often observed with increased levels of cytocan production.
Some autoimmune diseases, including diabetes and rheumatoid arthritis, have certain M
Associated with the HC haplotype. Other autoimmune disorders, such as reactive arthritis,
It has been shown to be induced by bacteria such as Ergenia and Shigella, and some other autoimmune diseases such as diabetes, multiple sclerosis, rheumatoid arthritis are also genetically susceptible There is evidence to suggest that in individuals of this nature, it can be triggered by viral or bacterial infection.

Current strategies for treatment generally include anti-inflammatory drugs such as NSAIDs or cyclosporine and anti-proliferative drugs such as methotrexate. These treatments are non-specific, and there is a need for treatments with higher specificity and means to direct the immune response in a direction that inhibits the autoimmune process.

The present invention provides several strategies that can meet those needs. First, the present invention provides a method for obtaining an antigen having greater tolerogenicity and / or improved antigenicity. In a preferred embodiment, antigens prepared according to the present invention exhibit improved tolerance induction by oral delivery. Oral tolerance is characterized by induction of immunological tolerance after oral administration of large amounts of antigen. In animal models, this approach has proven to be a very promising approach for treating autoimmune diseases, and clinical trials are in progress and humans such as rheumatoid arthritis and multiple sclerosis The effectiveness of this approach in treating autoimmune diseases has been positioned. It has also been suggested that the induction of oral resistance to viruses used in gene therapy can reduce the immunogenicity of gene therapy vectors. However, the amount of antigen required to induce oral tolerance is very high, and the methods of the present invention provide a means for obtaining antigens that show a significant improvement in the induction of oral tolerance.

Expression Library Immunization (Barry et al. (1995) Nature 377: 6)
32) is a particularly useful method for screening optimized antigens for use in genetic vaccines. For example, one can screen for induction of a T cell response in HLA-B27-positive individuals to identify self-antigens present in Ergenia, Shigella, and the like. A complex comprising an epitope of a bacterial antigen associated with an autoimmune disease and an MHC molecule (e.g., HLA-B27 for an Ergenia antigen) can be used to prevent reactive arthritis and ankylosing spondylitis in HLA-B27 positive individuals. .

Screening for optimized antigens can be performed in animal models known to those of skill in the art. Examples of models that are suitable for various conditions include: collagen-induced arthritis, an NFS / sld mouse model of human Sjogren's syndrome; a 120 kD organ-specific self recently identified as an analog of the human cytoskeletal protein. Antigen (α-fodrin (Haneji et al. (1997) Science 276)
: 604), New Zealand Black / White of human SLE
F1 hybrid mouse model, NOD mouse, mouse model of human diabetes mellitus, fas / fas ligand mutant mouse (naturally causes autoimmune and lymphoproliferative disorders) (Watanabe-Fukuunaga et al. (1992) Nature
356: 314), as well as experimental autoimmune encephalomyelitis (EAE) in which myelin basic protein induces a disease resembling human multiple sclerosis.

Self-antigens that can be shuffled according to the methods of the invention and used in vaccines to treat multiple sclerosis include, but are not limited to: myelin basic protein (Stinissen et al. (199)
6) J.I. Neurosci. Res. 45: 500-511) or a fusion protein of myelin basic protein and proteolipid protein (Ellio
TT et al. (1996) J. Am. Clin. Invest. 98: 1602-1612)
, Proteolipid protein (PLP) (Rosener et al. (1997) J.N.
euroimmunol. 75: 28-34) 2 ', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) (Rosener et al. (1997)
J. Neuroimmunol. 75: 28-34), Epstein Barr virus nuclear antigen-1 (EBNA-1) (Vaughan et al. (1996) J. Neuro.
immunol. 69: 95-102), HSP70 (Salvetti et al. (1
996) J.I. Neuroimmunol. 65: 143-153; Feldma
nn et al. (1996) Cell 85: 307).

[0085] Target antigens that can be used to treat scleroderma, systemic sclerosis and systemic lupus erythematosus after shuffling according to the methods of the invention include, for example: (-2-GPI , 50 kDa glycoprotein (Blank et al. (1
994). Autoimmun. 7: 441-455), Ku (p70 / p8)
0) autoantigen or its 80 kd subunit protein (Hong et al. (
1994) Invest. Ophthalmol. Vis. Sci. 35:40
23-4030; Wang et al. (1994) J. Am. Cell Sci. 107: 32
23-3233), nuclear autoantigens La (SS-B) and Ro (SS-A) (Hu
ang et al. (1997) J. Am. Clin. Immunol. 17: 212-219;
(Igarashi et al. (1995) Autoimmunity 22: 33-42).
Keech et al. (1996) Clin. Exp. Immunol. 104: 25
5-263; Manoussakis et al. (1995) J. Mol. Autoimmun.
8: 959-969; Topfer et al. (1995) Proc. Nat'l. Ac
ad. Sci. USA 92: 875-879), proteasome (-type
Subunit C9 (Feist et al. (1996) J. Exp. Med. 184: 1
313-1318), scleroderma antigen Rpp30, Rpp38 or Scl-70
(Eder et al. (1997) Proc. Nat'l. Acad. Sci. USA 9
4: 1101-1106; Hietarinta et al. (1994) Br. J. Rh
eumatol. 33: 323-326), centrosome autoantigen PCM-1 (Bao
(1995) Autoimmunity 22: 219-228), polymyositis-scleroderma autoantigen (PM-Scl) (Kho et al. (1997) J. Biol. C).
hem. 272: 13426-13431), scleroderma (and other systemic autoimmune diseases) autoantigen CENP-A (Muro et al. (1996) Clin. Immun).
ol. Immunopathol. 78: 86-89), U5, micronucleus ribonucleoprotein (snRNP) (Okano et al. (1996) Clin. Immunol.
Immunopathol. 81: 41-47), 10 of PM-Scl autoantigens.
0 kd protein (Ge et al. (1996) Arthritis Rheum. 39
: 1588-1595), U3- and Th (7-2) ribonucleoproteins of nucleoli (Verheijen et al. (1994) J. Immunol. Methods).
169: 173-182), ribosomal protein L7 (Neu et al. (1995)
Clin. Exp. Immunol. 100: 198-204), hPop1 (
Lygerou et al. (1996) EMBO J. 15: 5936-5948), and a 36-kd protein derived from nuclear matrix antigen (Deng et al. (1996)
Arthritis Rheum. 39: 1300-1307).

[0086] Liver autoimmune disorders can also be treated with the improved recombinant antigens prepared according to the methods described herein. Among the antigens useful in such treatments are cytochrome P450 and UDP glucuronosyltransferase (Obermayer-Straub and Manns (1996) Bai.
llieres Clin. Gastroenterol. 10: 501-53
2), cytochromes P450 2C9 and P450 1A2 (Bourdi et al. (
1996) Chem. Res. Toxicol. 9: 1159-1166; Cl
elemente et al. (1997) J. Am. Clin. Endocrinol. Metab
. 82: 1353-1361), LC-1 antigen (Klein et al. (1996) J. Am.
Pediatr. Gastroenterol. Nutr. 23: 461-46
5), and a 230 kDa Golgi-related protein (Funaki et al. (199)
6) Cell Struct. Funct. 21: 63-72).

Useful antigens for the treatment of autoimmune disorders of the skin include, but are not limited to: a 450 kD human epithelial autoantigen (Fujiwara et al. (1996) J. Invest. Dermatol. 106: 1125-113
0), 230 kD and 180 kD bullous pemphigoid antigens (Hashimoto
(1995) Keio J. et al. Med. 44: 115-123; Murakami
(1996) J. Am. Dermatol. Sci. 13: 112-117), pemphigus foliaceus antigen (desmoglein 1), pemphigus vulgaris antigen (desmog)
lein 3), BPAg2, BPAg1, and collagen VII (Bat
teux et al. (1997) J. Mol. Clin. Immunol. 17: 228-233
Hashimoto et al. (1996) J .; Dermatol. Sci. 12: 1
0-17), a 168 kDa mucosal antigen in a subset of patients with pemphigoid scarring (Ghohestani et al. (1996) J. Invest. Derm.
atol. 107: 136-139), as well as a 218 kd nuclear protein (2
18-kd Mi-2) (Seelig et al. (1995) Arthritis R)
heum. 38: 1389-1399).

The methods of the present invention are also useful for obtaining improved antigens for the treatment of insulin-dependent diabetes mellitus using one or more antigens, including but not limited to: insulin, Proinsulin, GAD65 and GAD67, heat shock protein 65 (hsp65), and islet cell antigen 69 (ICA69)
(French et al. (1997) Diabetes 46: 34-39; Roep
(1996) Diabetes 45: 1147-1156; Schroot et al. (1997) Doabetologia 40: 332-338), GAD65.
Viral proteins homologous to (Jones and Crosby (1996)
) Diabetologia 39: 1318-1324), islet cell antigen-related protein tyrosine phosphatase (PTP) (Cui et al. (1996) J. Bio.)
l. Chem. 271: 24817-24823), GM2-1 ganglioside (Cavallo et al. (1996) J. Endocrinol. 150: 113-).
120; Dotta et al. (1996) Diabetes 45: 1193-119.
6), glutamate decarboxylase (GAD) (Nepom (1995) C
urr. Opin. Immunol. 7: 825-830; Panina-Bo
rdignon et al. (1995) J. Mol. Exp. Med. 181: 1923-192
7), islet cell antigen (ICA69) (Karges et al. (1997) Biochim)
. Biophys. Acta. 1360: 97-101; Roep et al. (1996)
) Eur. J. Immunol. 26: 1285-1289), Tep69, a single T cell epitope recognized by T cells from diabetic patients (Karges
(1997) Biochim. Biophys. Acta 1360: 97-
101), ICA 512, a self-antigen of type I diabetes (Solimena et al. (
1996) EMBO J .; 15: 2102-2114), islet cell protein tyrosine phosphatase from type I diabetes and a 37 kDa autoantigen (IA-2, I
A-2) (La Gasse et al. (1997) Mol. Med. 3: 163).
173), a 64 kDa protein (I) derived from In-111 cells or human thyroid follicle cells immunoprecipitated with serum from a patient having islet cell surface antibodies (ICSA).
Gawa et al. (1996) Endocr. J. 43: 299-306), hogrin, a human transmembrane protein tyrosine phosphatase homolog, I
Autoantigens of type 2 diabetes (Kawasaki et al. (1996) Biochem. Bio).
phys. Res. Commun. 227: 440-447), 40 kD and 37 kDa tryptic fragments in IDDM and their precursors IA-2 and IA-2 (Lampasona et al. (1996) J. Immuno.
l. 157: 2707-2711; Notkins et al. Auto
immun. 9: 677-682), insulin or cholera toxin-insulin conjugates (Bergerot et al. (1997) Proc. Nat'l. Acad.
Sci. USA 94: 4610-4614), carboxypeptidase H, g
A human homolog of p330, which is a renal epithelial glycoprotein for Heymann nephritis induction in rats, and a 38 kD islet mitochondrial autoantigen (Ard
(1996) J. En. Clin. Invest. 97: 551-561).

[0089] Rheumatoid arthritis is another condition that can be treated with an optimized antigen prepared according to the present invention. Useful antigens for the treatment of rheumatoid arthritis include, but are not limited to: a 45 kDa DEK nuclear antigen specifically expressed in juvenile rheumatoid arthritis and iridocyclitis (Murray et al. (1997) J .
Rheumatol. 24: 560-567), human cartilage glycoprotein-39,
Autoantigen of rheumatoid arthritis (Verheijden et al. (1997) Arthr
itis Rheum. 40: 1115-1125), a 68k autoantigen in rheumatoid arthritis (Blas et al. (1997) Ann. Rheum. Dis.
56: 317-322), collagen (Rosloniec et al. (1995) J. Am.
Immunol. 155: 4504-4511), type II collagen (Cook)
(1996) Arthritis Rheum. 39: 1720-1727;
Trentham (1996) Ann. N. Y. Acad. Sci. 778: 3
06-314), cartilage link protein
tein) (Guerassimov et al. (1997) J. Rheumatol.
24: 959-964), ezulin, radixin and moesin (autoimmune antigen in rheumatoid arthritis) (Wagatsuma et al. (19)
96) Mol. Immunol. 33: 1171-1176), and mycobacterial heat shock protein 65 (Ragno et al. (1997) Arthri.
tis Rheum. 40: 277-283).

Also among conditions that can obtain improved antigens suitable for treatment are autoimmune thyroid disorders. Antigens useful for these applications include, but are not limited to, thyroid peroxidase and thyroid stimulating hormone receptor (Tandon and Weetman (1994) JR.
Coll. Physicians London. 28: 10-18), thyroid peroxidase from human graves thyroid tissue (Gardas et al. (1997) B
iochem. Biophys. Res. Commun. 234: 366-37
0; Zimmer et al. (1997) Histochem. Cell. Biol. 1
07: 115-120), a 64 kDa antigen associated with thyroid-associated ocular disorders (Zh
ang et al. (1996) Clin. Immunol. Immunopathol.
80: 236-244), human TSH receptor (Nicholson et al. (19)
96) J. Amer. Mol. Endocrinol. 16: 159-170), and In-11 immunoprecipitated with serum from patients with islet cell surface antibodies (ICSA).
One cell or a 64 kDa protein from human thyroid follicle cells (Igawa et al.
1996) Endocr. J. 43: 299-306).

Other conditions and related antigens include, but are not limited to: Sjogren's syndrome (-fodrin; Haneji et al. (1997) Scie
nce 276: 604-607), myasthenia gravis (human M2 acetylcholine receptor or a fragment thereof, particularly the second extracellular loop of human M2 acetylcholine receptor; Fu et al. (1996) Clin. Immunol. Immunol.
opolol. 78: 203-207), vitiligo (tyrosinase; Fishma)
(1997) Cancer 79: 1461-1464), a 450 kD human epithelial autoantigen recognized by serum from individuals with blistering skin disease and ulcerative colitis (chromosomal proteins HMG1 and HMG2; Sobajim).
a (1997) Clin. Exp. Immunol. 107: 135-140
).

6. Cancer Cancer has great promise for treating cancer and preventing metastasis. By inducing an immune response against cancerous cells, the body's immune system can be added to attenuate or eliminate cancer. The improved antigen obtained using the method of the present invention is currently
An immunotherapeutic method for cancer with improved efficacy compared to available antigens is provided.

One approach to immunotherapy of cancer involves the inclusion of antigens specific for tumor cells,
Or vaccination with an encoding vaccine, or vaccination by injecting a patient with purified recombinant cancer antigen. The methods of the present invention can be used to obtain antigens that exhibit an enhanced immune response to known tumor-specific antigens, and also to search for novel protective antigen sequences. Antigens with optimized expression, processing and presentation may be obtained as described herein. The approach used for each particular cancer can vary. For the treatment of hormone-sensitive cancers such as breast and prostate cancer, the methods of the invention can be used to obtain optimal hormone antagonists. For highly immunogenic tumors, including melanomas, one can screen for recombinant antigens that optimally boost the immune response to the tumor. In contrast, breast cancer is relatively less immunogenic and shows slow progression, so that individual treatments can be designed for each patient. Metastasis protection is also a goal in the design of cancer vaccines.

Among the tumor-specific antigens that can be used in the antigen shuffling method of the present invention are: Pemphigus pemphigoid antigen 2, prostate mucin antigen (PMA) (Beck
ett and Wright (1995) Int. J. Cancer 62:70
3-710), tumor-associated Thomsen-Friedenreich antigen (Da
hlenborg et al. (1997) Int. J. Cancer 70: 63-71
), Prostate specific antigen (PSA) (Dannul and Belldegrun (
1997) Br. J. Urol. 1: 97-103), luminal epithelial antigen (LEA.135) of breast and bladder transitional cell carcinoma (TCC) (Jones et al. (1997) A
nticancer Res. 17: 685-687), cancer-associated serum antigen (CA
SA) and cancer antigen 125 (CA125) (Kierkegaard et al. (199)
5) Gynecol. Oncol. 59: 251-254), epithelial glycoprotein 40 (EGP40) (Kievit et al. (1997) Int. J. Cancer).
71: 237-245), squamous cell carcinoma antigen (SCC) (Lozza et al. (199)
7) Anticancer Res. 17: 525-529), cathepsin E (
Mota et al. (1997) Am. J. Pathol. 150: 1223-1229
), Tyrosinase in melanoma (Fishman et al. (1997) Cancer 7).
9: 1461-1464), cell nucleus antigen of the cerebral cavity (PCNA) (Note
t et al. (1997) Surg. Neurol. 47: 364-370), DF3 /
MUC1 breast cancer antigen (Apostolopoulos et al. (1996) Immuno
l. Cell. Biol. 74: 457-464; Pandey et al. (1995)
Cancer Res. 55: 4000-4003), carcinoembryonic antigen (Pano)
e. (1996) J. Am. Cancer Res. Clin. Oncol. 122:
499-503; Schlom et al. (1996) Breast Cancer R.
es. Treat. 38: 27-39), tumor-associated antigen CA19-9 (Toll)
iver and O'Brien (1997) South Med. J. 90: 8
9-90; Tsuruta et al. (1997) Urol. Int. 58: 20-24
), Human melanoma antigens MART-1 / Melan-A27-35 and gp100
(Kawakami and Rosenberg (1997) Int. Rev. I.
mmunol. 14: 173-192; Zajac et al. (1997) Int. J.
Cancer. 71: 491-496), pan-cancer (CA) glycopeptide epitopes of T and Tn (Springer (1995) Crit. Rev. Oncog.
6: 57-85), 35 kD tumor-associated autoantigen (Lu) in papillary thyroid carcinoma
cas et al. (1996) Anticancer Res. 16: 2493-249
6), KH-1 adenocarcinoma antigens (Deshpande and Danishefsky (
1997) Nature 387: 164-166), A60 mycobacterial antigen (Maes et al. (1996) J. Cancer Res. Clin. Oncol).
. 122: 296-300), heat shock protein (HSP) (Blache
re and Srivastava (1995) Semin. Cancer Bi
ol. 6: 349-355), as well as MAGE, tyrosinase, melan (me).
lan) -A and gp75 and mutant oncogene products (eg, p53, ra
s and HER-2 / neu (Bueller and Mulligan (1996)
) Mol. Med. 2: 545-555; Lewis and Houghton (
1995) Semin. Cancer Biol. 6: 321-327; The
obald et al. (1995) Proc. Nat'l. Acad. Sci. USA
92: 11993-11997).

7. Contraception Genetic vaccines containing optimized antigens obtained according to the present invention are also useful for contraception. For example, a genetic vaccine that encodes a sperm cell-specific antigen and thus induces an anti-sperm immune response can be obtained. Vaccination can be achieved, for example, by administration of a recombinant bacterial strain that expresses sperm antigens (eg, Salmonella, etc.) and anti-hCG neutralizing antibodies by vaccination with a DNA vaccine encoding human chorionic gonadotropin (hCG) or a fragment thereof. Can be achieved.

