U.S. patent application number 10/383317 was filed with the patent office on 2004-01-01 for antigen library immunization.
This patent application is currently assigned to Maxygen, Inc., a Delaware corporation. Invention is credited to Bass, Steven H., Howard, Russell, Punnonen, Juha, Stemmer, Willem P.C., Whalen, Robert Gerald.
Application Number | 20040001849 10/383317 |
Document ID | / |
Family ID | 27372458 |
Filed Date | 2004-01-01 |
United States Patent
Application |
20040001849 |
Kind Code |
A1 |
Punnonen, Juha ; et
al. |
January 1, 2004 |
Antigen library immunization
Abstract
This invention is directed to antigen library immunization,
which provides methods for obtaining antigens having improved
properties for therapeutic and other uses. The methods are useful
for obtaining improved antigens that can induce an immune response
against pathogens, cancer, and other conditions, as well as
antigens that are effective in modulating allergy, inflammatory and
autoimmune diseases.
Inventors: |
Punnonen, Juha; (Belmont,
CA) ; Bass, Steven H.; (Hillsborough, CA) ;
Whalen, Robert Gerald; (Foster City, CA) ; Howard,
Russell; (Los Altos Hills, CA) ; Stemmer, Willem
P.C.; (Los Gatos, CA) |
Correspondence
Address: |
MAXYGEN, INC.
INTELLECTUAL PROPERTY DEPARTMENT
515 GALVESTON DRIVE
RED WOOD CITY
CA
94063
US
|
Assignee: |
Maxygen, Inc., a Delaware
corporation
|
Family ID: |
27372458 |
Appl. No.: |
10/383317 |
Filed: |
March 7, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10383317 |
Mar 7, 2003 |
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09724852 |
Nov 28, 2000 |
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6576757 |
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09724852 |
Nov 28, 2000 |
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09247890 |
Feb 10, 1999 |
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6541011 |
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60105509 |
Oct 23, 1998 |
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60074294 |
Feb 11, 1998 |
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Current U.S.
Class: |
424/186.1 ;
424/188.1; 424/189.1; 424/190.1; 530/350 |
Current CPC
Class: |
A61K 39/12 20130101;
Y02A 50/30 20180101; A61K 39/0011 20130101; A61K 39/35 20130101;
C12N 15/1037 20130101; A61K 39/015 20130101; A61K 39/085 20130101;
C07K 14/24 20130101; C07K 2319/74 20130101; A61K 39/00 20130101;
A61K 39/0291 20130101; A61K 2039/70 20130101; A61K 39/107 20130101;
C12N 15/62 20130101; A61K 39/105 20130101; A61K 39/0258 20130101;
C12N 2770/24234 20130101; C07K 14/005 20130101; C12N 2710/16634
20130101; C12N 2740/16134 20130101; C12N 2770/36134 20130101; C12N
15/1034 20130101; A61K 2039/53 20130101; A61K 39/0225 20130101;
C12N 2770/24134 20130101; C07K 2319/40 20130101; C12N 2730/10134
20130101; C40B 40/02 20130101; C12N 15/1027 20130101; C12N
2760/12134 20130101; C07K 2319/02 20130101 |
Class at
Publication: |
424/186.1 ;
424/188.1; 424/189.1; 424/190.1; 530/350 |
International
Class: |
A61K 039/12; A61K
039/21; A61K 039/29; A61K 039/02; C07K 014/16; C07K 014/02; C07K
014/195 |
Claims
What is claimed is:
1. An recombinant multivalent antigenic polypeptide that comprises
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 the
polypeptide comprises at least a third antigenic determinant from a
third polypeptide.
3. The multivalent antigenic polypeptide of claim 1, wherein the
first and second polypeptides are selected from the group
consisting of cancer antigens, antigens associated with
autoimmunity disorders, antigens associated with inflammatory
conditions, antigens associated with allergic reactions, and
antigens from infectious agents.
4. The multivalent antigenic polypeptide of claim 3, wherein the
antigens are from a virus, a parasite, or a bacteria.
5. The multivalent antigenic polypeptide of claim 4, wherein the
antigens are from a virus selected from the group consisting of a
Venezuelan equine encephalitis virus or a related alphavirus, a
virus of the Japanese encephalitis virus complex, a virus of the
tick-borne encephalitis virus complex, a Dengue virus, a Hanta
virus, an HIV, a hepatitis B virus, a hepatitis C virus, and a
Herpes simplex virus.
6. The multivalent antigenic polypeptide of claim 5, wherein the
antigens are envelope proteins.
7. The multivalent antigenic polypeptide of claim 4, wherein the
antigens are from a bacteria and are selected from the group
consisting of a Yersinia V antigen, a Staphylococcus aureus
enterotoxin, a Streptococcits pyogenes enterotoxin, a Vibrio
cholera toxin, an enterotoxigenic Escherichia coli heat labile
enterotoxin, a OspA and a OspC polypeptide from a Borrelia species,
an Antigen 85 polypeptide from a Mycobacterium species, a VacA and
a CagA polypeptide from Helicobacter pylori, and an MSP antigen
from Plasmodium falciparum.
8. The multivalent antigenic polypeptide of claim 1, wherein the
multivalent antigenic polypeptide exhibits reduced affinity to IgE
from a mammal compared to the first or second polypeptides.
9. The multivalent antigenic polypeptide of claim 1, wherein the
first antigenic determinant and the second antigenic determinant
are from different serotypes of a pathogenic organism.