Sperm antigens that can be used in genetic vaccines include, for example: lactate dehydrogenase (LDH-C4), galactosyltransferase (GT), SP-10, rabbit sperm autoantigen (RSA), guinea pig (g) )
PH-20, cleavage signal protein (CS-1), HSA-63, human (h)
PH-20, and AgX-1 (Zhu and Naz (1994) Arch. A
ndrol. 33: 141-144), synthetic sperm peptide, P10G (O'Ra
nd et al. (1993) J. Am. Reprod. Immunol. 25: 89-102)
, 135 kD, 95 kD, 65 kD, 47 kD, 41 kD and 23 kD of sperm
Protein and FA-1 antigen (Naz et al. (1995) Arch. Andr
ol. 35: 225-231) and a 35 kD fragment of cytokeratin 1 (Lukas et al. (1996) Anticancer Res. 16: 2493).
２４2496).

The method of the present invention can also be used to obtain a genetic vaccine that is specifically expressed in testis. For example, a polynucleotide sequence that directs the expression of a testis-specific gene can be used (eg, fertilized antigen 1). In addition to sperm antigen,
Oocytes expressed antigens or hormones that regulate reproduction may be useful targets for contraceptive vaccines. For example, genetic vaccines can be used to produce antibodies to gonadotropin-releasing hormone (GnRH) or zona pellucida proteins (Miller et al. (1997) Vaccine 15: 1858-1862.
). Vaccination with these molecules has been shown to be effective in animal models (Miller et al. (1997) Vaccine 15: 1858-
1862). Another example of a useful component of a genetic contraceptive vaccine is the ovarian zona pellucida glycoprotein ZP3 (Tung et al. (1994) Reprod. Fertil.
Dev. 6: 349-355).

(Method for Selection and Identification of Optimized Recombinant Antigen) Once shuffling of DNA is performed to obtain a library of polynucleotides encoding the recombinant antigen, the library can be selected and / or Screening identifies members of the library that encode antigenic peptides with improved ability to induce an immune response to the pathogenic agent. Selection and screening for recombinant polynucleotides encoding a polypeptide having an improved ability to elicit an immune response can include both in vivo and in vitro methods, but most often includes a combination of these methods. For example,
In an exemplary embodiment, the members of the library of recombinant nucleic acids are selected either separately or as a pool. This clone can be directly subjected to analysis or expressed to produce the corresponding polypeptide. In a presently preferred embodiment, an in vitro screen is performed to identify the best candidate sequences for in vivo studies. Alternatively, the library can be directly subjected to in vivo challenge studies. This analysis may use either the nucleic acid itself (eg, as a genetic vaccine) or the polypeptide encoded by the nucleic acid. A schematic of a representative strategy is shown in FIG. Both in vitro and in vivo methods are described in more detail below.

When the recombination cycle is performed in vitro, the products of the recombination (ie, the recombination segments) are sometimes introduced into the cells prior to the screening step. The recombinant segment can also be ligated to a suitable vector or other regulatory sequence prior to screening. Alternatively, products produced recombinantly in vitro are sometimes packaged in a virus (eg, bacteriophage) prior to screening. If the recombination is performed in vivo, the recombination products can sometimes be screened in cells in which the recombination occurs. In another application, the recombinant segments are extracted from the cells and optionally packaged as a virus prior to screening.

Often, improvements are obtained after a single recombination and selection. However, recursive sequence recombination can also be used to obtain yet further improvements in the desired properties or to bring about new (or "clear") properties. Inductive sequence recombination causes a continuous cycle of recombination to create molecular diversity. That is, a family of nucleic acid molecules that show some degree of sequence identity to one another, but differ in the presence of mutations, is created. In any given cycle, recombination can occur in vivo or in vitro, intracellularly or extracellularly. In addition, the diversity arising from recombination can be determined by applying previous mutagenesis methods (eg, error-prone PCR or cassette mutagenesis) to either the substrate or the product for recombination. It can be enhanced in any cycle.

In a presently preferred embodiment, the polynucleotide encoding the optimized recombinant antigen is subjected to backcrossing of the molecule. This backcross provides a means to cultivate shuffled chimeras / variants that revert to the parental or wild-type sequence while retaining mutations that are important for the phenotype that provides an optimized immune response. In addition to eliminating natural mutations, backcrossing of the molecules can also be used to characterize which large numbers of mutations in the improved variants contribute significantly to the improved phenotype. This cannot be achieved by any other method in an efficient library fashion. Backcrossing is performed by shuffling the improved sequence with the excess parent sequence of the macromolecule.

The nature of the screening or selection depends on which property or feature is obtained or which property or feature is sought for improvement, and a number of examples are discussed below. . Understanding the basis of a molecule in which a particular product of recombination has acquired new or improved properties or characteristics relative to the starting substrate is usually a matter of understanding.
Not necessary. For example, a gene encoding an antigenic polypeptide can have multiple component sequences, each with a different intended role (see, eg, FIG. 4).
checking). Each of these component sequences can be changed and recombined simultaneously. Screening / selection can then be performed, for example, on recombinant segments with an enhanced ability to induce an immune response against the virulence factor (such improvement due to any individual component sequence of the recombinant polynucleotide). Without having to do that).

[0103] Depending on the particular screening protocol used for the desired properties, the first round of screening can sometimes be performed with bacterial cells for high transfection efficiency and ease of culture. However, particularly for testing for immunogenic activity, test animals are used for library expression and screening. Similar other types of screening where screening is unacceptable in cells of a bacterial or simple eukaryotic library are performed on cells selected for use in an environment that is closer to the environment of the library's intended use Is done. The final round of screening may be performed on cells or organisms as close as possible to the exact cell type or organism intended for use.

If further improvements in properties are desired, at least one of the recombinant segments remaining in the first round of screening / selection, and the collection, are usually subjected to a further round of recombination. These recombinant segments can be recombined with each other or with exogenous segments that exhibit the original substrate or further modifications thereof.
Furthermore, recombination can proceed in vitro or in vivo. If the previous screening step identifies the desired recombinant segment as a component of the cell, this component can be subjected to further recombination in vivo, or to further recombination in vitro, or It can be isolated before performing the round. Conversely, if previous screening steps identified the desired recombinant segments in naked form or as components of the virus, these segments could be introduced into cells to perform a round of in vivo recombination. Regardless of how it is performed, the second round of recombination produces additional recombinant segments that include more diversity than exists in the recombinant segments obtained from previous rounds.

The second round of recombination may be followed by a further round of screening / selection according to the principles discussed above for the first round. Screening / selection stringency may increase from round to round. Also, the nature of the screening and the properties being screened may change from time to time, if a plurality of properties are desired to be improved, or if a plurality of new properties are desired to be obtained. Further rounds of recombination and screening may then be performed until the recombination segment has evolved sufficiently to acquire the desired new or improved property or function.

The practice of the present invention involves the construction of recombinant nucleic acids and the expression of genes in transformed host cells. Molecular cloning techniques to achieve these ends are known in the art. A wide variety of cloning and in vitro amplification methods suitable for the construction of recombinant nucleic acids (eg, expression vectors) are well known to those of skill in the art.
General text describing molecular biology techniques (including mutagenesis) useful herein include: Berger and Kimmel, G.
Uide to Molecular Cloning Technologies
, Methods in Enzymology, Volume 152, Academi
c Press, Inc. , San Diego, CA (Berger); Sa
mbrook et al., Molecular Cloning-A Laborato.
ry Manual (2nd edition), Volumes 1-3, Cold Spring Har
Bor Laboratory, Cold Spring Harbor, Ne
w York, 1989 ("Sambrook") and Current Pr.
otocols in Molecular Biology, F.C. M. Aus
ed. ubel et al., Current Protocols, Greene Publ
ising Associates, Inc. And John Wiley & So
ns, Inc. Joint venture (supplement from 1998) ("Ausubel"). Including polymerase chain reaction (PCR), ligase chain reaction (LCR), Q-replicase amplification and other RNA polymerase-mediated techniques (eg, NASBA),
Examples of techniques that are sufficient to direct one of skill through the in vitro amplification method are found below: Berger, Sambrook, and Ausubel, and Mullis et al. (1987) US Patent No. 4,683,202; PCR Pr
otocols A Guide to Methods and Appli
editions (edited by Innis et al.) Academic Press Inc. S
an Diego, CA (1990) (Innis); Arnheim and L.
evinson (October 1, 1990) C & EN 36-47; The Jo
urnal of NIH Research (1991) 3, 81-94;
Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86
Guatelli et al. (1990) Proc. Natl. Acad.
Sci. USA 87, 1874; Lomell et al. (1989) J. Am. Clin.
Chem 35, 1826; Landegren et al. (1988) Science.
241, 1077-1080; Van Brunt (1990) Biotec
hnology 8, 291-294; Wu and Wallace (1989).
Gene 4, 560: Barringer et al. (1990) Gene 89, 1.
17, and Sooknanan and Malek (1995) Biotech.
No. 13: 563-564. Improved methods for cloning in vitro amplified nucleic acids are described in Wallace et al., US Pat. No. 5,426,039. An improved method for amplifying large nucleic acids by PCR is described in Cheng et al. (1994).
Nature 369: 684-685 and references therein, where PCR amplicons of up to 40 kb are produced. One of skill in the art will appreciate any RNA
Can be essentially converted to double-stranded DNA suitable for restriction cleavage, PCR extension and sequencing using reverse transcriptase and polymerase. Ausu
See Bel, Sambrook and Berger (all supra).

For use as a probe (eg, in an in vitro amplification method)
Oligonucleotides for use as genetic probes or as shuffling targets (eg, synthetic genes or gene segments) are, for example, N
eedham-VanDevanter et al. (1984) Nucleic Aci.
ds Res. 12; 6159-6168, and using an automated synthesizer, typically in Beucage and Caruthers (1981) Tetra.
hedron Letts. , 22 (20): 1859-1862, chemically synthesized according to the solid-phase phosphoramidite triester method. Oligonucleotides can also be custom made and purchased from a variety of commercial sources known to those of skill in the art.

Indeed, any nucleic acid having a known sequence can be customized, essentially, from any of a variety of commercial sources, such as: The Midland Cer
tied Reagent Company (mcrc@oligos.c)
om), The Great American Gene Company (
http: // www. genco. com), ExpressGen Inc
. (Www.expressgen.com), Operon Technology
oils Inc. (Alameda, CA) and many others. Similarly,
Peptides and antibodies are described, for example, in PeptidoGenic (pkim @ ccn).
et. com), HTI Bio-products, Inc. (Http: //
/ Www. htbio. com), BMA Biomedicals Ltd
(UK), BioSynthesis, Inc. And many other various sources.

1. Purification and In Vitro Analysis of Recombinant Nucleic Acids and Polypeptides Once DNA shuffling has been performed, the resulting library of recombinant polynucleotides identifies the most promising candidate recombinant nucleic acids. To perform in vitro purification and preliminary analysis. Advantageously, the assay can be performed in a high-throughput format. For example, to purify individual shuffled recombinant antigens, clones are automatically selected into a 96-well format, expanded, and optionally frozen for storage.

Although whole cell lysates (V-antigen), periplasmic extracts or culture supernatants (toxins) can be analyzed directly by ELISA as described below,
Sometimes needed. Affinity chromatography (with immobilized metal affinity chromatography) using the incorporation of small non-immunogenic affinity tags, such as immobilized antibodies or hexahistidine peptides, allows for rapid protein purification. High binding capacity reagents with 96 well filter bottom plates provide a high throughput purification process. The size of the culture and purification depends on the protein yield, but initial studies require 50 μg
Less than a protein. Antigens that exhibit improved properties can be purified on a larger scale by FPLC for reassays and animal challenge studies.

In some embodiments, the shuffled antigen-encoding polynucleotides are assayed as a genetic vaccine. Genetic vaccine vectors containing shuffled antigen sequences can be prepared using automated colony selection and subsequent automated plasmid purification. An automated plasmid purification protocol is available that allows for purification of 600-800 plasmids per day. The amount and purity of the DNA can also be analyzed, for example, in a 96-well plate. In a presently preferred embodiment, the amount of DNA in each sample is automatically normalized, which can significantly reduce variability between different batches of vector.

Once the proteins and / or nucleic acids have been selected and purified as desired,
They can be subjected to any of a number of in vitro analytical methods. Such screens include, for example, phage display, flow cytometry, and ELISA assays to identify antigens that are efficiently expressed and have multiple epitopes and the proper folding pattern. In the case of bacterial toxins, the library can also be screened for reduced toxicity in mammalian cells.

As one example, a panel of monoclonal antibodies for screening can be used to identify cross-reactive recombinant antigens. A humoral immune response generally targets multiple regions of an antigenic protein. Thus, monoclonal antibodies can be raised against various regions of the immunogenic protein (Alving et al. (199).
5) Immunol. Rev .. 145: 5). In addition, there are several examples of monoclonal antibodies that recognize only one strain of a given pathogen. And, of course,
Different serotypes of the pathogen are recognized by different sets of antibodies. For example, a panel of monoclonal antibodies is raised against the VEE envelope protein, thus providing a means for recognizing different subtypes of the virus (R
Oehring and Bolin (1997) J. Am. Clin. Microbio
l. 35: 1887). Such antibodies (phage display and ELIS
A in combination with A screening) can be used to enrich recombinant antigens with epitopes from multiple pathogen strains. Cell sorting (cell sorting)
Based flow cytometry further allows the selection of the most efficiently expressed variants.

[0114] Phage display provides a powerful method of selecting proteins of interest from large libraries (Bass et al. (1990) Proteins: Struc).
t. Funct. Genet. 8: 309; Lowman and Wells (1
991) Methods: A Comparison to Methods
Enz. 3 (3); 205-216. Lowman and Wells (1993)
J.). Mol. Biol. 234; 564-578). Some current reviews on phage display technology include, for example: Mc;
Gregor (1996) Mol Biotechnol. 6 (2): 155
62; Dunn (1996) Curr. Opin. Biotechnol. 7 (
5): 547-53; Hill et al. (1996) Mol Microbiol 2
0 (4): 685-92; Phase Display of Peptide
s and Proteins: A Laboratory Manual. B
K. Kay, J .; Winter, J.M. Academi, edited by McCafferty
c Press 1996; O'Neil et al. (1995) Curr. Opin.
Struct. Biol. 5 (4): 443-9; Physicky et al. (199
5) Microbiol Rev. 59 (1): 94-123; Clackso
n et al. (1994) Trends Biotechnol. 12 (5): 173-
84; Felici et al. (1995) Biotechnol. Annu. Rev ..
1: 149-83; Burton (1995) Immunotechnology.
y1 (2): 87-94. ). See also Cwila et al., Proc. Natl.
Acad. Sci. USA 87: 6378-6382 (1990): Devl.
in et al., Science 249: 404-406 (1990), Scott and Smith, Science 249: 386-388 (1990); La.
See dner et al., US 5,571,698. Each phage particle displays a unique variant protein on its surface and packages the gene encoding that particular variant. The shuffled gene for the antigen
It is fused to a protein expressed on the phage surface (eg, gene III of phage M13) and cloned into a phagemid vector. In a presently preferred embodiment, a repressible stop codon (eg, an amber stop codon)
Isolates the gene, resulting in E. coli. coli. Antigen-g
A fusion of IIIp is produced upon infection of M13 helper phage and incorporated into the phage particle. The same vector has non-repressible E. coli for protein purification.
E. coli can direct the production of the unfused antigen alone.

The most commonly used genetic packages for display libraries are bacteriophages (in particular, filamentous phages), and especially phages M13, Fd and F1. Most studies have focused on the gII of these phages to form fusion proteins.
Inserting a library encoding a polypeptide displayed in either I or gVIII. For example, Dower, WO 91/1981
8; Devlin, WO 91/18989; MacCafferty, WO
92/01047 (gene III); Huse, WO 92/06204; Ka
ng, WO 92/18619 (gene VIII). Such fusion proteins (usually, but not necessarily) include phage coat proteins,
Includes the displayed polypeptide and signal sequences from either the gene III or gene VIII proteins or fragments thereof. The exogenous coding sequence is often inserted at or near the N-terminus of gene III or gene VIII, although other insertion sites are possible.

[0116] Eukaryotic viruses can be used to present polypeptides in a similar manner. For example, the presentation of human heregulin fused to the Moloney murine leukemia virus gp70 is described in Han et al., Proc. Natl. Aca
d. Sci. USA 92: 9747-9751 (1995). Spores can also be used as replicable genetic packages. In these cases, the polypeptide is presented from the outer surface of the spore. For example, B. subt
Spores from ilis have been reported to be suitable. The sequence of the coat protein of these spores is described in Donovan et al. Mol. Biol. 196, 1
To 1987 (1987). Cells can also be used as replicable genetic packages. The displayed polypeptide is inserted into a gene encoding a cellular protein expressed on the cell surface. Salmonella typhi
murium, Bacillus subtilis, Pseudomonas
aeruginosa, Vibrio cholerae, Klebsiel
la pneumonia, Neisseria gonorrhoeae, N
eisseria meningitidis, Bacteroides no
dosus, Moraxella bovis and especially Escherichi
Bacterial cells containing a coli are preferred. For more information on external surface proteins, see Lad
ner et al., US Pat. No. 5,571,698 and the references cited therein. For example, E. coli. The lamB protein is suitable.

The basic concept of a display method using a phage or other replicable genetic package is the establishment of a physical association between the DNA encoding the polypeptide to be screened and the polypeptide. This physical association is provided by a replicable genetic package. This genetic package displays the polypeptide encoded by the genome as part of a capsid containing the genome of a phage or other package. Establishing physical associations between polypeptides and their genetic material allows for the simultaneous population screening of a large number of phage carrying different polypeptides. Phage displaying the polypeptide with affinity for the target (eg, receptor) bind to the target, and these phage are enriched by affinity screening for the target. The identity of the polypeptides displayed from these phages can be determined from their respective genomes.
Using these methods, if the identified polypeptide has binding affinity for the desired target, it can then be synthesized in bulk by conventional means. Alternatively, the polynucleotide encoding the peptide or polypeptide can be used as part of a genetic vaccine.

Variants with specific binding properties (in this case, binding to family-specific antibodies) are readily enriched by panning with immobilized antibodies. Antibodies specific to one family are used in each round of panning to rapidly select variants with multiple eptoops from the antigen family. For example, A family-specific antibodies can be used to select shuffled clones that display A-specific epitopes in the first round of panning. The second round of panning with B-specific antibodies selects from the "A" clones those that display both A-specific and B-specific epitopes. The third round of panning with C-specific antibodies selects for variants with A, B, and C epitopes. E. FIG. For clones that express well in E. coli and are stable through selection, there is a continuous selection during this process. Improvements in factors such as transcription, translation, secretion, folding, and stability are often observed and enhance the utility of the selected clone for use in vaccine production.

The phage ELISA method can be used to rapidly characterize individual variants. These assays provide a rapid method for quantification of variants without requiring purification of each protein. Individual clones are placed in 96-well plates, expanded and frozen for storage. Cells in duplicate plates are infected with helper phage, grown overnight, and pelleted by centrifugation. The supernatant containing the phage displaying the particular variant is incubated with the immobilized antibody, and the bound clone is detected by an anti-M13 antibody conjugate. The titration series of phage particles, immobilized antigen, and / or soluble antigen competition binding studies are all highly effective tools for quantifying protein binding. Variant antigens displaying multiple epitopes are further studied in appropriate animal challenge models.

Several groups have reported in vitro ribosome display systems for screening and selecting for mutant proteins with desired properties from large libraries. This technique can also be used to phage display to select or enrich for variant antigens with improved properties (eg, improved cross-reactivity to antibodies and improved folding) (eg, Hanes
(1997) Proc. Nat'l. Acad. Sci. USA 94 (10
): 4937-42; Mattheakis et al., (1994) Proc. Nat
'l. Acad. Sci. ScL USA 91 (19): 9022-6; He et al. (19)
97) Nucl. Acids Res. 25 (24): 5132-4; Nemo
(1997) FEBS Lett. 414 (2): 405-8).

There are other display methods for screening antigens for improved properties (eg, increased expression levels, extensive cross-reactivity, folding and stability). These include intact E. coli. coli, or display of proteins in other cells, such as, but not limited to, F.
rancisco et al. (1993) Proc. Nat'l. Acad. Sci. U
SA 90: 1044-10448; Lu et al. (1995) Bio / Techno.
13: 366-372). Fusion of a shuffled antigen to a DNA binding protein can link the antigen protein to its gene with an expression vector (S
Chatz et al. (1996) Methods Enzymol. 267: 171-
91; Gates et al. Mol. Biol. 255: 373-86
).

A variety of display methods and ELISA assays may provide improved properties (eg, presentation of multiple epitopes, improved immunogenicity, increased expression levels, increased folding speed and efficiency, factors (eg, temperature, buffer, etc.)). , Solvents, improved purification properties, etc.) can be used to screen for shuffled antigens having increased stability. Selection of shuffled antigens with improved expression, folding, stability, and purification profile under various chromatographic conditions can be very important improvements to incorporate for the vaccine manufacturing process.

[0122] Flow cytometry is a useful technique for identifying recombinant antigenic polypeptides that exhibit improved expression in host cells. Flow cytometry is
It provides a way to efficiently analyze the functional properties of millions of individual cells. The expression levels of several genes can be analyzed simultaneously, and cell sorting based on flow cytometry allows the selection of cells that present antigen variants that are properly expressed on the cell surface or in the cytoplasm. A very large number (> 10 ⁷ ) of cells can be evaluated in a single vial experiment, and the best pool of individual sequences can be recovered from the sorted cells. These methods are particularly useful, for example, in the case of Hunter virus glycoprotein. It is generally very poorly expressed in mammalian cells. This approach provides a common solution to improve the expression levels of pathogenic antigens in mammalian cells, a phenomenon that is critical to the function of genetic vaccines.

To use flow cytometry to analyze polypeptides that are not expressed on the cell surface, a recombinant polynucleotide is
The polynucleotide can be engineered to be expressed as a fusion protein having a region of amino acids targeted to the cell membrane. For example, this region signals the attachment of the inositol-glycan (PIG) terminus on the expressed protein and directs the protein to be expressed on the surface of transfected cells. Stretch can be coded (Whitehorn
(1995) Biotechnology (NY) 13: 1215-9). When using antigens that are essentially soluble proteins, this method probably does not affect the three-dimensional folding of the protein in this engineered fusion with a novel C-terminus. When using antigens that are essentially transmembrane proteins (eg, surface membrane proteins in pathogenic viruses, bacteria, protozoa, or tumor cells), there are at least two possibilities. First, the extracellular domain can be engineered to be fused to a C-terminal sequence for PIG-linked signaling. Second, the protein can be expressed in toto dependent on host cell signaling to efficiently direct it to the cell surface. In a few cases, the antigen for expression has an endogenous PIG-terminal junction (eg, some antigens of the pathogenic protozoan subdivision).

[0125] Cells expressing the antigen can be identified using a fluorescent monoclonal antibody specific for the C-terminal sequence in the PIG-linked form of the surface antigen. FACS analysis allows a quantitative assessment of the level of expression of the correct form of the antigen in the cell population. Cells expressing the highest levels of antigen are selected, and standard molecular biology methods are used to recover the plasmid DNA vaccine vector that has given this reaction. An alternative procedure that allows purification of all cells expressing the antigen (and may be useful prior to addition to a cell sorter because antigen expressing cells can be a very small minority population) is a surface antigen Is rosetting or panning purification. Rosettes can be formed between antigen-expressing cells and red blood cells with covalent antibodies to the relevant antigen. These are rapidly purified by unit gravity settling. Panning of cell populations in petri dishes with immobilized monoclonal antibodies specific for the relevant antigen can also be used to remove unwanted cells.

With the high throughput assays of the present invention, it is possible to screen up to thousands of different shuffled variants in a single day. For example, each well of a microtiter plate can be used to perform a separate assay, or if observing the effect of concentration or incubation time, every 5-10 wells:
One variant may be tested. Thus, one standard microtiter plate can assay about 100 (eg, 96) reactions. If a 1536 well plate is used, one plate can easily assay from about 100 to about 1500 different reactions. It is possible to assay several different plates per day. Assay screens (i.e., including different nucleic acids, encoded proteins, concentrations, etc.) for up to about 6,000-20,000 different assays are possible with the integrated system of the present invention. More recently, microfluidic approaches to reagent manipulation have been developed (
For example, Caliper Technologies (Palo Alto, C
A)).

In one aspect, library members (eg, cells, viral plaques, etc.) are separated on a solid medium to generate individual colonies (or plaques). Automated colony collection devices (eg, Q-bot, Genetix, U
. K. ), Colonies or plaques are identified, lifted, and up to 10,000 different mutants are inoculated into 96-well microtiter dishes (if necessary, glass in wells to prevent clumping) Put the ball). Qb
It does not pick up the entire colony, but rather inserts a pin through the center of the colony and terminates with a small sample of cells (or virus in plaque smear). Each of the time that the pins are in the colony, the number of dips to inoculate the culture medium, and the time that the pins are in the medium affects the inoculation size, and each can be adjusted and optimized. The uniform process of Q-bot reduces human error and increases the rate of culture establishment (approximately 1
000/4 hours). These cultures are then shaken in a temperature and humidity controlled incubator. The glass spheres in the microtiter plate, like the blades in the fermenter, act to promote uniform cell aeration, cell dispersion, and the like. Clones from the culture of interest can be cloned by limiting dilution. The plaques or cells that make up the library can also be directly screened for protein production by detecting any of hybridization, protein activity, protein binding to the antibody, and the like.

The ability to detect that the performance of a shuffled library member has been slightly increased over that of the parent strain depends on the sensitivity of the assay. The chance of finding an organism with an improvement in the ability to elicit an immune response is increased by the number of individual variants that can be screened by the assay. To increase the chance of identifying a pool of sufficient size, a prescreen that increases the number of mutants processed by 10-fold may be used. The goal of pre-screening is to quickly identify mutants with equal or better production values compared to the parental strain, and to transfer only these mutants to liquid cell culture for subsequent analysis .

Many well-known robotic systems have also been developed for solution phase chemistry useful in assay systems. These systems are based on Takeda Chem
ical Industries, LTD. Automated workstations such as the automated synthesizer developed by (Osaka, Japan), and many robotic systems (Zymate II, Zymark Corporation, etc.) that utilize robotic arms to mimic the manual synthesis operations performed by scientists.
Hopkinton, Mass. Orca, Hewlett-Packard;
, Palo Alto, Calif). Any of the devices described above are suitable for use with the present invention, eg, for high-throughput screening of molecules encoded by codon-altered nucleic acids. As they can operate as discussed herein with respect to the integrated system,
The nature and implementation (if any) of the variants for these devices will be apparent to those skilled in the art.

[0130] High-throughput screening systems are commercially available (eg, Zymark).
Corp .. , Hopkinton, MA; Air Technical In
industries, Mentor, OH; Beckman Instrumentum
ts, Inc. Fullerton, CA; Precision System
s, Inc. , Natick, MA, etc.). These systems are
Typically, the entire procedure is automated, including all sample and reagent pipetting, liquid dispensing, periodic incubations, and final reading of the microplate in a detector appropriate for the assay. These deployable systems provide high throughput and fast startup, and a high degree of variability and customization.

Manufacturers of such systems provide detailed protocols for various high throughputs. Therefore, for example, Zymark Corp. Provides a technical publication describing a screening system for detecting modulation of gene transcription, ligand binding, and the like. Microfluidic approaches to reagent manipulation have also been developed (e.g., Caliper Technologies (Palo Al
to, CA)).

The optical image displayed (and, optionally, recorded) by a camera or other recording device (eg, a photodiode and a data storage device) may be used, if necessary, in accordance with the embodiments herein. In either case, the image is further processed, for example, by digitally processing the image and / or storing and analyzing the image on a computer. As mentioned above, in some applications, the signal resulting from the assay is fluorescent, and in these cases, an optical detection approach is appropriate. A variety of commercially available peripheral devices and software are available for digitizing, storing, and analyzing digitized video or digitized optical images (eg, PC (Intel x86 or Pentium chip compatible DOS, OS)
2 WINDOWS, WINDOWS NT or WINDOWS 95 based machines), MACINTOSH, or UNIX based (eg, using a SUN workstation) computer).

One conventional system carries light from an assay device to a cooled charge-coupled device (CCD) camera conventional in the art. A CCD camera includes an array of pixels. Light from the sample is imaged on the CCD. The area of the sample (for example,
Particular pixels corresponding to individual hybridization sites on an array of biological polymers) are sampled to obtain a light intensity reading at each location. Multiple pixels are processed in parallel, increasing speed. The devices and methods of the present invention are readily used to display any sample, for example, by fluorescence microscopy or darkfield microscopy techniques.

The analytical integration system of the present invention typically moves high-throughput liquid control software, image analysis software, data interpretation software, and solutions from a source to a receiver operably linked to a digitizing computer. Robotic liquid control armature, an input device (eg, a computer keyboard) for inputting data to a digital computer to control high-throughput liquid transfer by the robotic liquid control armature, and, optionally, a labeling assay Includes a digitizing computer with an image scanner to digitize the label signal from the components. The image scanner interfaces with image analysis software and provides optical intensity measurements. Typically, intensity measurements are interpreted by data interpretation software to indicate whether an optimized recombinant antigenic polypeptide product is produced.

2. Antigen Library Immunization In a presently preferred embodiment, antigen library immunization (ALI) is used to identify optimized recombinant antigens with improved immunogenicity. ALI
Involves introducing into a test animal a library of recombinant antigen-encoding nucleic acids, or a recombinant antigen encoded by the shuffled nucleic acids. This animal is
It is then subjected to an in vivo challenge using the live pathogen. Neutralizing antibodies and cross-protective immune responses are studied after immunization with the entire library, pool, and / or individual antigen variants.

[0136] Methods for immunizing test animals are well known to those of skill in the art. In a currently preferred embodiment, test animals are immunized twice or three times at two week intervals. 1 since last immunization
After a week, the animal is challenged with a live pathogen (or mixture of pathogens) and the animal continues to survive and condition. Immunization using a test animal challenge can be performed, for example, using R
Oggenkamp et al. (1997) Infect. Immun. 65: 446;
Woody et al., (1997) Vaccine 2: 133; Agren et al., (1
997). Immunol. 158: 3936; Konishi et al. (1992)
) Virology 190: 454; Kinney et al. (1988) J. Am. Vir
ol. 62: 4697; Iacono-Connors et al. (1996) Viru.
s Res. 43: 125; Kochel et al. (1997) Vaccine 15
: 547; and Chu et al. (1995) J. Am. Virol. 69: 6417.

Immunization can be performed either by injecting the recombinant polynucleotide itself (ie, as a genetic vaccine), or by immunizing an animal with the polypeptide encoded by the recombinant polynucleotide. Bacterial antigens are typically screened primarily as recombinant proteins, while
Viral antigens are preferably analyzed using genetic vaccination.

To dramatically reduce the number of experiments required to identify individual antigens with improved immunogenic properties, pooling and deconvolution as illustrated in FIG. Can be used. The recombinant nucleic acid or a pool of polypeptides encoded by the recombinant nucleic acid is used to immunize a test animal. The pool that produces protection against the challenge is then subdivided and subjected to further analysis. The high throughput in vitro approach described above can be used to identify the best candidate sequence for in vivo studies.

Challenge models that can be used to screen for protective antigens include pathogenic and toxin models (eg, Yersinia bacteria, bacterial toxins (eg, Staphylococcal and Streptococcal enterotoxins, E. coli / V. Cholerae enterotoxins), Venezuelan equine encephalitis virus (VEE), flavivirus genus (Japanese encephalitis virus, tick-borne encephalitis virus, dengue virus), hunter virus, herpes simplex, influenza virus (eg, influenza A virus), vesicular stomatitis virus, Pseudomonas aeruginosa (P
pseudomonas aeruginosa) and Salmonella typhimurium (Salmo)
nella typhimurium, Escherichia c
oli), Klebsiella pneumoniae, Toxoplasma gondii, Plasmodium (Plasm)
odium yoelii), Herpes simplex
, Influenza viruses (eg, influenza A virus), as well as vesicular stomatitis virus)). However, test animals can also be challenged with tumor cells to allow screening for antigens that effectively protect against malignancy. An individual shuffled antigen or pool of antigens is introduced into the animal intradermally, intramuscularly, intravenously, intratracheally, intraanally, intravaginally, orally, or intraperitoneally; and Antigens that can prevent the disease are selected for further rounds of shuffling and selection, if desired. Finally, based on in vivo data in test animals and in vitro studies in animals and humans, the most potent antigens were selected for human trials and their ability to prevent and treat human disease was investigated. You.

In some embodiments, immunization and pooling of individual clonal antigen libraries is used to immunize against pathogenic strains that were not included in the sequences used to generate the library. Is done. The level of cross-protection provided by different strains of a given pathogen can be significant. However, homogeneous titers are always higher than heterogeneous titers. Pooling and deconvolution are particularly efficient in models where minimal protection is provided by the wild-type antigen used as starting material for shuffling (eg, antigen A against strain C in FIG. 3B). And B). This approach has been used, for example, in the genus Yersinae.
Alternatively, it can be obtained when the V antigen of Hunter virus glycoprotein is evolved.

[0141] In some embodiments, the desired screening involves analysis of an immune response based on immunological assays known to those of skill in the art. Typically, test animals are immunized first, and blood or tissue samples are collected, for example, 1-2 weeks after the last immunization. These studies, in addition to determining whether an antigen or pool of antigens provide protective immunity, also include protective immunity (eg, induction of specific antibodies (especially IgG) and induction of specific T lymphocyte responses). ) Can be measured. The spleen cells or peripheral blood mononuclear cells can be isolated from the immune test animal,
The presence of antigen-specific T cells and induction of cytokine synthesis can then be measured. ELISA, ELISPOT, and cytoplasmic cytokine staining can provide such information at the single cell level in combination with flow cytometry.

Common immunological tests that can be used to identify immunity efficacy include antibody measurements, neutralization assays, and activation levels or frequencies of antigen-presenting cells or lymphocytes specific for the antigen or pathogen. Analysis. Test animals that can be used for such studies include, but are not limited to, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, pigs, and monkeys. Monkeys are a particularly useful test animal. This is because monkey and human MHC molecules are very similar.

Virus neutralization assays are useful for the detection of antibodies that not only specifically bind to the pathogen, but also neutralize the function of the virus. These assays are typically based on the detection of antibodies in the serum of immunized animals and analysis of those antibodies for their ability to inhibit viral growth in tissue culture cells. Such assays are known to those skilled in the art. One example of a virus neutralization assay is Dolin R (
J. Infect. Dis. 1995, 172: 1175-83). Virus neutralization assays provide a means to screen for antigens that also provide protective immunity.

[0144] In some embodiments, the shuffled antigens are screened for their ability to induce T cell activation in vivo. More specifically, peripheral blood mononuclear cells or spleen cells from injected mice can be isolated and the ability of cytotoxic T lymphocytes to lyse infected autologous target cells is studied. Spleen cells can be reactivated in vitro with a specific antigen. Furthermore, T-helper cell activation and differentiation, cell proliferation or T _H 1 (IL-2 and IFN-gamma) and T _H 2 (IL-4 and IL-5) the production of cytokines, by ELISA, and cytoplasmic cytokine It is analyzed by direct measurement in CD4 ⁺ T cells by staining and flow cytometry. Based on the cytokine production profiles, also changes in the ability of the antigen to direct T _H 1 / T _H 2 differentiation (
For example, IL-4 / IFN-γ, IL-4 / IL-2, IL-5 / IFN-γ,
(As indicated by changes in the ratio of IL-5 / IL-2, IL-13 / IFN-γ, IL-13 / IL-2). Analysis of T cell activation induced by antigen variants is a very useful screening method. This is because strong activation of specific T cells in vivo correlates with induction of protective immunity.

The frequency of antigen-specific CD8 ⁺ T cells in vivo can also be analyzed directly using tetramers of MHC class I molecules expressing specific peptides from the corresponding pathogenic antigen (Ogg and McMichael) , Curr.Opin, Im
munol. 1998, 10: 393-6); Altman et al., Science.
1996, 274: 94-6). Tetramer binding can be detected using flow cytometry and provides information on the potency of the shuffled antigen in inducing specific T cell activation. For example, flow cytometry and tetramer staining provide an efficient way to identify T cells that are specific for a given antigen or peptide. Another method involves panning using tetramer-coated plates with specific peptides. Although this method allows for the handling of large numbers of cells in a short period of time, the method only selects for the highest expression levels. The higher the frequency of antigen-specific T cells in vivo, the more efficient immunization will allow the identification of antigen variants with the strongest ability to elicit a protective immune response. These studies are particularly useful when performed on monkeys or other primates. This is because human MHC class I molecules are more similar to other primates than to mice.

Measurement of activation of antigen presenting cells (APCs) in response to immunization with antigen variants is another useful screening method. Induction of APC activation is based on changes in surface expression levels of activating antigens (eg, B7-1 (CD80), B7-2 (CD86), MHC Class I and II, CD14, CD23, and Fc receptor). Can be detected.

Shuffled cancer antigens that induce cytotoxic T cells capable of killing cancer cells can be identified by measuring the ability of T cells from immunized animals to kill cancer cells in vitro. . Typically, cancer cells are first labeled with a radioisotope, and the release of radioactivity is indicative of tumor cell killing after incubation in the presence of T cells from the immunized animal. Such cytotoxicity assays are known in the art.

For example, an indication of the efficacy of an antigen that activates T cells that are specific for a cancer antigen, allergen, or autoantigen is also indicative of the degree of skin inflammation when the antigen is injected into the skin of a patient or test animal. It is. Strong inflammation correlates with strong activation of antigen-specific T cells. Improved activation of tumor-specific T cells may lead to enhanced tumor killing. For self-antigens, but may be added immunomodulator that distort the response to T _H 2, the case of allergens, T _H 1 response is desired. Skin biopsies can be taken, which allows for a detailed study of the type of immune response generated at each injection site (in mice and monkeys, multiple injections / antigens can be analyzed). Such studies involve detecting changes in the expression of cytokines, chemokines, accessory molecules, etc. by cells upon injection of the antigen into the skin.