10. The multivalent antigenic polypeptide of claim 1, wherein the
first antigenic determinant and the second antigenic determinant
are from different species of pathogenic organism.
11. The multivalent antigenic polypeptide of claim 1, wherein the
first polypeptide and the second polypeptide are allergens.
12. The multivalent antigenic polypeptide of claim 11, wherein the
allergens are dust mite allergens, grass pollen allergens, birch
pollen allergens, ragweed pollen allergens, hazel pollen allergens,
cockroach allergens, rice allergens, olive tree pollen allergens,
fungal allergens, mustard allergens, and bee venom.
13. The multivalent antigenic polypeptide of claim 1, wherein the
first polypeptide and the second polypeptide are associated with an
inflammatory or autoimmune disease.
14. The multivalent antigenic polypeptide of claim 13, wherein the
first polypeptide and the second polypeptide are autoantigens
associated with a disease selected from the group consisting of
multiple sclerosis, scleroderma, systemic sclerosis, systemic lupus
erythematosus, hepatic autoimmune disorder, skin autoimmune
disorder, insulin-dependent diabetes mellitus, thyroid autoimmune
disorder, and rheumatoid arthritis.
15. The multivalent antigenic polypeptide of claim 1, wherein the
first polypeptide and the second polypeptide are cancer antigens or
sperm antigens.
16. A recombinant antigen library comprising recombinant nucleic
acids that encode antigenic polypeptides, wherein the library is
obtained by recombining at least first and second forms of a
nucleic acid which comprises a polynucleotide sequence that encodes
a disease-associated antigenic polypeptide, wherein the first and
second forms differ from each other in two or more nucleotides, to
produce a library of recombinant nucleic acids.
17. The recombinant antigen library of claim 16, wherein the first
and second polypeptides are toxins.
18. A method of obtaining a polynucleotide that encodes a
recombinant antigen having improved ability to induce an immune
response to a disease condition, the method comprising: (1)
recombining at least first and second forms of a nucleic acid which
comprises a polynucleotide sequence that encodes an antigenic
polypeptide that is associated with the disease condition, wherein
the first and second forms differ from each other in two or more
nucleotides, to produce a library of recombinant nucleic acids; and
(2) screening the library to identify at least one optimized
recombinant nucleic acid that encodes an optimized recombinant
antigenic polypeptide that has improved ability to induce an immune
response to the disease condition.
19. The method of claim 18, wherein the method further comprises:
(3) recombining at least one optimized recombinant nucleic acid
with a farther form of the nucleic acid, which is the same or
different from the first and second forms, to produce a further
library of recombinant nucleic acids; (4) screening the further
library to identify at least one further optimized recombinant
nucleic acid that encodes a polypeptide that has improved ability
to induce an immune response to the disease condition; and (5)
repeating (3) and (4), as necessary, until the further optimized
recombinant nucleic acid encodes a polypeptide that has improved
ability to induce an immune response to the disease condition.
20. The method of claim 18, wherein the disease-associated
polypeptides are selected from the group consisting of cancer
antigens, antigens associated with autoimmunity disorders, antigens
associated with inflammatory conditions, antigens associated with
allergic reactions, and antigens associated with infectious
agents.
21. The method of claim 18, wherein the disease condition is an
infectious disease and the first and second forms of the nucleic
acid each encode an antigen of a different serotype of a pathogenic
agent.
22. The method of claim 21, wherein the first and second forms of
the nucleic acid are each from a different species of pathogen.
23. The method of claim 21, wherein the screening is accomplished
by: introducing into a test animal either: a) the library of
recombinant nucleic acids, or b) recombinant polypeptides encoded
by the library of recombinant nucleic acids; introducing the
pathogenic agent into the test animal; and determining whether the
test animal is resistant to challenge by the pathogenic agent.
24. The method of claim 23, wherein the pathogenic agent introduced
into the test animal is of a different serotype than that used as a
source of the first and second forms of the nucleic acid.
25. The method of claim 23, wherein the library is subdivided into
a plurality of pools, each of which pools is introduced into a test
animal to identify those pools that include an optimized
recombinant nucleic acid that encodes a polypeptide which has
improved ability to induce an immune response to the pathogenic
agent.
26. The method of claim 25, wherein the pools that include an
optimized recombinant nucleic acid are further subdivided into a
plurality of subpools, each of which subpools is introduced into a
test animal to identify those pools that include an optimized
recombinant nucleic acid that encodes a polypeptide which has
improved ability to induce an immune response to the pathogenic
agent.
27. The method of claim 18, wherein the optimized recombinant
nucleic acid encodes a multivalent antigenic polypeptide and the
screening is accomplished by: expressing the library of recombinant
nucleic acids in a phage display expression vector such that the
recombinant antigen is expressed as a fusion protein with a phage
polypeptide that is displayed on a phage particle surface;
contacting the phage with a first antibody that is specific for a
first serotype of the pathogenic agent and selecting those phage
that bind to the first antibody; contacting those phage that bind
to the first antibody with a second antibody that is specific for a
second serotype of the pathogenic agent and selecting those phage
that bind to the second antibody; wherein those phage that bind to
the first antibody and the second antibody express a multivalent
antigenic polypeptide.
28. The method of claim 27, wherein the screening further comprises
contacting those phage that bind to the first and second antibodies
with one or more additional antibodies, each of which is specific
for an additional serotype of the pathogenic agent, and selecting
those phage that bind to the respective additional antibodies.
29. The method of claim 27, wherein the phage display expression
vector comprises a suppressible stop codon between the recombinant
nucleic acid and the phage polypeptide, whereby expression in a
host cell which comprises a corresponding suppressor tRNA results
in production of the fusion protein and expression in a host cell
which lacks a corresponding suppressor tRNA results in production
of the recombinant antigen not as a fusion protein.
30. The method of claim 18, wherein the optimized recombinant
antigen exhibits an enhanced expression level in a host cell and
the screening is accomplished by expression of each recombinant
nucleic acid in the host cell and subjecting the host cells to flow
cytometry-based cell sorting to obtain those host cells that
display the recombinant antigen on the host cell surface.
31. The method of claim 18, wherein the improved property is
selected from the group consisting of: improved immunogenicity;
enhanced cross-reactivity against different forms of the
disease-associated antigenic polypeptide; reduced toxicity;
improved adjuvant activity in vivo; and improved production of the
immunogenic polypeptide.
32. The method of claim 31, wherein the improved property is
enhanced cross-reactivity against different forms of the
disease-associated polypeptide and the first and second forms of
the nucleic acid are from a first and a second form of the
disease-associated polypeptide.
33. The method of claim 32, wherein the first and second forms of
the disease-associated polypeptide are obtained from at least a
first and second species of a pathogenic agent and the optimized
recombinant nucleic acid encodes a recombinant polypeptide that
induces a protective response against both species of the
pathogenic agent.
34. The method of claim 33, wherein the recombinant polypeptide
induces a protective response against at least one additional
species of the pathogenic agent.
35. The method of claim 33, wherein the pathogenic agent is a
toxin.
36. The method of claim 33, wherein the pathogenic agent is a virus
or a cell.
37. The method of claim 33, wherein the disease-associated
polypeptide is a Yersinia V-antigen.
38. The method of claim 37, wherein the at least first and second
forms of a nucleic acid are obtained from at least a first and
second species of Yersinia.
39. The method of claim 38, wherein the Yersinia species are
selected from the group consisting of Y. pestis, Y. enterocolitica,
and Y. pseudotuberculosis.
40. The method of claim 33, wherein the pathogenic agent is a
bacterial toxin.
41. The method of claim 18, wherein the disease condition is cancer
and the screening step involves introducing the optimized
recombinant nucleic acids into a genetic vaccine vector and testing
library members for ability to inhibit proliferation of cancer
cells or inducing death of cancer cells.
42. The method of claim 41, wherein the optimized recombinant
nucleic acid comprises a nucleotide sequence that encodes a tumor
specific antigen.
43. The method of claim 41, wherein the optimized recombinant
nucleic acid comprises a nucleotide sequence that encodes a
molecule which is capable of inhibiting proliferation of cancer
cells.
44. The method of claim 18, wherein the disease condition is an
inflammatory response which has an unknown or no antigen
specificity and the screening step involves one or more of the
following: a) determining the ability of the genetic vaccine vector
to induce cytokine production by PBMC, synovial fluid cells,
purified T cells, monocytes/macrophages, dendritic cells, or T cell
clones; b) determining the ability of the genetic vaccine vector to
induce T cell activation or proliferation; and c) determining the
ability of the genetic vaccine vector to induce T cell
differentiation to T.sub.H1 or T.sub.H2 cells.
45. The method of claim 18, wherein the disease condition is an
autoimmune response.
46. The method of claim 45, wherein the optimized recombinant
antigenic polypeptide shifts the immune response from a
T.sub.H1-mediated response to a T.sub.H2-mediated response.
47. The method of claim 18, wherein the disease condition is an
allergic immune response.
48. The method of claim 47, wherein the optimized recombinant
antigenic polypeptide shifts the immune response from a
T.sub.H2-mediated response to a T.sub.H1-mediated response.
49. The method of claim 47, wherein the optimized recombinant
antigenic polypeptide induces an immune response characterized by
predominant IgG and IgM expression and reduced IgE expression.
50. The method of claim 47, wherein the optimized recombinant
antigenic polypeptide is not recognized by pre-existing IgE
molecules present in sera of atopic mammals.
51. The method of claim 50, wherein the optimized recombinant
antigenic polypeptide retains T cell epitopes that are involved in
modulating a T cell response.
52. A method of obtaining a recombinant viral vector which has an
enhanced ability to induce an antiviral response in a cell, the
method comprising the steps of: (1) recombining at least first and
second forms of a nucleic acid which comprise a viral vector,
wherein the first and second forms differ from each other in two or
more nucleotides, to produce 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 Ix protein; and (4) isolating recombinant viral
vectors from cells which stain positive for Mx protein, wherein
recombinant viral vectors from positive staining cells exhibit
enhanced ability to induce an antiviral response.
53. The method of claim 52, wherein the viral vector comprises an
influenza viral genomic nucleic acid.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Provisional
Application No. 60/074,294, filed Feb. 11, 1998, and U.S.
Provisional Application No. 60/105,509, filed Oct. 23, 1998, which
applications are incorporated herein by reference for all
purposes.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention pertains to the field of methods for
developing immunogens that can induce efficient immune responses
against a broad range of antigens.
[0004] 2. Background
[0005] The interactions between pathogens and hosts are results of
millions of years of evolution, during which the mammalian immune
system has evolved sophisticated means to counterattack pathogen
invasions. However, bacterial and viral pathogens have
simultaneously gained a number of mechanisms to improve their
virulence and survival in hosts, providing a major challenge for
vaccine research and development despite the powers of modem
techniques of molecular and cellular biology. Similar to the
evolution of pathogen antigens, several cancer antigens are likely
to have gained means to downregulate their immunogenicity as a
mechanism to escape the host immune system.