[0149] Peripheral blood mononuclear cells from a previously infected or immunized human individual can be used to screen for antigens with optimal ability to activate antigen-specific T cells. This is a particularly useful method. This is because the MHC molecule presenting the antigenic peptide is a human MHC molecule. Peripheral blood mononuclear cells or purified professional antigen presenting cells (APCs) can be isolated from previously vaccinated or infected individuals, or from patients with acute infection with the pathogen of interest. These individuals increase the frequency of circulating pathogen-specific T cells, and antigens expressed in PBMCs or purified APCs of these individuals increase proliferation and cytokine production by antigen-specific CD4 ⁺ and CD8 ⁺ T cells. Induce. Thus, antigens having epitopes from several antigens simultaneously are recognized by their ability to stimulate T cells from various patients infected or immunized with different pathogenic antigens, cancer antigens, autoantigens, or allergens Can be done. One buffy coat from a blood supplier contains lymphocytes from 0.5 liters of blood,
The resulting PBMC were obtained up to 10 ^4, to allow very large screening experiments that use T cells from one of the suppliers.

When healthy vaccinated individuals (experimental volunteers) are studied, EBV transformed B cell lines can be generated from these individuals. These cell lines can be used as antigen-specific cells in subsequent experiments using blood from the same donor; this reduces inter-assay and inter-donor variability. Furthermore, when the antigen variant is EBV
After introduction into transformed B cells, antigen-specific T cell clones can be generated. The efficiency of the transformed B cells to induce the expansion of specific T cell clones is then studied. When studied with specific T cell clones, the proliferation and cytokine synthesis response is significantly higher than with whole PBMC. This is because the frequency of antigen-specific T cells between PBMCs is very low.

[0151] CTL epitopes can be presented by most cell types. This is because class I major histocompatibility complex (MHC) surface glycoproteins are widely expressed. Thus, transfection of cells in culture with a library of shuffled antigen sequences in a suitable expression vector can result in presentation of class I epitopes. If a specific CTL directed to a given epitope is isolated from an individual, co-culture of CTL with the transfected presenting cells will result in cytokines (eg, IL-2, IFN-γ) if the epitope is presented. , Or TNF) by CTL. Higher amounts of released TNF correspond to more efficient processing and presentation of class I epitopes from shuffled and evolved sequences. Shuffled antigens that induce cytotoxic T cells capable of killing infected cells can also be identified by measuring in vitro the ability of T cells from immunized animals to kill infected cells. Typically, target cells are first labeled with a radioisotope, and the release of radioactivity is indicative of target cell killing after incubation in the presence of T cells from the immunized animal. Such cytotoxicity assays are known in the art.

The second method for identifying optimized CTL epitopes does not require isolation of CTL that reacts with the epitope. In this approach, class I MHC
Cells expressing the surface glycoprotein are transfected with the library of evolved sequences as described above. After appropriate incubation to allow processing and presentation, detergent soluble extracts are prepared from each cell culture, and after partial purification of the MHC-epitope complex, the (possibly optional) product is transferred to a mass spectrometer. (Henderson et al. (1993) Proc. Nat'l.A.
cad. Sci. ScL USA 90: 10275-10279). Since the sequence of the epitope whose presentation is to be increased is known, mass spectrometry can be calibrated to identify this peptide. In addition, cellular proteins can be used for internal calibration to obtain quantitative results; cellular proteins used for internal calibration are M
It can be the HC molecule itself. Thus, the amount of bound peptide epitope can be measured as a percentage of MHC molecules.

(Use of Multivalent Recombinant Antigen) The multivalent antigen of the present invention is useful for treating and / or preventing various diseases and conditions associated with each antigen. For example, a multivalent antigen can be expressed in a suitable host cell line and administered in a polypeptide form. Suitable formulations and dosing regimes for vaccine delivery are well known to those skilled in the art.

In a currently preferred embodiment, the optimized recombinant polynucleotide encoding the improved allergen is used in combination with a genetic vaccine vector. The choice of vector and components can also be optimized for the specific purpose of treating allergies. For example, a polynucleotide encoding a recombinant antigenic polypeptide can be placed under the control of a promoter, such as a highly active or tissue-specific promoter. The promoter used to express the antigenic polypeptide can itself be optimized using recombination and selection methods similar to those described herein. The vector is a co-pending U.S. patent application Ser. No., "Optimization of Immunomodul
Attribution Molecules ", TTC Agent Reference Number 18097-030
It may include an immunostimulatory sequence as described in the 300 US application filed February 10, 1999. T _H 1 responses is operated to direct vectors are preferred for many immune responses mediated by antigens described herein (e.g., U.S. patents commonly assigned co-pending Application number Issue, "Genetic Vaccine
Vector Engineering ”, TTC Agent Reference Number 18097
(See application filed February 10, 1999 as -030100US). It is sometimes advantageous to use a genetic vaccine that is targeted for a particular target cell type (eg, antigen presenting cells or antigen processing cells); appropriate targeting methods have been assigned to co-pending individuals. U.S. Patent Application No. No., "Targeti
ng of Genetic Vaccine Vectors, "TTC Attorney Docket No. 18097-030200US, filed on February 10, 1999.

[0155] Genetic vaccines encoding the multivalent antigens described herein can be delivered to mammals (including humans) to induce a therapeutic or prophylactic immune response.
Vaccine delivery vehicles can be delivered in vivo by administration to an individual patient (typically systemic administration (eg, intravenous, intraperitoneal, intramuscular, subcutaneous, intracranial, intraanal, intravaginal, oral, By the buccal route, or they can be inhaled) or they can be administered by topical application). Alternatively, the vectors can be delivered to the cells ex vivo. For example, cells can be explanted from individual patients (eg, lymphocytes, bone marrow aspirate, tissue biopsy) or universal donor hematopoietic stem cells, and then these cells can be re-transplanted into the patient (usually taking up the vector After selection of cells).

A number of delivery methods are well known to those skilled in the art. Such methods are described, for example, in liposome-based gene delivery (Debs and Zhu (1993) WO 93/24).
640; Mannino and Gould-Fogerite (1988) Bi
oTechniques 6 (7): 682-691; Rose U.S. Pat. No. 5,2.
79,833; Brightham (1991) WO 91/06309; and F
elner et al. (1987) Proc. Natl. Acad. Sci. USA
84: 7413-7414), as well as the use of viral vectors (e.g., adenovirus (e.g., for a review, see Berns et al. (1995) Ann. NY).
. Acad. Sci. 772: 95-104; Ali et al. (1994) Gene.
Ther. 1: 367-384; and Haddada et al. (1995) Curr.
. Top. Microbiol. Immunol. 199 (Pt3): 297-
306), papillomaviruses, retroviruses (eg, Buc
hscher et al. (1992) J. Mol. Virol. 66 (5) 2731-2739;
Johan et al. (1992) J. Am. Virol. 66 (5) 1635-1640 (
1992); Sommerfeld et al. (1990) Virol. 176: 58-
59; Wilson et al. (1989) J. Am. Virol. 63: 2374-2378
Miller et al. Virol. 65: 2220-2224 (1991);
Wong-Staal et al., PCT / US94 / 05700, and Rosenb
urg and Fauci (1993) Fundamental Immunol
org., Third Edition, Paul (eds.), Raven Press, Ltd. , New
York and references therein, and Yu et al., Gene Therap.
y (1994) supra), and adeno-associated virus vectors (AA
For a review of V vectors, see West et al. (1987) Virology 160.
: 38-47; Carter et al. (1989) U.S. Patent No. 4,797,368; Carter et al., WO 93/24641 (1993); Kotin (1994).
) Human Gene Therapy 5: 793-801; Muzycz
ka (1994) J. Mol. Clin. Invst. 94: 1351 and Samul
See, ski, supra; also, Lebkowski, US Pat. No. 5,17.
No. 3,414; Tratschin et al. (1985) Mol. Cell. Biol
. 5 (11): 3251-3260; Tratschin et al. (1984) Mol.
. Cell. Biol. 4: 2072-2081; Hermonat and M
uzyczka (1984) Proc. Natl. Acad. Sci. USA,
81: 6466-6470; McLaughlin et al. (1988) and Sam.
ulski et al. (1989) J. Am. Virol. , 63: 03822-3828) and the like.

“Naked” DNA and / or RNA, including genetic vaccines, can be introduced directly into tissues (eg, muscle). See, for example, USPN 5,580,859. Other methods (eg, “biolistic” or particle-mediated transformation (eg, Sanford et al., USPN 4,945,050; USPN 5,036,0)
06) are also suitable for introducing a genetic vaccine into mammalian cells according to the invention. These methods are useful not only for in vivo introduction of DNA into mammals, but also for ex vivo modification of cells for reintroduction to mammals. With respect to other methods for delivering genetic vaccines, if necessary, vaccine administration is repeated to maintain the desired level of immunomodulation.

[0158] Gene vaccine vectors (eg, adenovirus, liposomes, papillomavirus, retrovirus, etc.) can be administered directly to mammals for transduction of cells in vivo. The genetic vaccine obtained using the method of the present invention can be administered in any suitable manner (eg, parenterally (eg, subcutaneously, intramuscularly, intradermally, or intravenously) for prophylactic and / or therapeutic treatment. ), Topical, oral, rectal, endotracheal,
It may be formulated as a pharmaceutical composition for buccal (eg, sublingual) or topical (eg, via aerosol or transdermal) administration. Pretreatment of the skin (eg, by use of a depilatory) may be useful in transdermal delivery. Suitable methods of administration (eg, packaged nucleic acids) are available and well known to those of skill in the art, and more than one route may be used to administer a particular composition. This route can often provide a more immediate and more effective reaction than another route.

[0159] Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical compositions of the present invention. A variety of aqueous carriers can be used (eg, buffered saline and the like). These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well-known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions (eg, pH and buffering agents, toxicity modifiers, etc. (eg, sodium acetate, sodium chloride, Potassium chloride, calcium chloride, sodium lactate, etc.)). The concentration of the gene vaccine vector in these formulations can vary widely and is chosen primarily based on liquid volume, viscosity, body weight, etc., according to the particular chosen mode of administration and the needs of the patient.

Formulations suitable for oral administration include (a) liquid solutions (eg, diluents (eg, water,
(B) capsules, sachets or tablets (each containing a predetermined amount of the active ingredient in a liquid, solid, granule, Or as gelatin); (c) a suspension in a suitable liquid; and (d) a suitable emulsion. The tablet form is 1
One or more lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, tragacanth, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other Excipients, colorants, fillers, binders, diluents, buffers, wetting agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers can be included. Lozenge forms may contain the active ingredient in flavor, usually sucrose and acacia or tragacanth. Then, the troches include the active ingredient in an inert base (eg, gelatin and glycerin or sucrose and acacia emulsions, gels, and the like).
These include, in addition to the active ingredient, carriers known in the art. It is recognized that when administered orally, a genetic vaccine must be protected from digestion. This is typically done by complexing the vaccine vector with a composition that renders it resistant to acidic and enzymatic hydrolysis, or by rendering the vector into a suitable resistant carrier (
(Eg, liposomes). Means of protecting vectors from digestion are well-known in the art. Pharmaceutical compositions can be encapsulated, for example, in liposomes or in a formulation that provides for slow release of the active ingredient.

[0161] The packaged nucleic acids can be made into aerosol formulations, alone or in combination with other suitable components, for administration by inhalation (eg, they can be "nebulized"). Aerosol formulations can be placed into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.

[0162] Suitable formulations for rectal administration may include, for example, suppositories. It consists of a nucleic acid packaged with a suppository base. Suitable suppository bases include neutral or synthetic triglycerides or paraffin hydrocarbons. Further, the base (for example,
It is also possible to use gelatin rectal capsules consisting of a combination of nucleic acids packaged with liquid triglycerides, including polyethylene glycol and paraffin hydrocarbons).

Formulations suitable for parenteral administration (eg, by intra-articular (intra-articular), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes) include aqueous and non-aqueous isotonic Sterile injectable solutions, which may contain antioxidants, buffers, bacteriostats and solutes that render the formulation isotonic with the blood of the intended recipient, and sterile aqueous and non-aqueous suspensions Liquids, which can include suspending agents, solubilizing agents, thickening agents, stabilizers, and preservatives. In the practice of the present invention, the compositions may be administered orally, topically, intraperitoneally, intravesically, or intratracheally, for example, by intravenous injection. Parenteral administration and intravenous administration are preferred modes of administration. Formulations of packaged nucleic acids may be presented in unit-dose or multi-dose sealed containers (eg,
Ampoules and vials).

Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. Cells transduced with the packaged nucleic acid can also be administered intravenously or parenterally.

The dose administered to a patient, in the context of the present invention, should be sufficient to effect a beneficial therapeutic response in the patient over time. The dose will be determined by the efficacy of the particular vector used and the condition of the patient, as well as the weight or vascular surface area of the patient being treated. The size of the dose will also be determined by the presence, nature, and extent of any side effects that accompany the administration of the particular vector, or transduced cell type, in the particular patient.

In determining the effective amount of the vector to be administered in the treatment or prevention of an infection or other condition, a physician will evaluate the toxicity of the vector, the progression of the disease, and the production of anti-vector antibodies, if any. I do. Generally, an equivalent dose of naked nucleic acid from the vector is about 1 μg to 1 mg for a typical 70 kg patient,
And the dose of the vector used to deliver the nucleic acid is calculated to obtain an equivalent amount of the therapeutic nucleic acid. Administration can be accomplished via single or divided doses.

For therapeutic applications, the composition may be a disease (eg, an infectious disease or an autoimmune disorder).
Is administered to a patient suffering from an amount sufficient to cure or at least partially prevent the disease and its complications. An amount sufficient to accomplish this is defined as "therapeutically effective amount." Effective amounts for this use will depend on the severity of the disease and the general state of the patient's health. Single or multiple administrations of the compositions can be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient amount of a protein of the invention to effectively treat the patient.

In prophylactic applications, the compositions are administered to humans or other mammals to induce an immune response that can help protect against the establishment of an infectious disease or other condition.

The toxicity and therapeutic efficacy of the genetic vaccine vectors provided by the present invention are determined in cell cultures or experimental animals using standard pharmaceutical procedures. Using the procedures presented herein and others known to those of skill in the art,
The LD ₅₀ (the dose that is lethal to 50% of the population) and the ED ₅₀ (the dose that is therapeutically effective in 50% of the population) can be determined.

A typical pharmaceutical composition for intravenous administration is about 0.1 per patient per day.
〜１０10 mg. Dosages from 0.1 to about 100 mg per patient per day may be used, especially when the drug is administered to sites where it has been blocked and when it is not administered in the bloodstream (eg, in a body cavity or lumen of an organ). Can be used. For topical administration, substantially higher dosages are possible. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and
on's Pharmaceutical Science, 15th edition, Mac
k Publishing Company, Easton, Pennsylv
Publications such as Ania (1980) describe in more detail.

The multivalent antigenic polypeptides of the invention, and genetic vaccines expressing the polypeptides, can be packaged in packs, dispenser devices, and kits for administering the genetic vaccines to mammals. For example, a pack or dispenser device is provided that includes one or more unit dosage forms. Typically, instructions for administration of the compound will be provided in the packaging, along with appropriate instructions for indicating that the compound is suitable for treatment of the indicated condition.
For example, the label may indicate that the active compound in packaging is a particular infectious disease,
It can be shown to be useful for treating autoimmune disorders, tumors, or for preventing or treating other diseases or conditions mediated by or potentially susceptible to a mammalian immune response.

EXAMPLES The following examples are provided to illustrate the present invention, but not to limit the present invention.

Example 1 Development of a Broad Spectrum Vaccine Against Bacterial Pathogens and Toxins A. Evolution of Yersinia V Antigen This example develops an immunogen that produces a strong cross-protective immune response against various Yersinia strains. The use of DNA shuffling is described. Passive immunization with an anti-V antigen antibody or active immunization with a purified V antigen is effective for pathogenic autologous Yersini.
It may provide protection from challenge with species a. However, protection against heterologous species is limited (Motin et al. (1994) Infect. Immun. 62:41).
92).

V antigen genes from various Yersinia strains (Y. pestis, Y. en
terocolitica, and Y. pseudotuberculosis
Serotypes) are subjected to DNA shuffling as described herein. For example, the Yersinia pestis V antigen coding sequence is used as a query in database searches to identify homologous genes that can be used in a family shuffling format to obtain improved antigens. Table 1 shows the results of the BLAST search of the GenBank and EMBL databases. Here, each row represents a unique sequence entry listing the database, accession number, locus name, bit score, and E value. For an overview of the search algorithm, see Altschul et al. (1997) Nucleic.
Acids Res. 25: 3389-3402). Homologous antigens have been cloned and sequenced from a number of related but different Yersinia strains, and additional natural diversity can be obtained by cloning antigen genes from other strains. These and other genes or fragments thereof are cloned, shuffled, and screened for improved antigens by methods such as PCR.

[0175]

[Table 1] Select shuffled clones by phage display or
And / or screening by ELISA to identify those recombinant nucleic acids that encode polypeptides having multiple epitopes corresponding to different serotypes. The shuffled antigen gene is cloned into a linear phage genome for multivalent phage display or into a suitable phagemid vector for monovalent phage display. A typical protocol for panning an antigen by phage display is as follows.

• Apply an appropriate surface (eg, Nunc Maxisorp tube or multiwell plate) to the target antibody (usually 1-10 in PBS or other appropriate buffer).
(at a concentration of μg / ml) at 4 ° C. overnight Rinse with PBSM (PBS + 3% non-fat dry milk) and block for 1-2 hours at 37 ° C. Pre-block phage if necessary (PBSM, RT 1 hour) Rinse tubes and bind phage (typically 1 hour at 37 ° C.) Change time, temperature, buffers, and add competitive inhibitors and the like Thorough washing with PBST (PBS + 0.1% TWEEN20) (15 times)
And then wash with PBS. • Bound phages are at low pH (eg 10 mM glycine), 100 mM
Elute with triethylamine, competitive ligand, protease, etc., then neutralize pH if necessary E. coli is then infected with the eluted phage and a new host is transduced with the expressed phagemid. Titrate and plate colonies on drug plate • Pool colonies in media, grow cells, and infect helper phage to produce phage for the next step.

The phage ELISA assay is a useful method for rapidly assessing single colonies after panning a library. Single colonies are picked into individual wells of a multiwell plate containing 2YT media and grown as master plates. Replicate plates are infected with helper phage and grown so that phage from a single well display a single antigen variant. Phage E
A suitable protocol for the LISA assay is as follows.

Coat the microtiter plate with 50 μl of 1 μg / ml target antibody overnight at 4 ° C. Rinse with PBSM and block for 2 hours at 37 ° C. Rinse and pre-block phage Add and allow to bind for 1 hour at 37 ° C. Wash plate 3 times with PBST, then wash 3 times with PBS for 2 minutes soak HRP (or AP) conjugated anti-M13 antibody at 37 ° C. Add for 1 hour at <RTIgt; C. </ RTI> Add substrate and measure absorbance. Positive clones are identified for further evaluation.