[0006] Efficient vaccine development is also hampered by the
antigenic heterogeneity of different strains of pathogens, driven
in part by evolutionary forces as means for the pathogens to escape
immune defenses. Pathogens also reduce their immunogenicity by
selecting antigens that are difficult to express, process and/or
transport in host cells, thereby reducing the availability of
immunogenic peptides to the molecules initiating and modulating
immune responses. The mechanisms associated with these challenges
are complex, multivariate and rather poorly characterized.
Accordingly, a need exists for vaccines that can induce a
protective immune response against bacterial and viral pathogens.
The present invention fulfills this and other needs.
SUMMARY OF THE INVENTION
[0007] The present invention provides recombinant multivalent
antigenic polypeptides that include a first antigenic determinant
from a first disease-associated polypeptide and at least a second
antigenic determinant from a second disease-associated polypeptide.
The disease-associated polypeptides can be selected from the group
consisting of cancer antigens, antigens associated with
autoimmunity disorders, antigens associated with inflammatory
conditions, antigens associated with allergic reactions, antigens
associated with infectious agents, and other antigens that are
associated with a disease condition.
[0008] In another embodiment, the invention provides a recombinant
antigen library that contains recombinant nucleic acids that encode
antigenic polypeptides. The libraries are typically obtained by
recombining at least first and second forms of a nucleic acid which
includes a polynucleotide sequence that encodes a
disease-associated antigenic polypeptide, wherein the first and
second forms differ from each other in two or more nucleotides, to
produce a library of recombinant nucleic acids.
[0009] Another embodiment of the invention provides methods of
obtaining a polynucleotide that encodes a recombinant antigen
having improved ability to induce an immune response to a disease
condition. These methods involve: (1) recombining at least first
and second forms of a nucleic acid which comprises a polynucleotide
sequence that encodes an antigenic polypeptide that is associated
with the disease condition, wherein the first and second forms
differ from each other in two or more nucleotides, to produce a
library of recombinant nucleic acids; and (2) screening the library
to identify at least one optimized recombinant nucleic acid that
encodes an optimized recombinant antigenic polypeptide that has
improved ability to induce an immune response to the disease
condition.
[0010] These methods optionally further involve: (3) recombining at
least one optimized recombinant nucleic acid with a further form of
the nucleic acid, which is the same or different from the first and
second forms, to produce a further library of recombinant nucleic
acids; (4) screening the further library to identify at least one
further optimized recombinant nucleic acid that encodes a
polypeptide that has improved ability to induce an immune response
to the disease condition; and (5) repeating (3) and (4), as
necessary, until the further optimized recombinant nucleic acid
encodes a polypeptide that has improved ability to induce an immune
response to the disease condition.
[0011] In some embodiments, the optimized recombinant nucleic acid
encodes a multivalent antigenic polypeptide and the screening is
accomplished by expressing the library of recombinant nucleic acids
in a phage display expression vector such that the recombinant
antigen is expressed as a fusion protein with a phage polypeptide
that is displayed on a phage particle surface; contacting the phage
with a first antibody that is specific for a first serotype of the
pathogenic agent and selecting those phage that bind to the first
antibody; and contacting those phage that bind to the first
antibody with a second antibody that is specific for a second
serotype of the pathogenic agent and selecting those phage that
bind to the second antibody; wherein those phage that bind to the
first antibody and the second antibody express a multivalent
antigenic polypeptide.
[0012] The invention also provides methods of obtaining a
recombinant viral vector which has an enhanced ability to induce an
antiviral response in a cell. These methods can include the steps
of: (1) recombining at least first and second forms of a nucleic
acid which comprise a viral vector, wherein the first and second
forms differ from each other in two or more nucleotides, to produce
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 Mx protein; and
(4) isolating recombinant viral vectors from cells which stain
positive for Mx protein, wherein recombinant viral vectors from
positive staining cells exhibit enhanced ability to induce an
antiviral response.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1 shows a schematic representation of a method for
generating a chimeric, multivalent antigen that has immunogenic
regions from multiple antigens. Antibodies to each of the
non-chimeric parental immunogenic polypeptides are specific for the
respective organisms (A, B, C). After carrying out the
recombination and selection methods of the invention, however, a
chimeric immunogenic polypeptide is obtained that is recognized by
antibodies raised against each of the three parental immunogenic
polypeptides.
[0014] FIG. 2 shows the principle of family DNA shuffling. A family
of antigen genes from related pathogens are subjected to shuffling,
which results in a library of chimeric and/or mutated antigens.
Screening methods are employed to identify those recombinant
antigens that are the most immunogenic and/or cross-protective.
These can, if desired, be subjected to additional rounds of
shuffling and screening.
[0015] FIG. 3A-FIG. 3B shows a schematic for a method by which one
can obtain recombinant polypeptides that can induce a
broad-spectrum immune response. In FIG. 3A, wild-type immunogenic
polypeptides from the pathogens A, B, and C provide protection
against the corresponding pathogen from which the polypeptide is
derived, but little or no cross-protection against the other
pathogens (left panel). After shuffling, an A/B/C chimeric
polypeptide is obtained that can induce a protective immune
response against all three pathogen types (right panel). In FIG.
3B, shuffling is used with substrate nucleic acids from two
pathogen strains (A, B), which encode polypeptides that are
protective only against the corresponding pathogen. After
shuffling, the resulting chimeric polypeptide can induce an immune
response that is effective against not only the two parental
pathogen strains, but also against a third strain of pathogen
(C).
[0016] FIG. 4 diagrams some of the possible factors that can
determine whether a particular polynucleotide encodes an
immunogenic polypeptide having a desired property, such as enhanced
immunogenicity and/or cross-reactivity. Those sequence regions that
positively affect a particular property are indicated as plus signs
along the antigen gene, while those sequence regions that have a
negative effect are shown as minus signs. A pool of related antigen
genes are shuffled and screened to obtain those that recombinant
nucleic acids that have gained positive sequence regions and lost
negative regions. No pre-existing knowledge as to which regions are
positive or negative for a particular trait is required.
[0017] FIG. 5 shows a schematic representation of the screening
strategy for antigen library screening.
[0018] FIG. 6 shows a schematic representation of a strategy for
pooling and deconvolution as used in antigen library screening.
[0019] FIG. 7 is an alignment of the nucleotide sequences of
glycoprotein D (gD) from HSV-1 (SEQ ID NO: 1) and HSV-2 (gD-I (SEQ
ID NO: 2) and gD-2.(SEQ ID NO: 3)).
[0020] FIG. 8A shows a diagram of a method for expressing HIV gp120
using genetic vaccine vectors and generation of a library of
shuffled gp120 genes. FIG. 8B shows PCR primers that are useful for
obtaining gp120 nucleic acid substrates for DNA shuffling
reactions. Primers suitable for generating substrates include 6025F
(SEQ ID NO: 4), 7773R (SEQ ID NO: 5), and primers suitable for
amplifying the shuffled nucleic acids include 6196F (SEQ ID NO: 6)
and 7746R (SEQ ID NO: 7). The primer BssH2-6205F (SEQ ID NO: 8) can
be used to clone the resulting fragment into a genetic vaccine
vector.
[0021] FIG. 9 shows the domain structure of hepadnavirus envelope
genes.
[0022] FIG. 10 shows a schematic representation of the use of
shuffling to obtain hepadnavirus proteins in which the
immunogenicity of one antigenic domain is improved.
[0023] FIG. 11 shows a strategy in which genes that encode the
hepadnavirus proteins having one antigenic domain that has improved
immunogenicity are shuffled to obtain recombinant proteins in which
all three domains have improved immunogenicity.
[0024] FIG. 12 shows the transmembrane organization of the HBsAg
polypeptide.
[0025] FIG. 13 shows a method for using phage display to obtain
recombinant allergens that are not bound by pre-existing IgE.
[0026] FIG. 14 shows a strategy for screening of recombinant
allergens to identify those that are effective in activating
T.sub.H cells. PBMC or T cell clones from atopic individuals are
exposed to antigen-presenting cells that display the antigen
variants obtained using the methods of the invention. To identify
those allergen variants that are effective in activating T cells,
the cultures are tested for induction of T cell proliferation or
for a pattern of cytokine synthesis that is indicative of the
particular type of T cell activation that is desired. If desired,
the allergen variants that test positive in the in vitro assay can
be subjected to in vivo testing.
[0027] FIG. 15 shows a strategy for screening of recombinant cancer
antigens to identify those that are effective in activating T cells
of cancer patients.
[0028] FIG. 16A and FIG. 16B show two different strategies for
generating vectors that contain multiple T cell epitopes obtained,
for example, by DNA shuffling. In FIG. 16A, each individual
shuffled epitope-encoding nucleic acid is linked to a single
promoter, and multiple promoter-epitope gene constructs can be
placed in a single vector. The scheme shown in FIG. 16B involves
linking multiple epitope-encoding nucleic acids to a single
promoter.
[0029] FIG. 17 shows the sequences of PreS2-S coding regions and
corresponding amino acid sequences of different hepatitis B surface
antigen (HBsAg) or woodchuck hepatitis B (WHV) proteins. Primers
suitable for amplification of this region are also shown.
[0030] FIG. 18 shows primers that are suitable for amplification of
large fragments that contain the S2S coding sequences. The primers
hybridize to regions that are approximately 200 bp outside the
desired sequences.
[0031] FIG. 19 shows an alignment of the amino acid sequences of
surface antigens from different HVB and WHV subtypes.
[0032] FIG. 20 shows a diagram of multimeric particles that
assemble when an appropriate number of chimeric polypeptides and
native HBsAg S monomers are mixed.
DETAILED DESCRIPTION
[0033] Definitions
[0034] The term "screening" describes, in general, a process that
identifies optimal antigens. Several properties of the antigen can
be used in selection and screening including antigen expression,
folding, stability, immunogenicity and presence of epitopes from
several related antigens. Selection is a form of screening in which
identification and physical separation are achieved simultaneously
by expression of a selection marker, which, in some genetic
circumstances, allows cells expressing the marker to survive while
other cells die (or vice versa). Screening markers include, for
example, luciferase, beta-galactosidase and green fluorescent
protein. Selection markers include drug and toxin resistance genes,
and the like. Because of limitations in studying primary immune
responses in vitro, in vivo studies are particularly useful
screening methods. In these studies, the antigens are first
introduced to test animals, and the immune responses are
subsequently studied by analyzing protective immune responses or by
studying the quality or strength of the induced immune response
using lymphoid cells derived from the immunized animal. Although
spontaneous selection can and does occur in the course of natural
evolution, in the present methods selection is performed by
man.