[0179] ELISA assays can also be used to screen for individual epitopes with multiple epitopes or increased expression levels. Single colonies are picked into individual wells of a multi-well plate containing the appropriate medium and grown as a master plate such that the antigen produced from a single well is a single antigen variant. Replicate plates are grown, for example, for a Lac repressor-based system, protein production is induced by the addition of 0.5 mM IPTG, and grown for a suitable time period during which antigen is produced. At this point, a crude antigen preparation is made, which depends on the antigen and where the antigen is produced. Secreted proteins can be assessed by assaying the cell supernatant after centrifugation. Periplasmic proteins are often readily released from cells by simple extraction into hypertonic or hypotonic buffers. Proteins produced intracellularly require some form of cell lysis, such as detergent treatment, to release them. A suitable protocol for the ELISA assay is as follows.

Coat the microtiter plate with 50 μl of 1 μg / ml target antibody overnight at 4 ° C. Rinse with PBSM and block for 2 hours at 37 ° C. Rinse and add antigen prep Wash plate three times with PBST. Add HRP (or AP) conjugated secondary antibody and incubate at 37 ° C. for 1 hour. Add appropriate substrate. Measure absorbance. Identify positive clones for further evaluation.

Antibodies specific for many different antigens are commercially available (eg, T
oxin Technology, Inc, Sarasota, FL), or by immunizing a suitable animal with a purified antigen. Immunoglobulins can be purified from serum using Protein A or Protein G Sepharose (Pharmacia). A variety of affinity purification schemes can be used to further purify the family-specific antibodies, if necessary (eg, NHS
-, CNBr-, or immobilization of specific antigens on epoxy activated sepharose beads). Other related antigens may be included in soluble form to prevent binding and immobilization of cross-reactive antibodies.

The multivalent polypeptide identified by the initial screening protocol is purified and submitted for in vivo screening. For example, purifying a shuffled antigen selected by or in combination with any of these methods, and using it to immunize an animal (primarily a mouse), Are evaluated for improved immune response. Typically, 10 μg of protein is added to a suitable adjuvant (eg, Alhydrogel (EM Se)
Argent Pulp and Chemical, Inc. Injections at the appropriate sites, with or without)), and animals are boosted with additional doses 2-4 weeks later. At this point, a serum sample is taken and assessed by ELISA assay for the presence of antibodies that cross react against multiple parental antigens. In this ELISA assay format, the antigen is coated on a multiwell plate, and then a serial dilution of each serum is bound. After washing the unbound antibody, a secondary antibody conjugated to HRP or AP conjugated against the constant region of the appropriate test antibody (eg, goat anti-mouse IgG Fc (Sigma)). After further washing, an appropriate substrate is added (eg, O-phenylenediamine (Sigma)). The absorbance of each well is read by a plate reader at the appropriate wavelength (eg, 490 nm for OPD),
Wells that produce high antibody titers against multiple antigens are then selected for further evaluation.

Further, the ability of an antigen to produce neutralizing antibodies can be assessed in a suitable system. Antigen variants that elicit a broad cross-reactive response are further evaluated in a pathogenic challenge model using appropriate pathogenic organisms. For example, a mouse is immunized with a multivalent polypeptide, which is then challenged with live Yersinia. These multivalent polypeptides that protect against the challenge are identified and purified.

B. Evolution of Broad Spectrum Vaccines Against Bacterial Toxins This example demonstrates the use of DNA shuffling to obtain multivalent polypeptides that are effective in eliciting an immune response against a broad spectrum of bacterial toxins. Is described.

1. Staphylococcus Group A Streptococci, which can cause diseases such as food poisoning, toxic shock syndrome, and autoimmune disorders, are very toxic by inhalation. The group A Streptococcus toxin family counts about 30 related members, making this group a suitable target for family shuffling. Thus, this example describes the use of family DNA shuffling to generate chimeric proteins that can elicit broad spectrum protection.

[0186] Nucleic acids encoding many different attenuated toxins are subjected to DNA shuffling, as described herein. Table 2 shows S.D. to identify homologous genes. Using Aureus enterotoxin B protein, GenBank, PDL,
The result of BLAST search of EMBL and SwissProt is shown. this is,
It can be used in a family shuffling format to obtain improved antigens.

[0187]

[Table 2] Shuffled recombinant clones are first selected by phage display for the presence of multivalent epitopes from different families and / or screened by ELISA. Variant proteins with multiple epitopes were purified and used for in vivo screening as described above. Mouse sera were analyzed for antibodies specific for different toxin subtypes, and variants that elicit a broad cross-reactive response were further evaluated in a challenge model.

(2. Escherichia coli and Vibrio choler)
ae) This example is based on The use of DNA shuffling to obtain cross-reactive multivalent polypeptides that induce an immune response against E. coli heat labile toxin (LT), cholera toxin (CT), and vero toxin (VT) is described. The nucleic acids encoding the cholera toxin B chain and the LT toxin B chain are subjected to DNA shuffling. Table 3 shows
V. to identify homologous genes. BLAS using cholerae toxin B chain
This shows the result of the T search. This can be used in a family shuffling format to obtain improved antigens. Homologous antigens have been identified in many related and different Vibrio and E. coli. E. coli strains can be cloned and sequenced, and additional natural diversity can be obtained by cloning antigen genes from other strains. These and other genes or fragments thereof can be cloned, shuffled, and screened for improved antigens by methods such as PCR.

[0189]

[Table 3] Those chimeric toxins that elicit high levels of neutralizing antibodies to both toxins and have improved adjuvant properties are identified. For example, shuffled clones are selected for the presence of epitopes from different parent B chains by phage display and / or screened by an ELISA assay.
Variants with multiple epitopes are purified and studied for their ability to serve as adjuvants in a challenge model and elicit a cross-protective immune response.

Example 2: Evolution of a Broad Spectrum Vaccine Against Borrelia burgdorferi Lyme disease is currently one of the fastest increasing infections in the United States. This is due to the spirochete bacterium Borrelia burgdorferi.
This bacterium is carried and transmitted by infected mite bites. Early signs of infection include skin rash and flu-like symptoms. If Lyme disease is left untreated, it can cause arthritis, cardiac abnormalities, and facial paralysis. Treating Lyme disease early with antibiotics may stop the infection, but persistent immunity may not improve enabling re-infection. Current vaccines require three immunizations over a year to acquire immunity.

The purified B. burgdorferi outer surface protein A (OspA) protein, both passive and active immunizations are described by B. burgdorferi. burgdorferi, but has no effect on ongoing infection, since this antigen is not expressed in vertebrate hosts. OspA is usually attached to the outside of the cell by covalent attachment of a lipid moiety through an amino-terminal cysteine residue. In contrast, outer surface protein C (OspC) is highly expressed by spirochetes in vertebrate hosts. And vaccination of infected individuals with OspC may be an effective treatment in curing infection (Zhong et al. (1)
997) Proc. Nat'l. Acad. Sci. USA 94 12533
-12538).

Non-overlapping GenBank, PDB, SwissProt, Spupdate
, And a recent BLAST search of the PIR database (Altschul et al. (1).
997) Nucleic Acids Res. 25: 3389-3402) was used to identify homologs of the OspA outer surface protein gene. This is O
Over 200 entries have been identified that are related to spA. B. burgdor
feri, B .; Garinii, B .; afzelii, B .; tanukii, and B. 100 entries from different strains of turdi are shown in Table 4 below.
It shares at least 83% DNA sequence identity with the Borrelia burgdorferi OspA protein. The ospA genes from these and other strains provide a source of diversity for family shuffling to obtain improved antigens for Lyme disease protection. These genes are converted to PC
Clones, shuffles, and screens for improved antigens by methods such as R.

[0193]

[Table 4] B. BLAST using burgdorferi OspC protein gene
The search showed more than 200 relevant entries. At least 82
Entries for 100 sequences sharing% DNA sequence identity are shown in Table 5 below. This provides a source of diversity for family shuffling to obtain improved therapeutics in the treatment of Lyme disease. These genes are cloned, shuffled, and screened for improved antigen by methods such as PCR.

[0194]

[Table 5] Example 3: Evolution of a Broad Spectrum Vaccine Against Mycobacterium Tuberculosis is an ancient bacterial disease caused by Mycobacterium tuberculosis. This continues as a significant public health problem worldwide and there is a need for improved efforts to eradicate (Morb. Mortal Wkly Rep (August 21, 1998; 47 (RR-13)): 1-6), which infects more than 50 million people and kills more than 3 million people during the year with tuberculosis The currently available vaccine, the Bacillus Calmette-Guerin vaccine (BCG) Has been found to be less effective in developing countries, and a number of increasing multidrug resistances (
(MDR) strain has been isolated.

M. The major immunodominant antigen of tuberculosis is 30-35 kDa (antigen 85, also known as α-antigen), which is usually a lipoglycoprotein on the cell surface. Other protective antigens include the 65 kDa heat shock protein, and the 36 kDa proline-enriched antigen (Tascon et al. (199)
6) Nat. Med. 2: 888-92).

Table 6 shows the major M. cerevisiae of 30-35 kDa to identify homologous genes. Tub
FIG. 3 shows the results of a BLAST search using the erculosis antigen (antigen 85, also known as α antigen) coding sequence. This can be used in a family shuffling format to obtain improved antigens. Many homologous antigens have been cloned and sequenced from many related and different mycobacterial strains. These genes are cloned, shuffled, and screened for improved antigen by methods such as PCR.

[0197]

[Table 6] Example 4: Evolution of a Broad Spectrum Vaccine Against Helicobacter pylori Chronic infection of the gastroduodenal mucosa with Helicobacter pylori bacteria can result in chronically active gastritis, peptic ulcers, and gastric cancer (eg, adenocarcinoma and low malignant B cells) Lymphoma). The increasing presence of antibiotic resistant strains has limited this treatment. The use of vaccines to both prevent and treat progressive infection is being actively explored (Crabtree JE (1
998) Gut 43: 7-8; Axon AT (1998) Gut 43: Aug. 1: S70-3; Dubois et al. (1998) Infect. Immun. 6
6: 4340-6; Tygat GN (1998) Alignment. Phar
macol. Ther. 12 Supplement 1: 123-8; Blaster MJ (1
998) BMJ 316: 1507-10; Marchetti et al. (1998).
Vaccine 16: 33-7; Kleanthous et al. (1998) Br.
Med. Bull. 54: 229-41; Wermeille et al. (1998) P.
harma. World Sci. 20: 1-17).

Helicobacters suitable for use in prophylactic and therapeutic vaccines
Identification of r antigens may include two-dimensional gel electrophoresis, sequence analysis, and serum profiling (McAtee et al. (1998) Clin. Diagn. L.
ab. Immunol. 5: 537-42; McAtee et al. (1998) Hel.
icobacter 3: 163-9). Related antigenic differences between Helicobacter species and strains may limit the use of vaccines for prevention and treatment of infection (Keenan et al. (1998) FEMS Microbiol. L.
ett. 161: 21-7).

In this example, further DNA family shuffling of immunologically distinct related antigens allows for the isolation of complex chimeric antigens. This composite chimeric antigen may provide extensive cross-reactive protection against many related strains and species of Helicobacter. H. adapted mice were used to evaluate the therapeutic use of the vaccine against infection. A shuffled antigen was evaluated using a mouse model of persistent infection with S. pylori strains (Crabtree J
E (1998) Gut 43: 7-8; Axon AT (1998) Gut 4
3: Supplement 1: S70-3).

[0200] Vacuolating cytotoxin (VacA) and a cytotoxin-related gene product (CagA) were isolated from H. coli in animal models. pylori infection. This supports the application of this approach in humans.

[0201] Table 7 shows that H. to identify homologous genes. The result of BLAST search using the pylori VacA gene is shown. This can be used in a family shuffling format to obtain improved antigens. Homologous antigens have been identified in many related, even different H. pylori strain has been cloned and sequenced. Additional natural diversity can be obtained by cloning antigen genes from other strains. These and other genes or fragments thereof are cloned, shuffled, and screened for improved antigen by methods such as PCR.

[0202]

[Table 7] Table 8 shows that H. to identify homologous genes. The result of BLAST search using the pylori CagA gene is shown. This can be used in a family shuffling format to obtain improved antigens. Homologous antigens have been identified in many related, even different H. pylori strain has been cloned and sequenced. And further natural diversity can be obtained by cloning antigen genes from other strains. These and other genes or fragments thereof are cloned, shuffled, and screened for improved antigen by methods such as PCR.

[0203]

[Table 8] Example 5 Development of a Broad Spectrum Vaccine Against Malaria This example demonstrates the use of D to produce an improved vaccine against malaria infection.
Describes the use of NA shuffling. An excellent target for evolution by DNA shuffling is the Plasmodium falciparum merozoite surface protein MSP1 (Hui et al. (1996) Infect. Immun. 64).
: 1502-1509). MSP1 is expressed on the surface of merozoites as an essential membrane protein. It is cleaved shortly before by a parasitic protease and involves rupture and release from infected cells. Cleavage appears to be essential for full function in MSP1 binding to the RBC receptor. The cleaved fragment remains attached to the merozoite membrane. Other membrane proteins on merozoites are also involved in connections and are specific for invasive events. MSP1 is a proven candidate for inclusion in vaccines against the asexual blood stage of malaria.

The gene encoding MSP1 was replaced with Plasmodium falciparou.
m merozoites can be isolated from various isolates by PCR techniques. Related naturally occurring genes can be further used to increase the diversity of the starting gene. A library of shuffled MAP1 genes is generated by DNA shuffling, and the library is screened for induction of an effective immune response.

This screening can be performed by injecting test animals (eg, mice or monkeys) with individual variants. Inject either the purified recombinant protein or a DNA vaccine or viral vector encoding the relevant gene. Typically, a booster injection is given 2-3 weeks after the first injection. afterwards,
The sera of the test animals were collected and these sera were used as uninfected red blood cells (RB
C) Analyze for the presence of antibodies that reduce infiltration of merozoites into. RBC,
Infected by merozoites just inside the RBC, the merozoites differentiate into rings, which mature into a reproductive body containing some newborn daughter merozoites, and then burst out of the infected cells, Break it down and proceed to adhere and invade another RBC. In vivo, this merozoite appears to be extracellular for only a few seconds. In vitro, any blockade of this event can dramatically reduce the level of reinfection. Antibodies against MSP1 bind to the surface of the merozoites and, when ruptured, cause RBCs to infect propagules.
, Thereby reducing the ability of these merozoites to attach and bind the cognate RBC receptor on uninfected RBC surfaces. Merozoite attachment is reduced and merozoite entry into new RBCs is reduced, and therefore the number of newly infiltrated cells detected in the early ring stage will test the culture several hours after blocking the invasion test If it decreases. In some assay formats, a surrogate for inhibition of merozoite invasion is to note the appearance of aggregated merozoites, which is an indirect measurement of antibodies that result in reduced infiltration.

[0206] Shuffled antigens that induce most potent antibody responses to reduce infiltration of merozoites into uninfected erythrocytes can be selected for further testing and subjected to a new round of shuffling and selection. . Subsequent studies will investigate the ability of these antigens to induce antibodies in humans. Again, either a purified recombinant antigen, or a DNA vaccine or viral vector encoding the relevant gene is injected and the protective immune response is analyzed.

Example 6 Development of a Broad Spectrum Vaccine Against a Viral Pathogen This example demonstrates the use of DNA shuffling to obtain a vaccine capable of inducing an immune response against multiple isolates of a viral pathogen. Describe.

A. Venezuelan equine encephalitis virus (VEE) VEE belongs to the genus Alphavirus, which is commonly transmitted by mosquitoes. However, VEE is an abnormal alphavirus in that it is also highly infectious by air transmission to both humans and rodents. The disease manifests in humans, ranging from asymptomatic or mild febrile illness to severe infection and central nervous system inflammation. Viral clearance (elimination) coincides with the production of specific anti-VEE antibodies, which are believed to be the primary mediator of the protective immune response (Schmaljohn et al. (1982) Nature 297: 70). . VEE
It is also an aberrant virus because its primary target outside the central nervous system is lymphoid tissue, and therefore replication deficient variants are useful for targeting vaccines or pharmaceutically useful proteins to the immune system. Means may be provided.

[0209] At least seven subtypes of VEE are known and can be identified genetically and serologically. Based on epidemiological data, virus isolates fall into two main categories: I-AB and IC strains (these are VEE veterinary /
Infectious, and associated with the remaining serotypes, which are mainly associated with veterinary vertebrate-mosquito cycles and circulation in certain ecological zones) (Jhon
Ston and Peters, Fields Virology, 3rd edition, B.S.
N. Edited by Fields et al., Lippincott-Raven Publishe.
rs, Philadelphia, 1996).

The envelope protein protein (E) appears to be the major antigen in the induction of neutralizing Abs. Thus, a library of recombinant E proteins is obtained by shuffling the corresponding genes from various strains of VEE using DNA shuffling. These libraries and their individual chimeras /
Mutants are subsequently screened for their ability to induce broad cross-reactivity and protective Ab responses.

B. Flaviviruses The Japanese encephalitis virus (JE), tick-borne encephalitis virus (TBE) and dengue virus are members of the flavivirus family, which includes 69 related viruses.
Is an arthropod-borne virus belonging to Heterogeneity of the viruses within the family is a major challenge for vaccine development. For example, there are four major serotypes of dengue virus, and there is a need for a tetravalent chromosome vaccine that induces neutralizing Abs for all four serotypes. In addition, non-neutralizing antibodies induced by infection with one dengue virus or vaccination can cause disease enhancement during subsequent infection with another serotype. Therefore, a broad spectrum of cross-protective vaccines for TBE and JE provides a significant improvement over existing vaccines. In this example, the ability to efficiently generate DNA shuffling chimeras and mutated genes is used to generate cross-protective vaccines.

(1. Japanese Encephalitis Virus) Japanese encephalitis virus (JE) is the prototype of the JE antigenic complex and is described in St. Lou
is encephalitis virus, Murray Valley encephalitis virus, Kunjin virus, and West Nile virus (Monath and Hei
nz, Fields Virology, 3rd ed. N. Edited by Fields et al.
Lippincott-Raven Publishers, Philadel
pia, 961-1034, 1996). Although infections caused by JE are relatively rare, the mortality in this case is 5% because no specific treatment is available.
４０40%. JE is widely distributed in China, Japan, the Philippines, Far East Russia and India and poses a serious threat to travel in these regions. Currently available JE vaccines are produced from brain tissue of mice infected with a single virus isolate. Side effects are observed in 10% to 30% of vaccinees.

[0213] DNA shuffling is performed on the viral envelope genes to obtain chimeric and / or mutated antigens that provide a protective immune response against all or most viruses within the JE complex. Amino acid identity within the JE complex varies between 72% and 93%. In addition, significant antigenic variability has been observed among JE strains by neutralization assays, agar gel diffusion, antibody absorption, and monoclonal antibody analysis (Oda (1976) Kobe J. Med. Sc.
i. 22: 123; Hobyashi et al. (1984) Infect. Immun
. 44: 117). Furthermore, the amino acid diversity of the envelope protein gene among 13 strains from different Asian countries is of the order of 4.2% (Ni and B
arrett (1995) J. Am. Gen. Virol. 76: 401). The resulting library of recombinant polypeptides encoded by the shuffled genes is screened to identify those that provide a cross-protective immune response.

2. Tick-borne encephalitis virus The tick-borne encephalitis virus complex comprises 14 antigenically related viruses, 8 of which cause human disease, including Powassan, Loop
ing ill and tick-borne encephalitis virus (TBE) (Mo)
Nath and Heinz, Fields Virology, 3rd ed. N
. Edited by Fields et al., Lippincott-Raven Publisher.
s, Philadelphia, pp. 961-1034, 1996). TBE is
Powassan occurs in Russia, Canada, and the United States, whereas it is recognized in all Central and Eastern European countries, Scandinavia and Russia. The symptoms vary from influenza-like illness to severe meningitis, meningoencephalitis and meningoencephalitis with 1% to 2% mortality (Gresikova).
And Calisher, Monath, eds., The arboviruses:
Ecology and epidemiology, Vol. IV, Boca R
aton, FL, CRC Press, 177-203, 1988).

[0215] Family DNA shuffling is used to generate chimeric envelope proteins from the TBE complex to generate cross-protective antigens. Envelope proteins within the family are 77-96% homologous, and viruses can be distinguished by specific mAbs (Holzmann et al., Vaccine,
10, 345, 1992). The Powasan envelope protein is 78% identical to the TBE envelope protein at the amino acid level and does not appear to be cross-protective, but epidemiological data is limited.

The in vivo protective immune response is analyzed using the Langat virus as a model system (Iacono-Connors et al. (1996) Virus Re.
s. 43: 125). Langat virus belongs to the TBE complex and
Can be used for challenge studies on L3 function. Serological studies based on recombinant envelope proteins are performed to identify immunogenic variants that elicit high levels of antibodies to envelope proteins from most or all viruses of the TBE complex.

3. Dengue Virus Dengue virus is transmitted by mosquito bites and poses a serious threat to military and civilian populations, especially in the tropics. There are four major serotypes of dengue virus, Dengue 1, 2, 3 and 4. D
A tetravalent vaccine that induces neutralizing antibodies against all four strains of N. engue should avoid antibody-mediated enhancement of the disease when individuals encounter other strains of the virus.

The envelope protein of dengue virus has been shown to provide an immune response that protects against further challenge with the same strain of virus. However, the levels of neutralizing antibodies produced are relatively low, and protection from live virus challenge is not always observed. For example, mice injected with a genetic vaccine encoding the envelope protein of Dengue-2 virus produced neutralizing antibodies when analyzed by an in vitro neutralization assay, but the mice were challenged with live Dengue-2 virus. Did not survive (Koc
hel et al. (1997) Vaccine 15: 547-552). However, a protective immune response was observed in mice immunized with a recombinant vaccine virus expressing Dengue 4 viral structural proteins (Bray et al. (1989) J. Am.
Virol. 63: 2853). These studies show that vaccination with the E protein works, but requires a significant improvement in the immunogenicity of the protective antigen.

In this example, DNA shuffling is performed on the gene encoding the envelope (E) protein from all four dengue viruses and antigenic variants thereof. Using family DNA shuffling, Dengue
Produce chimeric E protein variants that induce high titers of neutralizing antibodies against all serotypes of The E protein of different dengue viruses contains 6 of their amino acids.
Share 2% -77%. Dengue 1 and Dengue 3 are most closely related (77% homology), followed by Dengue 2 (69%) and Dengue 4
(62%). These homologies are sufficient to allow efficient family shuffling (Crameri et al. (1998) Nature 39).
1: 288-291).

The shuffled antigen sequence is incorporated into a genetic vaccine vector, the plasmid is purified and subsequently injected into mice. Serum is collected from the mice and analyzed for the presence of high levels of cross-reactive antibodies. The best antigens are selected for further study using an in vivo challenge model and screened for chimeras / variants that induce cross protection against all strains of Dengue.

C. Improved Expression and Immunogenicity of Hantaan Virus Glycoproteins One of the advantages of genetic vaccines is that vectors expressing pathogen antigens cannot be isolated in culture for a given pathogen. Even that can be generated.
An example of such a potential situation is the previously unknown hantavirus Sin N
There was a surge of severe respiratory illness among rural residents in the southwestern United States caused by the Ombre virus (Hjelle et al. (1994) J. Virol.
68: 592). A great deal of viral RNA sequence information was available before the virus could be isolated and characterized in vitro. In these situations, genetic vaccines can provide a means to produce vaccines in a short time and efficiently by making vectors encoding antigens encoded by the pathogen. However, genetic vaccines can only work if these antigens can be properly expressed in the host.

The Hantaan virus belongs to the Bunyavirus family. A characteristic property of this family is that their glycoproteins typically accumulate on the membrane of the Golgi apparatus when expressed by cloned cDNA, thereby reducing the efficiency of the corresponding genetic vaccine (Matsuook
a et al. (1991) Curr. Top. Microb. Immunol. 169:
161-179). Poor expression of the Hantaan virus glycoprotein on the cell surface is also one explanation for the poor immune response following injection of the Hantaan virus gene vaccine.

In this example, family DNA shuffling is used to generate a recombinant Hantaan virus-derived glycoprotein that can be efficiently expressed in human cells and elicit a protective immune response against wild-type pathogens I do. Hant
Aan virus glycoprotein-encoding nucleic acid can be transferred to other homologous Bunyavir
Shuffle with the gene encoding the us glycoprotein. The resulting library is screened to identify proteins that are readily expressed in human cells. This screening is performed using a dual marker expression vector that allows for simultaneous analysis of transfection efficiency and expression of a fusion protein that is PIG-bound to the cell surface (Whitehorn et al. (1995) Biotech).
nology (NY) 13: 1215-9).

[0224] Flow cytometry-based cell sorting is used to select for Hantaan virus glycoprotein variants that are efficiently expressed in mammalian cells. The corresponding sequence is then obtained by PCR or plasmid recovery. These chimeras / variants are further analyzed for their ability to protect wild-type mice against Hantaan virus infection.

Example 7 DNA Shuffling of HSV-1 and HSV-2 Glycoproteins B and / or D as a Means of Inducing an Enhanced Protective Immune Response This example demonstrates the administration of Described is the use of DNA shuffling to obtain HSV glycoprotein B (gB) and glycoprotein D (gD) polypeptides that exhibit improved ability to induce a protective immune response. Epidemiological studies
Prior infection with HSV-1 has been shown to result in partial protection against infection with HSV-2, indicating the presence of a cross-reactive immune response. Based on previous vaccination studies, the major immunogenic glycoproteins in HSV appear to be gB and gD encoded by the 2.7 kb and 1.2 kb genes, respectively. The gB and gD genes of HSV-1 are about 85% identical to the corresponding genes of HSV-2, and each gB gene shares little sequence identity with the gD gene. Baboon HSV-2 gB is human HSV-1 gB or human HV.
It is about 75% identical to SV-2 gB and almost 90% identical to a slightly longer stretch. In addition, 60-75% identity is found in portions of the horse and bovine herpesvirus genes.

Family shuffling is used using nucleic acids encoding gB and / or gD from HSV-1 and HSV-2 as substrates. Preferably, homologous genes are obtained from various strains of HSV. The gD nucleotide sequence from HSV-1 and an alignment of the two strains of HSV-2 are shown in FIG. The antigen encoded by the shuffled nucleic acid is expressed and analyzed in vivo. For example, one may screen for a neutralizing antibody against HSV-1 / HSV-2 and / or improved induction of a CTL response. Protective immunity can also be detected by challenge mice or guinea pigs with this virus. Screening can be performed with pools or individual clones.

Example 8: HIV Gp1 for induction of a broad spectrum neutralizing Ab response
This example describes the use of DNA shuffling to generate immunogens that cross-react with different strains of virus (different from wild-type immunogens). Shuffling of the two envelope sequences can generate an immunogen that induces neutralizing antibodies against a third strain.

Antibody-mediated neutralization of HIV-1 is strictly type-specific. Although neutralizing activity spreads over time in infected individuals, induction of such antibodies by vaccination is
It has been shown to be very difficult. Antibody-mediated protection from HIV-1 infection in vivo correlates with antibody-mediated neutralization of the virus in vitro.

FIG. 8 illustrates the generation of a library of shuffled gp120 genes. Gp from HIV-1DH-12 and HIV-1 IIIB (NL43)
Shuffle 120 genes. The chimeric / variant gp120 genes are then analyzed for their ability to elicit antibodies with a broad spectrum of ability to neutralize different strains of HIV. Each shuffled gp120 gene is incorporated into a genetic vaccine vector, which is then introduced into mice by injection into skin or topical application. These antigens can also be delivered as a purified recombinant protein. The immune response is measured by analyzing the ability of mouse serum to neutralize HIV growth in vitro. Neutralization assays were performed using HIV-1
Perform on DH-12, HIV-1IIIB and HIV-189.6. Chimeras / mutants that demonstrate broad spectrum neutralization are selected for further rounds of shuffling and selection. Further studies are performed in monkeys to illustrate the ability of the shuffled gp120 gene to provide protection for subsequent infection with the immunodeficiency virus.

Example 9 Antigen Shuffling of the Hepadnavirus Envelope Protein Hepatitis B virus (HBV) is one member of a family of viruses called hepadnaviruses. This example describes that the use of genomes and individual genes from this family are used for DNA shuffling, resulting in antigens with improved properties.

A. Shuffling of the Hepadnavirus Envelope Protein Gene The envelope protein of the HBV assembly that forms particles possessing antigenic structure is the hepatitis B virus surface antigen (HBsAg; the term also (Used to indicate). The major antigenic site ("a
"Epitope (found in the envelope domain called S))
Antibodies can neutralize the virus. Immunization with the HBsAg-carrying protein thus acts as a vaccine against viral infection. The HBV envelope also contains other antigenic sites that can protect against viral infection and are potential viral components of improved vaccines. The epitope is part of the envelope protein domain known as preS1 and preS2 (FIG. 9).

[0232] DNA shuffling of envelope genes from several members of the hepadnavirus family is used to obtain more immunogenic proteins. Specifically, genes from the following hepatitis viruses are shuffled: human HBV virus, subtypes ayw and adw2; hepatitis virus isolated from chimpanzees; hepatitis virus isolated from gibbon; hepatitis virus isolated from marmot.

If desired, genes from other genotypes of the human virus are available for inclusion in a DNA shuffling reaction. Similarly, other animal hepadnaviruses are available.

To enhance the efficiency of the formation of chimeras selected from DNA shuffling, several artificial genes are created: • In one case, create a synthetic gene containing the HBV envelope sequence (chimpanzee and gibbon genes) Excluding codons specifying the amino acids found in). For these codons, a chimpanzee or gibbon sequence is used.

• In the second case, synthesize a synthetic gene in which the preS2 gene sequence from the human HBVadw2 strain is fused to the marmot S region.

• In a third case, mix in approximately equal amounts all oligonucleotides that require chemically synthesizing each of the hepadnavirus envelope genes,
It can then be annealed to form a library of sequences.

After DNA shuffling of the hepadnavirus envelope gene, either or both of the two strategies are used to obtain an improved HBsAg antigen. (Strategy A): Antigens are screened by immunization of mice using two possible methods. These genes are injected in the form of a DNA vaccine (ie, a shuffled envelope gene carried on a plasmid that contains the gene regulatory elements necessary for expression of the envelope protein). Alternatively, the protein is prepared from the shuffled gene and used as an immunogen.

PreS1-mediated HBV antigen, preS2-mediated HBV antigen or S-mediated H
Sequences that are more immunogenic for any of the BV antigens are selected for a second round of shuffling (FIG. 10). For the second round, the best candidates are selected on the basis of their improved antigenicity and their other properties (eg, higher expression levels or more efficient secretion). Screening and further rounds of shuffling continue until maximal optimization is obtained for one of the antigenic regions.

The individually optimized genes were then replaced with the preS1-mediated epitope, preS
Used as a combination vaccine for induction of an optimal response to the 2-mediated epitope and the S-mediated epitope.

(Strategy B): After isolation of individually optimized genes as in strategy A, preS1, preS2 and S candidates were shuffled together or in a pairwise fashion in additional rounds, and HBsAg A gene encoding a protein that demonstrates improved immunogenicity for at least two regions containing the epitope is obtained (FIG. 11).

B. Use of HBsAg Carrying Epitopes from Unrelated Antigens Several features of HBsAg make HBsAg a useful protein that carries epitopes from other unrelated antigens. This epitope is of class I (
B epitopes (antibodies from CD8 ⁺ T lymphocytes and induced cytotoxic cells) or from class II (which induce helper T lymphocytes and are important in providing an immunological memory response) Induce) or a T epitope.

1. B Cell Epitope The amino acid sequence of a potential B epitope is selected from any pathogen. Such sequences are often known to induce antibodies, but their immunogenicity is weak or not sufficient for vaccine preparation. These sequences can also be mimotopes, which have been selected on the basis of their ability to have a particular antigenicity or immunogenicity.

An amino acid coding sequence is added to the hepadnavirus envelope gene. A heterologous sequence can either replace a particular envelope sequence or be added in addition to all envelope sequences. The heterologous epitope sequence can be substituted at any position in the envelope gene. The preferred position is HBsAg
Region of the envelope gene encoding the major "a" epitope of
2). This region appears to be exposed on the outside of the particle formed by the envelope protein, thus exposing the heterologous epitope.

[0244] DNA shuffling is performed on the envelope gene sequence to maintain the sequence of certain heterologous epitopes. Screening is performed to select candidates secreted into the culture medium after transfection of the plasmid into cells in tissue culture from the shuffled library.

The clones encoding the secreted protein are then tested for immunogenicity in mice, as either a DNA vaccine or protein antigen, as described above. Clones that result in improved induction of antibodies to the heterologous epitope are selected for further rounds of DNA shuffling. This process is continued until the immunogenicity of the heterologous epitope is sufficient to be used as a vaccine against the pathogen (from which the heterologous epitope is derived).

2. Class I Epitopes MHC class I epitopes are relatively short linear peptide sequences, generally between 6 and 12 amino acids in length, most often 9 amino acids in length. is there. These epitopes are processed by antigen presenting cells either after synthesis of the epitope within the cell (usually as part of a larger protein) or after uptake of soluble protein by the cell.

A polynucleotide sequence encoding one or more class I epitopes is inserted into the sequence of a hepadnavirus envelope gene, either by replacing a specific envelope sequence or by inserting an epitope sequence into the envelope gene. I do. This is typically achieved by modifying the gene prior to DNA shuffling, or in shuffling reactions of specific oligonucleotide fragments encoding heterologous epitopes and sufficiently adjacent hepadnavirus sequences to be incorporated into the shuffled product. Do by including.

Preferably, the heterologous class I epitope is located at different locations on some of the hepadnavirus genes used in the DNA shuffling reaction. This means
Optimize the opportunity to find chimeric genes that carry the epitope at the optimal location for effective presentation.

3. Class II Epitopes MHC class II epitopes are generally required to be part of a protein that is taken up by antigen presenting cells rather than synthesized intracellularly. Preferably, such epitopes are incorporated into a carrier protein such as the HBV envelope, which can be produced in a soluble form, or which can be secreted when the gene is delivered in the form of a DNA vaccine.

A polynucleotide encoding a heterologous class II epitope is inserted into a region of the hepadnavirus envelope gene that does not participate in the transmembrane structure of the protein.
Perform DNA shuffling to obtain a secreted protein that also carries the class II epitope. When injected as a protein, or when delivering the gene as a DNA vaccine, the protein can be taken up by antigen presenting cells for processing of class II epitopes.

Example 10 Evolution of a Broad Spectrum Vaccine Against Hepatitis C Virus Antigen heterogeneity of different strains of hepatitis C virus (HCV) is a major problem in developing an effective vaccine against HCV . Antibodies or CTLs specific for one strain of HCV typically do not protect against other strains. Multivalent vaccine antigens that protect against several strains of HCV simultaneously are of major importance when developing an effective vaccine against HCV.

The HCV envelope gene, which contains the envelope proteins E1 and E2
) Have been shown to induce both antibodies and lymphoproliferative responses to these antigens (Lee et al. (1998) J. Virol. 72:
8430-6), and these responses can be optimized by DNA shuffling. The hypervariable region 1 (HVR1) of the envelope protein E2 of HCV is the most variable antigenic fragment in the entire viral genome and is a major cause of large inter- and intra-individual heterogeneity of the infecting virus (
(1998) EMBO J. Puntoriero et al. 17: 3521-33).
Therefore, the gene encoding E2 is a particularly useful target for evolution by DNA shuffling.

DNA shuffling of HCV antigens (eg, nucleocapsids or envelope proteins E1, E2) provides a means to generate multivalent HCV vaccines that protect against several strains of HCV simultaneously. These antigens are shuffled using a family DNA shuffling approach. The starting gene is obtained from various natural isolates of HCV. In addition, related genes from other viruses can be used to increase the number of different recombinants produced. Next, HCV
Generating a related library of chimeric variants of the antigen, and
Screen for induction of a broad cross-reactive immune response. This screening can be performed directly in vivo by injecting test animals (eg, mice or monkeys) with the individual variants. Inject either the purified recombinant protein encoding the relevant gene or the DNA vaccine. Typically, a booster injection is given 2-3 weeks after the first injection. Thereafter, test animal sera are collected and these sera are tested for the presence of antibodies reactive against multiple HCV virus isolates.

Prior to in vivo testing, the antigen may be pre-enriched in vitro for antigen recognized by polyclonal antisera from previously infected patients or test animals. Alternatively, monoclonal antibodies specific for various strains of HCV are used. Screening can be performed by phage display or ELISA.
Performed using an assay. For example, antigen variants are expressed on bacteriophage M13, and the phage are then incubated on plates coated with antisera from patients or test animals infected with various HCV isolates. The phage that bind to the antibody are then eluted and for induction of cross-reactive antibodies,
Further analysis in test animals.

Example 11 Evolution of Chimeric Allergens That Induce a Broad Immune Response and Reduce the Risk of Inducing an Anaphylactic Response Injecting a patient with an allergic specific immunotherapy with increasing amounts of a given allergen Done by This therapy is typically dominant T from the helper 2 (T _H 2) type response to the dominant T-helper 1 (T _H 1) type response, changing the type of allergen-specific immune responses. However, since allergic patients have increasing levels of IgE antibodies specific for the allergen, immunotherapy of allergy involves the risk of IgE receptor-mediated anaphylactic reactions.

T helper (T _H ) cells can produce a number of different cytokines, and based on their cytokine synthesis pattern, T _H cells are divided into two subsets (Paul and Seder ( 1994) Cell 76: 241-2.
51). T _H 1 cells, IL-2 and IFN-gamma in the high level produced, IL- 4, IL-5 and IL-13 at all or does not produce, or produce at a minimum level. In contrast, T _H 2 cells produce high levels of IL-4, IL-5 and IL- 13, whereas the production of IL-2 and IFN-gamma are either or absence is minimal. T _H 1 cells activate macrophages, dendritic cells, and CD8 ⁺ cytotoxic T lymphocytes and enhances the cytolytic activity of NK cells (Id.), Whereas T _H 2 cells, for B cells provide sufficient assistance in, and they also of T _H 2 cells, due to the ability to induce differentiation into IgE isotype switching, and B cell IgE secreting cells, mediating allergic responses (De Vrie
and Punnonen (1996) Cytokine regulatio
no of humoral immunity: basic and clin
ical aspects. Snapper, C.I. M. Hen, John Wile
y & Sons, Ltd. , West Sussex, UK, 195-215).
.