[0035] A "exogenous DNA segment", "heterologous sequence" or a
"heterologous nucleic acid", as used herein, is one that originates
from a source foreign to the particular host cell, or, if from the
same source, is modified from its original form. Thus, a
heterologous gene in a host cell includes a gene that is endogenous
to the particular host cell, but has been modified. Modification of
a heterologous sequence in the applications described herein
typically occurs through the use of DNA shuffling. Thus, the terms
refer to a DNA segment which is foreign or heterologous to the
cell, or homologous to the cell but in a position within the host
cell nucleic acid in which the element is not ordinarily found.
Exogenous DNA segments are expressed to yield exogenous
polypeptides.
[0036] The term "gene" is used broadly to refer to any segment of
DNA associated with a biological function. Thus, genes include
coding sequences and/or the regulatory sequences required for their
expression. Genes also include nonexpressed DNA segments that, for
example, form recognition sequences for other proteins. Genes can
be obtained from a variety of sources, including cloning from a
source of interest or synthesizing from known or predicted sequence
information, and may include sequences designed to have desired
parameters.
[0037] The term "isolated", when applied to a nucleic acid or
protein, denotes that the nucleic acid or protein is essentially
free of other cellular components with which it is associated in
the natural state. It is preferably in a homogeneous state although
it can be in either a dry or aqueous solution. Purity and
homogeneity are typically determined using analytical chemistry
techniques such as polyacrylamide gel electrophoresis or high
performance liquid chromatography. A protein which is the
predominant species present in a preparation is substantially
purified. In particular, an isolated gene is separated from open
reading frames which flank the gene and encode a protein other than
the gene of interest. The term "purified" denotes that a nucleic
acid or protein gives rise to essentially one band in an
electrophoretic gel. Particularly, 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.
[0038] The term "naturally-occurring" is used to describe an object
that can be found in nature as distinct from being artificially
produced by man. For example, a polypeptide or polynucleotide
sequence that is present in an organism (including viruses,
bacteria, protozoa, insects, plants or mammalian tissue) that can
be isolated from a source in nature and which has not been
intentionally modified by man in the laboratory is
naturally-occurring.
[0039] The term "nucleic acid" refers to deoxyribonucleotides or
ribonucleotides and polymers thereof in either single- or
double-stranded form. Unless specifically limited, the term
encompasses nucleic acids containing known analogues of natural
nucleotides which have similar binding properties as the reference
nucleic acid and are metabolized in a manner similar to naturally
occurring nucleotides. Unless otherwise indicated, a particular
nucleic acid sequence also implicitly encompasses conservatively
modified variants thereof (e.g. degenerate codon substitutions) and
complementary sequences and as well as the sequence explicitly
indicated. Specifically, degenerate codon substitutions may be
achieved by generating sequences in which the third position of one
or more selected (or all) codons is substituted with mixed-base
and/or deoxyinosine residues (Batzer 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 is used interchangeably with gene,
cDNA, and mRNA encoded by a gene.
[0040] "Nucleic acid derived from a gene" refers to a nucleic acid
for whose synthesis the gene, or a subsequence thereof, has
ultimately served as a template. Thus, an mRNA, a cDNA reverse
transcribed from an mRNA, an RNA transcribed from that cDNA, a DNA
amplified from the cDNA, an RNA transcribed from the amplified DNA,
etc., are all derived from the gene and detection of such derived
products is indicative of the presence and/or abundance of the
original gene and/or gene transcript in a sample.
[0041] A nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
instance, a promoter or enhancer is operably linked to a coding
sequence if it increases the 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. However, since
enhancers generally function when separated from the promoter by
several kilobases and intronic sequences may be of variable
lengths, some polynucleotide elements may be operably linked but
not contiguous.
[0042] A specific binding affinity between two molecules, for
example, a ligand and a receptor, means a preferential binding of
one molecule for another in a mixture of molecules. The binding of
the molecules can be considered specific if the binding affinity is
about 1.times.10.sup.4 M.sup.-1 to about 1.times.10.sup.6 M.sup.-1
or greater.
[0043] The term "recombinant" when used with reference to a cell
indicates that the cell replicates a heterologous nucleic acid, or
expresses a peptide or protein encoded by a heterologous nucleic
acid. Recombinant cells can contain genes that are not found within
the native (non-recombinant) form of the cell. Recombinant cells
can also contain genes found in the native form of the cell wherein
the genes are modified and re-introduced into the cell by
artificial means. The term also encompasses cells that contain a
nucleic acid endogenous to the cell that has been modified without
removing the nucleic acid from the cell; such modifications include
those obtained by gene replacement, site-specific mutation, and
related techniques.
[0044] A "recombinant expression cassette" or simply an "expression
cassette" is a nucleic acid construct, generated recombinantly or
synthetically, with nucleic acid elements that are capable of
effecting expression of a structural gene in hosts compatible with
such sequences. Expression cassettes include at least promoters and
optionally, transcription termination signals. Typically, the
recombinant expression cassette includes a nucleic acid to be
transcribed (e.g., a nucleic acid encoding a desired polypeptide),
and a promoter. Additional factors necessary or helpful in
effecting expression may also be used as described herein. For
example, an expression cassette can also include nucleotide
sequences that encode a signal sequence that directs secretion of
an expressed protein from the host cell. Transcription termination
signals, enhancers, and other nucleic acid sequences that influence
gene expression, can also be included in an expression
cassette.