This example describes a method of producing a chimeric allergen that can widely modulate an allergic immune response. This can be achieved by generating a chimeric gene by DNA shuffling of the relevant allergen gene. In addition, chimeric / mutated allergens are less likely to be recognized by pre-existing patient IgE antibodies. Importantly, allergen variants that are not recognized by the IgE antibody can be selected using patient serum and negative selection (FIG. 13).

As one example, Der p2, Der f2, Tyr p2 Lep d2
And chimeric allergen variants of the Gly d2 allergen. These house dust mite allergens are very common in exacerbating allergic and asthmatic symptoms, and improved means of down-regulating such allergic immune responses are desired. House dust mites can be used as a source of genes. The corresponding genes are shuffled using family DNA shuffling, and a shuffled library is generated. Phage display is used to eliminate allergens recognized by antibodies from allergic individuals. It is particularly important to exclude variants that are recognized by IgE antibodies. Phage expressing the allergen variant are incubated with a pool of serum from an allergic individual. Phages recognized by IgE antibodies are removed and the remaining allergens are further tested in vitro and in vivo for their ability to activate allergen-specific human T cells (FIG. 14). Immunotherapy of allergy, because compared with allergic T _H 2 responses are considered to function through the induction of dominant T _H 1 responses, efficient T cell activation by allergen variants and T _H 1 Induction of a type response is used as a measure of allergen efficiency to modulate allergic T cell responses.

The optimal allergen variants are then further tested in vivo by studying the skin response after injection into the skin. A strong inflammatory response around the injection site is an indicator of efficient T cell activation and induces the most efficient delayed T cell response (typically observed at 24 hours post injection. Allergen variants are the best candidates for further studies in vivo to identify allergens that effectively down-regulate the allergic immune response. Accordingly, these allergen variants are analyzed for their ability to inhibit allergic responses in allergic, atopic and asthmatic individuals. Screening for allergen variants is further illustrated in FIGS. 13 and 14.

Example 12 Evolution of Cancer Antigens That Induce an Efficient Anti-Tumor Immune Response Some cancer cells express antigens present on malignant cells at significantly higher levels in the body than others. I do. Such antigens provide an excellent target for protective cancer vaccines and immunotherapy of cancer. The immunogenicity of such antigens can be improved by DNA shuffling. In addition, DNA shuffling provides a means to improve expression levels of cancer antigens.

This example describes a method of producing a cancer antigen that can effectively induce an anti-tumor immune response by DNA shuffling of the relevant cancer antigen gene. Library of shuffled melanoma-associated glycoprotein (gp100 / pme117) genes (Huang et al. (1998) J. Invest. Dermatol. 111
: 662-7). Genes can be isolated from melanoma cells from various patients that may have mutations in the gene to increase the diversity of the starting gene. Furthermore, gp
One hundred genes can be isolated from other mammalian species to further increase the diversity of the starting gene. A typical method for gene isolation is RT-PCR. The corresponding gene is shuffled using single-gene DNA shuffling or family DNA shuffling, and a shuffled library is generated.

The shuffled gp100 variants (either pool or individual clones) are subsequently injected into test animals and the immune response is studied (FIG. 15). The shuffled antigen was used as E. coli. E. coli and injected with recombinant purified protein, or the antigen gene is used as a component of a DNA vaccine. The immune response can be analyzed, for example, by measuring anti-gp100 antibodies. Because previous studies have shown that antigens can induce specific antibody responses (Huang et al., Supra). Alternatively, test animals that can be challenged by malignant cells express gp100. Efficiently immunized animals produce gp100-specific cytotoxic T cells and aid in challenge, while in non-immunized or slightly immunized animals, malignant cells proliferate efficiently, Eventually results in lethal expansion of the cells. In addition, antigens that induce cytotoxic T cells capable of killing cancer cells can be identified by measuring in vitro the ability of T cells from immunized animals to kill cancer cells. Typically, the cancer cells are first labeled with a radioactive isotope, and the release of radioactivity is indicative of tumor cell killing after incubation in the presence of T cells from the immunized animal. Such cytotoxicity assays are known in the art.

Antigens that induce the highest levels of specific antibodies and / or can protect against the highest number of malignant cells can be selected for further rounds of shuffling and screening. Mice are useful test animals. Because a large number of antigens can be studied. However, monkeys are preferred as test animals. This is because monkey MHC molecules are very similar to human MHC molecules.

Peripheral blood mononuclear cells from previously infected or immunized human individuals can be used to screen for antigens with optimal ability to activate antigen-specific T cells. This is a particularly useful method. This is because the MHC molecule presenting the antigenic peptide is a human MHC molecule. Shuffled cancer antigens that induce cytotoxic T cells capable of killing cancer cells can be identified by measuring the ability of T cells from immunized animals to kill cancer cells in vitro. Typically, cancer cells are first labeled with a radioactive isotope, and the release of radioactivity is indicative of tumor cell killing after incubation in the presence of T cells from the immunized animal. Such cytotoxicity assays are known in the art.

Example 13 Evolution of Self-Antigens to Induce an Effective Immune Response Autoimmune diseases are characterized by an immune response directed against self-antigens expressed by the host. Autoimmune response is generally mediated by T _H 1 cells that produce high levels of IL-2 and IFN-gamma. Increasing levels of IL- 4 and vaccines that give oriented autoantigen-specific T cells of the IL-5 to T _H 2 phenotype that produces is advantageous. For such vaccines for research, the vaccine antigen must be able to efficiently activate specific T cells. DNA shuffling can be used to generate antigens with such properties. The T _H 2 cell fraction of to induce optimally, T _H 2 cytokines cells to enhance the activation and differentiation of being shown (e.g., IL-4) be a benefit to co-administer (Racke et al. (1994) J. Exp. Med. 180: 1961-66).
).

This example describes a method for producing a self-antigen that can effectively elicit an immune response. DNA shuffling is performed on the relevant autoantigen gene. For example, a library of shuffled myelin basic proteins, or fragments thereof (Zamvil and Steinman (1990) Ann. Rev. I)
mmunol. 8: 579-621; Brooke et al. (1996) Nature.
379: 343-46). MBP is thought to be an important autoimmunogen in patients with multiple sclerosis (MS). At least genes encoding MBP from bovine, mouse, rat, guinea pig and human have been isolated and provide an excellent starting point for family shuffling. A typical method for gene isolation is RT-PCR. Shuffled MB
P gene variants (either pool or individual clones) are subsequently injected into test animals and the immune response is studied. The shuffled antigen was used as E. coli. col
i and injected with the recombinant purified protein or
Used as a component of NA vaccine or viral vector. The immune response can be analyzed, for example, by measuring anti-MBP antibodies by ELISA. Alternatively, lymphocytes from immunized test animals are activated with MBP and T cell proliferation or cytokine synthesis is studied. A sensitivity assay for cytokine synthesis is ELISPOT (McCutcheon et al. (1997)
J. Immunol. Methods 210: 149-66). Mice are useful as test animals. Because a large number of antigens can be studied, monkeys are the preferred test animals. Because monkey MHC molecules are human MH
This is because it is very similar to the C molecule.

Peripheral blood monocytic cells from patients with MS can also be used to screen for antigens with optimal ability to activate MBP-specific T cells. This is a particularly useful method. This is because the MHC molecule presenting the antigenic peptide is a human MHC molecule. Shuffled antigens that activate MBP-specific T cells can be identified by measuring the ability of T cells from MS patients to proliferate or produce cytokines when cultured in the presence of antigen variants. . Such assays are known in the art. One such assay is ELISPOT (McCutcheon et al., Supra). An indicator of the efficiency of an MBP variant to activate specific T cells is also the degree of skin inflammation when the antigen is injected into the skin of patients with MS. Strong inflammation correlates with strong activation of antigen-specific T cells. Improved activation of MBP-specific T cells (in particular, IL
In the presence -4) is enhanced T _H 2 cell responses (which is for the chromatic treatment of MS patients) likely to produce.

Example 14 Method for Optimizing the Immunogenicity of Hepatitis B Surface Antigen This example demonstrates that the envelope protein sequence of hepatitis B virus provides a more immunogenic surface antigen. Describes methods that can be evolved. Such proteins are important for vaccination of low responders and immunotherapy of chronic hepatitis B.

BACKGROUND The current HBV vaccine (Merck, SKB) is based on the immunogenicity of the viral envelope protein and is a Major (or Small) form of the envelope protein produced as particles in yeast. including. These particles contain the major surface antigen (HBsAg), which has an antibody level of 1
Elicits antibodies that can protect against infection when at least 10 milli-international units per milliliter (mU / ml). These recombinant protein preparations cannot induce humoral immunity in chronic carriers (approximately 300 million worldwide), and their induction is important for controlling viral transmission. In addition, certain individuals respond slightly to the vaccine (up to 30-50% of those vaccinated in some groups) and do not develop protective levels of antibody. Inclusion of natural epitope sequences contained in intermediate or large forms of the viral envelope protein has been used as a way to increase the immunogenicity of vaccine preparations. An alternative is to introduce a new (ie, not present in the native viral sequence) helper T cell epitope into the HBsAg sequence using DNA shuffling techniques.

Methods HBsAg from different subtypes of HBV (eg, ayw and adr)
DNA sequences and the related marmot hepatitis virus are prepared for shuffling. Comparison of the genes encoding these proteins suggests that when shuffling ayw and Marmot Hepatitis Virus (WHV) DNA sequences, recombination occurs at least 10 times within 850 base pairs. The nucleotide and amino acid sequences of parts of the different subtypes of HBV are shown in FIG.

The sequence of the major HBsAg B cell antigen site (the “a” epitope) can be retained in the protein sequence by including the coding sequence of the external “a” loop in the final protein preparation. Peptide analogs for the “a” epitope of HBsAg have been described (Neurath et al. (1984) J. Virol. Method).
s 9: 341-346), and the immunogenicity of the "a" epitope has been demonstrated (Bhatnagar et al. (1982) Proc. Nat'l. Acad. Sc).
i. ScL USA 79: 4400-4404). HBsAg and WHsAg share a major "a" determinant, and chimpanzees can be protected by both antigens (Cote et al. (1986) J. Virol. 60: 895-901).
Similarly, important CTL epitopes can be included in a protein in a defined manner.

[0272] B or T (helper or CTL) epitopes from other antigens can also be easily derived into shuffled HBsAg sequences. This can focus the immune response on a particular epitope, independent of other potentially predominant epitopes from the same protein. In addition, the availability of the "a" loop in HBsAg may provide a region of the envelope protein where other artificial antigens or mimotopes may be included.

In all cases where a novel HBV envelope sequence is prepared to contain a specific epitope (HBV, from another pathogen or tumor cell), shuffling of surrounding sequences in the HBV envelope will result in increased protein expression. It acts to optimize and helps ensure that the immune response is directed to the desired epitope.

Several methods for analyzing and utilizing shuffled HBsAg sequences are described below.

A. Modulation of HBsAg Expression Levels The ability to introduce shuffled HBsAg sequences into cells in culture and direct expression of secreted HBsAG (measured using a clinical kit for HBsAg expression) ) To evaluate. This can be used to identify shuffled HBsAg sequences that exhibit optimized HBsAg expression levels. Such coding sequences are of particular interest for DNA vaccination.

B. Avoiding Low Responsiveness to HBsAg Shuffled HBsAg sequences are evaluated for their ability to elicit an immune response to a clinically relevant HBsAg epitope. This can be done using mice of the H-2s and H-2f haplotypes, which respond more or less to HBsAg protein immunity. In these experiments, the antibody was the major "a" epitope of the S protein,
And a second protective epitope of the PreS2 region (linear sequence).

The HBV ayw subtype (plasmid pCAG-M-Kan; Whalen
PreS2 and S coding sequences for the envelope protein (HBsAg) from WHV (plasmid pWHV8 from ATCC) are amplified from the two plasmids by PCR and shuffling. Examples of suitable primers for PCR amplification are shown in FIG. The shuffled library of sequences is cloned into an HBsAg expression vector, and individual clones are selected for plasmid DNA preparation. The DNA is administered to a test animal to identify and recover a vector that induces the desired immune response.

C. Presentation of Natural HBsAg CTL Epitope by Evolved HBsAg Protein This example describes a method using an HBsAG protein evolved to present a native HBsAg CTL epitope. Using shuffling,
As described above, it increases the overall immunogenicity of the HBsAg protein. However, some of the evolved HBsAg sequences are replaced with class I or class II epitope sequences from the native HBsAg protein in order to stimulate immunoreactivity specifically against these native viral epitopes. Alternatively, natural viral epitopes can be added to the evolved protein without compromising the immunogenicity of the evolved HBsAg.

D. Expression of Tumor-Derived CTL Epitope by Evolved HBsAg Protein This example describes a method of expressing a tumor-derived CTL epitope using an evolved HBsAg protein. The overall immunogenicity of the HbsAg protein is increased by shuffling. However, some of the evolved HBsAg sequences are replaced with class I or class II epitope sequences from tumor cells to specifically stimulate immunoreactivity against these natural viral epitopes. Alternatively, tumor cell epitopes can be added to the evolved protein without compromising the immunogenicity of the evolved HBsAg.

E. Expression of Mimotope Sequence by HBsAg This example describes the use of the evolved HBsAg protein for expression of mimotope sequences. Again, the evolved HbsAg protein is used to increase the overall immunogenicity of this protein. However, some of the evolved HBsAg sequences are replaced with mimotope sequences in order to stimulate immunoreactivity specifically to native sequences that cross-react with mimotopes. Alternatively, a mimotope sequence can be added to the evolved protein without compromising the immunogenicity of the evolved HBsAg.

Example 15 Fusion Protein of HbsAg Polypeptide and HIV gp120 Protein This example demonstrates the preparation of a fusion protein (“chimera”) formed from the extracellular fragment gp120 of the HBs polypeptide and the HIV envelope protein. As well as its use as a vaccine.

Background When used as a vaccine, recombinant monomeric gp120 is unable to elicit antibodies with potent neutralizing activity with HIV isolate primary isolates. It has been suggested that oligomeric forms of the HIV envelope protein that expose specific regions of the tertiary structure may better elicit virus-neutralizing antibodies (Parrin et al.
1997) Immunol. Lett. 57: 105-112; VanCott.
(1997) J. Am. Virol. 71: 4319-4330).

In this example, DNA shuffling is applied to this problem in order to obtain a gp120 polypeptide that conforms to a configuration that is slightly different from the configuration of a previous preparation of recombinant gp120. To allow the individual gp120 molecules to interact as oligomers, the fusion is made up of the gp120 sequence (N-
(On the end) and the HBsAg sequence (on the C-terminus of the fusion).

The N-terminal peptide sequence of the S region of the HBsAg polypeptide is a transmembrane structure that is locked to the endoplasmic reticulum membrane. The actual N-terminus of the S region and preS2 sequence is located in the luminal part of the ER. They are found outside the final HBsAg particles. By placing the gp120 sequence on the N-terminus of the HBsAg preS2 or S sequence, the gp120 sequence is also located outside the particle. Therefore, g
p120 molecules can be matched in three-dimensional space to interact as in viruses.

DNA shuffling is used because the exact configuration of the final chimera with the most appropriate immunogenicity cannot be predicted. The HBsAg polypeptide, which functions as a scaffold, and the sequence of gp120 are both shuffled. Screening of the shuffled products was performed by ELISA using antibodies (polyclonal or monoclonal) previously determined to have virus neutralizing activity.
A assay.

(Method) A sequence encoding the gp120 fragment of the HIV envelope protein is prepared, preferably, as a synthetic gene containing codons optimal for gene expression in mammals. The gp120 sequence typically contains a signal sequence on its N-terminus.

[0287] The gp120 sequence is inserted into the preS2 region of the HBsAg expression plasmid.
In the preS2 region of plasmid pMKan and its derivatives, EcoRI
The site and the XhoI site are available for cloning. gp120
The sequence can be inserted between these two sites (leading gp120 near the start of the S coding sequence) or the EcoRI site alone (about 50 amino acids between the gp120 sequence and the start of the S region of HBsAg). To leave a spacer sequence). These two different cloning strategies result in chimeric molecules, where the gp120 sequence is located at different distances from the transmembrane region of the HBsAg sequence.
This may be advantageous in adapting the gp120 sequence to a configuration that is a more suitable immunogen than the monomeric gp120.

Perform DNA shuffling of the entire chimeric sequence. Family shuffling is preferred; this involves the preparation of several gp120-HBsAg fusion proteins, where different gp120 and HBsAg (or WHV) sequences are used. An alignment of the HBsAg nucleotide sequence is shown in FIG. After shuffling the different sequences, the product is cloned into an expression vector such as pMKan. The pool of clones from the library of shuffled products was transfected into cultured cells, and secretion of the chimeric protein was determined by gp1
Assay with a broadly reactive antibody to 20. Positive clones were
V. Antibodies exhibiting neutralizing activity (eg, anti-CD4 binding domain recombinant human monoclonal antibody IgGlb12 (Kessler et al. (1997) AIDS Re
s. Hum. Retroviruses. 1: 13: 575-582; Robe
n, et al. Virol. 68: 4821-4828)). The candidate clones can then be used to immunize mice, and the resulting antisera evaluated for HIV virus neutralizing activity in an in vitro assay.

The gp120 molecule (about 1100 amino acids) contains the monomer HBsAg preS2
Because of the larger size than the + S protein (282 amino acids), none of the HBsAg monomers in the aggregated particles appear to contain the gp120 sequence. The internal initiation of protein synthesis is based on HBs with an initiation methionine that marks the beginning of the S region
It can occur with Ag coding sequences. Thus, the chimeric molecule, which contains the gp120 sequence, mixes with the S region in the cell, and the multimeric particles should assemble with the appropriate number of chimeric polypeptides and native HBsAg S monomers. Alternatively, the S expression plasmid can be mixed with a plasmid expressing the chimera, or a single plasmid expressing the chimera, and the S form can be constructed. FIG. 20 shows a diagram of the obtained particles.

Example 16 HSV-1 as a means of inducing an enhanced protective immune response
DNA shuffling of HSV-2 and HSV-2 glycoproteins B and / or D This example demonstrates that HSV glycoprotein B (gB) and glycoprotein B (gB) exhibit improved ability to induce a protective immune response upon administration to a mammal. The use of DNA shuffling to obtain a protein D (gD) polypeptide is described. Epidemiological studies
Prior infection with HSV-1 has been shown to confer partial protection against infection with HSV-2, indicating the presence of a cross-reactive immune response. Based on previous vaccination studies, the major immunogenic glycoproteins in HSV were gB and gD
Which are encoded by the 2.7 kb and 1.2 kb genes, respectively. The gB and gD genes of HSV-1 are about 85% identical to the corresponding genes of HSV-2, and each gB gene shares little sequence identity with the gD gene. Baboon HSV-2 gB is human HSV-1 g
It is about 75% identical to B or human HSV-2 gB and has a rather long stretch that is nearly 90% identical. In addition, 60-75% identity is found in portions of the horse and bovine herpesvirus genes.