[0045] A "multivalent antigenic polypeptide" or a "recombinant
multivalent antigenic polypeptide" is a non-naturally occurring
polypeptide that includes amino acid sequences from more than one
source polypeptide, which source polypeptide is typically a
naturally occurring polypeptide. At least some of the regions of
different amino acid sequences constitute epitopes that are
recognized by antibodies found in a mammal that has been injected
with the source polypeptide. The source polypeptides from which the
different epitopes are derived are usually homologous (i.e., have
the same or a similar structure and/or function), and are often
from different isolates, serotypes, strains, species, of organism
or from different disease states, for example.
[0046] The terms "identical" or percent "identity," in the context
of two or more nucleic acid or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same, when compared and aligned for maximum correspondence, as
measured using one of the following sequence comparison algorithms
or by visual inspection.
[0047] The phrase "substantially identical," in the context of two
nucleic acids or polypeptides, refers to two or more sequences or
subsequences that have at least 60%, preferably 80%, most
preferably 90-95% nucleotide or amino acid residue identity, when
compared and aligned for maximum correspondence, as measured using
one of the following sequence comparison algorithms or by visual
inspection. Preferably, the substantial identity exists over a
region of the sequences that is at least about 50 residues in
length, more preferably over a region of at least about 100
residues, and most preferably the sequences are substantially
identical over at least about 150 residues. In some embodiments,
the sequences are substantially identical over the entire length of
the coding regions.
[0048] For sequence comparison, typically one sequence acts as a
reference sequence to which test sequences are compared. When using
a sequence comparison algorithm, test and reference sequences are
input into a computer, subsequence coordinates are designated, if
necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
percent sequence identity for the test sequence(s) relative to the
reference sequence, based on the designated program parameters.
[0049] Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by visual
inspection (see generally Ausubel et al., infra).
[0050] One example of an algorithm that is suitable for determining
percent sequence identity and sequence similarity is the BLAST
algorithm, which is described in Altschul et al., J. Mol. Biol.
215:403-410 (1990). Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information (http://wvww.ncbi.nlm.nih.g- ov/). This algorithm
involves first identifying high scoring sequence pairs (HSPs) by
identifying short words of length W in the query sequence, which
either match or satisfy some positive-valued threshold score T when
aligned with a word of the same length in a database sequence. T is
referred to as the neighborhood word score threshold (Altschul et
al., supra). These initial neighborhood word hits act as seeds for
initiating searches to find longer HSPs containing them. The word
hits are then extended in both directions along each sequence for
as far as the cumulative alignment score can be increased.
Cumulative scores are calculated using, for nucleotide sequences,
the parameters M (reward score for a pair of matching residues;
always >0) and N (penalty score for mismatching residues; always
<0). For amino acid sequences, a scoring matrix is used to
calculate the cumulative score. Extension of the word hits in each
direction are halted when: the cumulative alignment score falls off
by the quantity X from its maximum achieved value; the cumulative
score goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters 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, M=5, N=-4, and a comparison
of both strands. For amino acid sequences, the BLASTP program uses
as defaults a wordlength (W) of 3, an expectation (E) of 10, and
the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989)
Proc. Natl. Acad. Sci. USA 89:10915).
[0051] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin & Altschul
(1993) Proc. Nat'l. Acad. Sci. USA 90:5873-5787). One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a nucleic acid is considered
similar to a reference sequence if the smallest sum probability in
a comparison of the test nucleic acid to the reference nucleic acid
is less than about 0.1, more preferably less than about 0.01, and
most preferably less than about 0.001.
[0052] Another indication that two nucleic acid sequences are
substantially identical is that the two molecules hybridize to each
other under stringent conditions. The phrase "hybridizing
specifically to", refers to the binding, duplexing, or hybridizing
of a molecule only to a particular nucleotide sequence under
stringent conditions when that sequence is present in a complex
mixture (e.g., total cellular) DNA or RNA. "Bind(s) substantially"
refers to complementary hybridization between a probe nucleic acid
and a target nucleic acid and embraces minor mismatches that can be
accommodated by reducing the stringency of the hybridization media
to achieve the desired detection of the target polynucleotide
sequence.
[0053] "Stringent hybridization conditions" and "stringent
hybridization wash conditions" in the context of nucleic acid
hybridization experiments such as Southern and northern
hybridizations are sequence dependent, and are different under
different environmental parameters. Longer sequences hybridize
specifically at higher temperatures. An extensive guide to the
hybridization of nucleic acids is found in Tijssen (1993)
Laboratory Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Acid Probes part I chapter 2
"Overview of principles of hybridization and the strategy of
nucleic acid probe assays", Elsevier, New York. Generally, highly
stringent hybridization and wash conditions are selected to be
about 5.degree. C. lower than the thermal melting point (T.sub.m)
for the specific sequence at a defined ionic strength and pH.
Typically, under "stringent conditions" a probe will hybridize to
its target subsequence, but to no other sequences.