Family shuffling is used as a substrate nucleic acid encoding gB and / or gD from HSV-1 and HSV-2. Preferably, homologous genes are obtained from various strains of HSV. An alignment of gD nucleotide sequences from two strains, HSV-1 and HSV-2, is shown in FIG. The antigen encoded by the shuffled nucleic acid is expressed and analyzed in vivo. For example, one may screen for a neutralizing antibody against HSV-1 / HSV-2 and / or improved induction of a CTL response. Protective immunity can also be detected by challenging mice or guinea pigs with the virus. Screening can be performed using pools or individual clones.

Example 17: HIV gp for induction of a broad spectrum neutralizing Ab response
This example describes the use of DNA shuffling to generate immunogens that cross-react between different strains of the virus (different from wild-type immunogens). Shuffling of the two envelope sequences can generate an immunogen that induces neutralizing antibodies against a third strain.

Antibody-mediated neutralization of HIV-1 is tightly type-specific. Although neutralizing activity spreads over time in infected individuals, induction of such antibodies by vaccination has proven to be very difficult. Antibody-mediated protection from HIV-1 infection in vivo correlates with antibody-mediated neutralization of the virus in vitro.

FIG. 8 illustrates the generation of a library of shuffled gp120 genes. Gp from HIV-1DH12 and HIV-1 IIIB (NL43)
Shuffle 120 genes. The chimeric / variant gp120 gene is then analyzed for its ability to elicit antibodies with a broad spectrum of ability to neutralize different strains of HIV. Each shuffled gp120 gene is
It is incorporated into a genetic vaccine vector, which is then introduced into mice by injection or topical application to the skin. These antigens can also be delivered as a purified recombinant protein. This immune response is measured by analyzing the ability of mouse serum to neutralize HIV growth in vitro. Neutralization assays were performed using HIV-1DH1.
2, for HIV-1IIIB and HIV-189.6. Chimeras / mutants showing broad spectrum neutralization are selected for further rounds of shuffling and selection. Further studies are performed in monkeys to illustrate the ability of the shuffled gp120 gene to provide protection against subsequent infection with the immunodeficiency virus.

The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes will be suggested to those skilled in the art in light of this and It is understood that they fall within the spirit and scope of the appended claims. All publications, patents, and patent specifications cited herein are hereby incorporated by reference for all purposes.

[Brief description of the drawings]

FIG. 1 shows a schematic diagram of a method for generating a chimeric multivalent antigen having immunogenic regions from multiple antigens. Antibodies to each of the non-chimeric parental immunogenic polypeptides are specific for the respective organism (A, B, C). However, after performing the methods of recombination and selection of the present invention, a chimeric immunogenic polypeptide that is recognized by antibodies raised against each of the three parent immunogenic polypeptides is obtained.

FIG. 2 shows the principle of family DNA shuffling. A family of antigen genes from the relevant pathogen is subjected to shuffling, resulting in a library of chimeric and / or mutant antigens. Using screening methods, the most immunogenic and / or cross-protective
e) identifying the recombinant antigen. If desired, they can be subjected to further rounds of shuffling and screening.

FIGS. 3A-3B show schematics of how to obtain recombinant polypeptides capable of inducing a broad immune response. In FIG. 3A, wild-type immunogenic polypeptides from pathogens A, B, and C provide protection against the corresponding pathogen from which the polypeptide is derived, but little or no protection against other pathogens Does not provide cross protection (left panel). After shuffling, an A / B / C chimeric polypeptide capable of eliciting a protective immune response against all three pathogen types is obtained (right panel). In FIG. 3B, shuffling is used on substrate nucleic acids from two pathogen strains (A, B), which encode polypeptides that are only protective against the corresponding pathogen. After shuffling, the resulting chimeric polypeptide can induce an immune response that is effective not only against the two parental pathogen strains, but also against a third strain (C) of the pathogen.

FIG. 4 can determine whether a particular polynucleotide encodes an immunogenic polypeptide with the desired properties (eg, enhanced immunogenicity and / or cross-reactivity). Some of the possible factors are illustrated. Sequence regions that positively affect a particular property are indicated as a plus sign along the antigen gene, while sequence regions that have a negative effect are indicated as a minus sign. The pool of related antigen genes is shuffled and screened to obtain a positive sequence region and a recombinant nucleic acid lacking the negative region. No prior knowledge is needed about which regions are positive or negative for a particular trait.

FIG. 5 shows a schematic of a screening strategy for antigen library screening.

FIG. 6 shows a schematic of the pool and de-rotation strategy used in antigen library screening.

FIG. 7 shows HSV-1 (SEQ ID NO: 1) and HSV-2 (gD-1 (SEQ ID NO: 2)
2) and gD-2 (SEQ ID NO: 3)) are alignments of nucleotide sequences of glycoprotein D (gD).

FIG. 8A shows a diagram of a method for expression of HIV gp120 using a genetic vaccine vector and generation of a library of shuffled gp120 genes.

FIG. 8B shows PCR primers that are useful for obtaining a gp120 nucleic acid substrate for a DNA shuffling reaction. Suitable primers for generating the substrate include 6025F (SEQ ID NO: 4), 7773R (SEQ ID NO: 5), and primers suitable for amplifying shuffled nucleic acid include 6196F (
SEQ ID NO: 6) and 7746R (SEQ ID NO: 7). Primer Bss
H2-6205F (SEQ ID NO: 8) can be used to clone the resulting fragment into a gene vaccine vector.

FIG. 9 shows the domain structure of the hepadnavirus envelope gene.

FIG. 10 shows a schematic diagram of the use of shuffling to obtain hepadnavirus proteins, wherein the immunogenicity of one antigenic domain is improved.

FIG. 11 Shuffles the gene encoding a hepadnavirus protein with one antigenic domain with improved immunogenicity, with all three domains having improved immunogenicity. 1 shows a strategy for obtaining a recombinant protein.

FIG. 12 shows the transmembrane mechanism of HBsAg polypeptide.

FIG. 13 shows a method for using phage display to obtain a recombinant allergen that is not bound by pre-existing IgE.

FIG. 14 shows a strategy for screening recombinant allergens to identify those that are effective in activating _TH cells. A PBMC or T cell clone from an atopic individual is exposed to antigen presenting cells presenting an antigen variant obtained using the method of the invention. To identify allergen variants that are effective in activating T cells, cultures may be used to induce T cell proliferation or for patterns of cytokine synthesis indicative of the particular type of T cell activation desired. To be tested. If desired, allergen variants that test positive in an in vitro assay can be subjected to in vivo testing.

FIG. 15 shows a strategy for screening recombinant cancer antigens to identify those that are effective in activating T cells in cancer patients.

FIG. 16A and FIG. 16B show two different strategies for generating a vector containing multiple T cell epitopes obtained, for example, by DNA shuffling. In FIG. 16A, each individual shuffled epitope-encoding nucleic acid is ligated to a single promoter, and multiple promoter-epitope gene constructs can be placed in a single vector. The schematic shown in FIG. 16B involves linking multiple epitope-encoding nucleic acids to a single promoter.

FIG. 17 shows the sequence of the PreS2-S coding region of the different hepatitis B surface antigen (HBsAg) or woodchuck hepatitis B (WHV) protein and the corresponding amino acid sequence. Primers suitable for amplification of this region are also provided.

FIG. 18 shows primers that are suitable for the amplification of large fragments containing the S2S coding sequence. This primer hybridizes to a region located approximately 200 bp outside the desired sequence.

FIG. 19 shows an alignment of amino acid sequences of surface antigens from different HVB and WHV subtypes.

FIG. 20 shows a diagram of multimeric particles that assemble when the appropriate number of chimeric polypeptides and native HBsAgS monomers are mixed.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] August 19, 1999 (August 19, 1999)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0066

[Correction method] Change

[Correction contents]

3. Parasites Parasite-derived antigens can also be optimized by the methods of the present invention. These include Schistosoma mansoni, S.A. haematobium,
Or S. schistosome intestinal associated antigen CAA (circulating anodic antigen) and CCA (
Circulating cathodic antigen
(Deeler et al. (1996) Parasitology 112: 21-35.
); A multiple antigen peptide consisting of two different protective antigens from the parasite Schistosoma mansoni (multiple antigen pep)
ide) (MAP) (Ferru et al. (1997) Parasite Immu)
nol. 19: 1-11); Leishmania parasite surface molecule (Leza)
ma-Davila (1997) Arch. Med. Res. 28: 47-53
); loa third stage larva (L3) antigen (Akue et al. (1997) J. In.
fact. Dis. 175: 158-63); Theileria annul
The genes Tams1-1 and Tams1-2 encoding the 30 kDa and 32 kDa major merozoite surface antigens of ata (Ta) (d'Oliveira et al. (1)
996) Gene 172: 33-39); Plasmodium falci.
param merozoite surface antigen 1 or 2 (al-Yaman et al. (1995) T
rans. R. Soc. Trop. Med. Hyg. 89: 555-559; B
Eck et al. (1997) J. Am. Infect. Dis. 175: 921-926; R
Zepczyk et al. (1997) Infect. Immun. 65: 1098-1
100); a B epitope based on the circumsporozoite (CS) protein from Plasmodium berghei, (PPPPNPND) ₂ (SEQ ID NO: 23) and Plasmodium yoelii, (QGPGAP) ₃ QG (SEQ ID NO: 24). berghei T helper epitope KQIRDSITEEWS (SEQ ID NO: 25)
(Reed et al. (1997) Vaccine 15: 482-488); sporozoites (circum sporozoite protein and sporozoite surface protein 2).
Plasmodium falcipa encoded by NYVAC-Pf7
rum antigen, liver (liver stage antigen 1), blood (merozoite surface protein 1, serine repeat antigen, and apical membrane antigen 1), but are not limited thereto, and the sexual (25 kDa sex) The antigenic stage is inserted into a single NYVAC genome and NYVAC-Pf7 (Tine et al. (1996) Infect.
Immun. 64: 3833-3844); Plasmodium falci.
param antigen Pfs230 (Williamson et al. (1996) Mol. B
iochem. Parasitol. 78: 161-169); Plasmod
ium falciparum apical membrane antigen (AMA-1) (Lal et al. (199)
6) Infect. Immun. 64: 1054-1059); Plasmod
ium falciparum proteins Pfs28 and Pfs25 (Duf
fy and Kaslow (1997) Infect. Immun. 65: 110
9-1113); Plasmodium falciparum merozoite surface protein, MSP1 (Hui et al. (1996) Infect. Immun. 64
: 1502-1509); Malaria antigen Pf332 (Ahlborg et al. (199)
6) Immunology 88: 630-635); Plasmodium
falciparum erythrocyte membrane protein 1 (Baruch et al. (1995) Pr
oc. Nat'l. Acad. Sci. USA 93: 3497-3502; B
aruch et al. (1995) Cell 82: 77-87); Plasmodiu.
m falciparum merozoite surface antigen, PfMSP-1 (Egan et al. (
1996). Infect. Dis. 173: 765-769); Plasm.
odium falciparum antigens SERA, EBA-175, RAP1 and RAP2 (Riley (1997) J. Pharm; Pharmacol.
49: 21-27); Schistosoma japonicum paramyosin (Sj97) or a fragment thereof (Yang et al. (1995) Bioche).
m. Biophys. Res. Commun. 212: 1029-1039);
And Hsp70 (Maresca and Kobayash) in parasites
i (1994) Experientia 50: 1067-1074).

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] FIG.

[Correction method] Change

[Correction contents]

FIG. 17 shows the sequence of the PreS2-S coding region of different hepatitis B surface antigens (HBsAg) (SEQ ID NOS: 9 and 11) and the corresponding amino acid sequence (SEQ ID NO: 10).
And 12), or PreS of woodchuck hepatitis B (WHV) protein
The sequence of the 2-S coding region and the corresponding amino acid sequence (SEQ ID NOS: 15 and 16)
). Primers suitable for amplification of this region are also provided (HBV, SEQ ID NO: 1).
3 and 14; WHV, SEQ ID NOs: 17 and 18).

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] FIG.

[Correction method] Change

[Correction contents]

FIG. 18 shows primers that are suitable for amplification of a large fragment containing the S2S coding sequence (SEQ ID NOs: 19-22). This primer hybridizes to a region located approximately 200 bp outside the desired sequence.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] FIG.

[Correction method] Change

[Correction contents]

FIG. 19 shows an alignment of the amino acid sequences of surface antigens from different HVB subtypes (SEQ ID NOs: 10 and 12).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 14/24 C07K 14/245 14/245 14/28 14/28 14/31 14/31 14/315 14 / 315 14/35 14/35 19/00 19/00 G01N 33/15 Z G01N 33/15 33/50 Z 33/50 37/00 103 37/00 103 C12N 15/00 ZNAA (31) Priority claim number 60 / 105,509 (32) Priority date October 23, 1998 (Oct. 23, 1998) (33) Priority claiming country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE) , LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG , BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD , SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW (71) Applicant 3410 Central Expressway, Santa Clara, California 95051 U.S.A. S. A. (72) Inventor Bass, Stephen H. United States California 94010, Hillsborough, Parrot Drive 950 (72) Inventor Fahren, Robert Gerald France F-75015 Paris, Lour le Coeurb, 332 (72) Inventor Howard, Russell United States California 94022, Los Altos Hills, Bisqueino Drive 12700 (72) ) Inventor Stemmer, Willem P. C. United States California 95030, Los Gatos, Cathy Court 108 F Term (Reference) 2G045 AA34 AA35 AA40 CB01 CB21 DA12 DA13 DA14 DA36 FB03 4B024 AA01 AA11 BA31 CA02 CA05 CA07 DA02 EA03 EA04 GA11 HA03 HA17 4H045 AA11 CA03 CA01 CA02 EA50 FA74

Claims (16)

[Claims] 1. A recombinant multivalent antigenic polypeptide comprising a first antigenic determinant of a first polypeptide and at least a second antigenic determinant from a second polypeptide. 2. The multivalent antigenic polypeptide of claim 1, wherein said polypeptide comprises at least a third antigenic determinant from a third polypeptide. 3. The method of claim 1, wherein the first and second polypeptides are a cancer antigen, an antigen associated with an autoimmune disorder, an antigen associated with an inflammatory condition, an antigen associated with an allergic reaction,
The polyvalent antigenic polypeptide according to claim 1, which is selected from the group consisting of an antigen derived from an infectious agent. 4. The multivalent antigenic polypeptide of claim 3, wherein said antigen is derived from a virus, a parasite and a bacterium. 5. The method according to claim 1, wherein the antigen is Venezuelan equine encephalomyelitis virus, or a related alphavirus, a virus of the Japanese encephalitis virus complex, a virus of the tick-borne encephalitis virus complex, dengue virus, hantavirus, HIV, hepatitis B. The multivalent antigenic polypeptide according to claim 4, which is derived from a virus selected from the group consisting of a virus, a hepatitis C virus, and a herpes simplex virus. 6. The multivalent antigenic polypeptide according to claim 5, wherein the antigen is an envelope protein. 7. The antigen of claim 1, wherein said antigen is of bacterial origin and said
tapylococcus aureus enterotoxin, Strepto
coccus pyogenes enterotoxin, Vibrio chole
ratoxin, enterotoxin-producing Escherichia coli heat-labile enterotoxin, OspA and OspC polypeptides from Borrelia sp., antigen 85 polypeptide from Mycobacterium sp.
The multivalent antigenic polypeptide according to claim 4, which is selected from the group consisting of MSP antigens derived from rum. 8. The multivalent antigen of claim 1, wherein said multivalent antigenic polypeptide exhibits reduced affinity for mammalian-derived IgE as compared to said first or second polypeptide. Sex polypeptide. 9. The multivalent antigenic polypeptide of claim 1, wherein said first antigenic determinant and said second antigenic determinant are from different serotypes of a pathogenic organism. 10. The multivalent antigenic polypeptide of claim 1, wherein said first antigenic determinant and said second antigenic determinant are from different species of a pathogenic organism. 11. The multivalent antigenic polypeptide according to claim 1, wherein said first polypeptide and said second polypeptide are allergens. 12. A recombinant antigen library comprising a recombinant nucleic acid encoding an antigenic polypeptide, said library comprising a disease-associated antigenic polypeptide to produce a library of recombinant nucleic acids. Obtained by recombining at least the first and second forms of the nucleic acid comprising the encoding polynucleotide sequence, wherein said first and second forms differ from each other in two or more nucleotides;
Recombinant antigen library. 13. A method for obtaining a polynucleotide encoding a recombinant antigen having improved ability to induce an immune response to a disease state, comprising the following steps: (1) Producing a library of recombinant nucleic acids Recombining at least the first and second forms of the nucleic acid comprising a polynucleotide sequence encoding an antigenic polypeptide associated with the disease state, wherein the first and second forms are Different from each other in two or more nucleotides; and (2) at least one optimized recombinant nucleic acid encoding an optimized recombinant antigenic polypeptide with improved ability to induce an immune response against said disease state Screening the library to identify the library. 14. The method further comprises the following steps: (3) a further form of the nucleic acid that is the same as or different from the first and second forms to produce a further library of recombinant nucleic acids. Recombination with at least one optimized recombinant nucleic acid; (4) identifying at least one further optimized recombinant nucleic acid encoding a polypeptide having improved ability to induce an immune response against said disease state (5) optionally, wherein said further optimized recombinant nucleic acid encodes a polypeptide having improved ability to induce an immune response to said disease state Until you do (3
14. The method of claim 13, comprising: repeating) and (4). 15. The phage polypeptide wherein the optimized recombinant nucleic acid encodes a multivalent antigenic polypeptide and the screening comprises the following steps: wherein the recombinant antigen is displayed on the surface of a phage particle Expressing said library of recombinant nucleic acids in a phage display expression vector such that it is expressed as a fusion protein with a first antibody specific for a first serotype of said virulence factor. Contacting with a phage and selecting a phage that binds to the first antibody; a second antibody that is specific for a second serotype of the virulence factor and a phage that binds to the first antibody And selecting those phages that bind to the second antibody; wherein the phages that bind to the first antibody and the second antibody are The step of expressing an antigenic polypeptide is achieved by method according to claim 13. 16. A method for obtaining a recombinant viral vector having an enhanced ability to induce an antiviral response in a cell, the method comprising the following steps: (1) Live recombinant virus vector Recombining at least the first and second forms of the nucleic acid, including the viral vector, to produce a rally,
Wherein said first and second forms differ from each other in two or more nucleotides; (2) transfecting said library of recombinant viral vectors into a population of mammalian cells; (3) Staining the cells for the presence of the Mx protein; and (4) isolating a recombinant viral vector from cells having a positive staining for the Mx protein, wherein the recombinant virus is derived from positively stained cells. Wherein the vector exhibits an enhanced ability to induce an antiviral response.