[0054] The T.sub.m is the temperature (under defined ionic strength
and pH) at which 50% of the target sequence hybridizes to a
perfectly matched probe. Very stringent conditions are selected to
be equal to the T.sub.m for a particular probe. An example of
stringent hybridization conditions for hybridization of
complementary nucleic acids which have more than 100 complementary
residues on a filter in a Southern or northern blot is 50%
formamide with 1 mg of heparin at 42.degree. C., with the
hybridization being carried out overnight. An example of highly
stringent wash conditions is 0.1 SM NaCl at 72.degree. C. for about
15 minutes. An example of stringent wash conditions is a
0.2.times.SSC wash at 65.degree. C. for 15 minutes (see, Sambrook,
infra., for a description of SSC buffer). Often, a high stringency
wash is preceded by a low stringency wash to remove background
probe signal. An example medium stringency wash for a duplex of,
e.g., more than 100 nucleotides, is 1.times.SSC at 45.degree. C.
for 15 minutes. An example low stringency wash for a duplex of,
e.g., more than 100 nucleotides, is 4-6.times.SSC at 40.degree. C.
for 15 minutes. For short probes (e.g., about 10 to 50
nucleotides), stringent conditions typically involve salt
concentrations of less than about 1.0 M Na.sup.+ ion, typically
about 0.01 to 1.0 M Na.sup.+ ion concentration (or other salts) at
pH 7.0 to 8.3, and the temperature is typically at least about
30.degree. C. Stringent conditions can also be achieved with the
addition of destabilizing agents such as formamide. In general, a
signal to noise ratio of 2.times.(or higher) than that observed for
an unrelated probe in the particular hybridization assay indicates
detection of a specific hybridization. Nucleic acids which do not
hybridize to each other under stringent conditions are still
substantially identical if the polypeptides which they encode are
substantially identical. This occurs, e.g., when a copy of a
nucleic acid is created using the maximum codon degeneracy
permitted by the genetic code.
[0055] 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 cross reactive
with, or specifically binds to, the polypeptide encoded by the
second nucleic acid. Thus, a polypeptide is typically substantially
identical to a second polypeptide, for example, where the two
peptides differ only by conservative substitutions.
[0056] The phrase "specifically (or selectively) binds to an
antibody" or "specifically (or selectively) immunoreactive with",
when referring to a protein or peptide, refers to a binding
reaction which is determinative of the presence of the protein, or
an epitope from the protein, in the presence of a heterogeneous
population of proteins and other biologics. Thus, under designated
immunoassay conditions, the specified antibodies bind to a
particular protein and do not bind in a significant amount to other
proteins present in the sample. The antibodies raised against a
multivalent antigenic polypeptide will generally bind to the
proteins from which one or more of the epitopes were obtained.
Specific binding to an antibody under such conditions may require
an antibody that is selected for its specificity for a particular
protein. A variety of immunoassay formats may 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 a protein. See Harlow
and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor
Publications, New York "Harlow and Lane"), for a description of
immunoassay formats and conditions that can be used to determine
specific immunoreactivity. Typically a specific or selective
reaction will be at least twice background signal or noise and more
typically more than 10 to 100 times background.
[0057] "Conservatively modified variations" of a particular
polynucleotide sequence refers to those polynucleotides that encode
identical or essentially identical amino acid sequences, or where
the polynucleotide does not encode an amino acid sequence, to
essentially identical sequences. Because of the degeneracy of the
genetic code, a large number of functionally identical nucleic
acids encode any given polypeptide. For instance, 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 variations are "silent variations," which are one species of
"conservatively modified variations.". Every polynucleotide
sequence described herein which encodes a polypeptide also
describes every possible silent variation, except where otherwise
noted. One of skill will recognize that each codon in a nucleic
acid (except AUG, which is ordinarily the only codon for
methionine) can be modified to yield a functionally identical
molecule by standard techniques. Accordingly, each "silent
variation" of a nucleic acid which encodes a polypeptide is
implicit in each described sequence.
[0058] Furthermore, one of skill will recognize that individual
substitutions, deletions or additions which alter, add or delete a
single amino acid or a small percentage of amino acids (typically
less than 5%, more typically less than 1%) in an encoded sequence
are "conservatively modified variations" where the alterations
result in the substitution of an amino acid with a chemically
similar amino acid. Conservative substitution tables providing
functionally similar amino acids are well known in the art. The
following five groups each contain amino acids that are
conservative substitutions for one another:
[0059] Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine
(L), Isoleucine (I);
[0060] Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan
(W);
[0061] Sulfur-containing: Methionine (M), Cysteine (C);
[0062] Basic: Arginine (R), Lysine (K), Histidine (H);
[0063] Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine
(N), Glutamine (Q). See also, Creighton (1984) Proteins, W. H.
Freeman and Company, for additional groupings of amino acids. In
addition, individual substitutions, deletions or additions which
alter, add or delete a single amino acid or a small percentage of
amino acids in an encoded sequence are also "conservatively
modified variations".
[0064] A "subsequence" refers to a sequence of nucleic acids or
amino acids that comprise a part of a longer sequence of nucleic
acids or amino acids (e.g., polypeptide) respectively.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0065] The invention provides a new approach to vaccine
development, which is termed "antigen library immunization." No
other technologies are available for generating libraries of
related antigens or optimizing known protective antigens. The most
powerful previously existing methods for identification of vaccine
antigens, such as high throughput sequencing or expression library
immunization, only explore the sequence space provided by the
pathogen genome. These approaches are likely to be insufficient,
because they generally only target single pathogen strains, and
because natural evolution has directed pathogens to downregulate
their own immunogenicity. In contrast, the immunization protocols
of the invention, which use shuffled antigen libraries, provide a
means to identify novel antigen sequences. Those antigens that are
most protective can be selected from these pools by in vivo
challenge models. Antigen library immunization dramatically expands
the diversity of available immunogen sequences, and therefore,
these antigen chimera libraries can also provide means to defend
against newly emerging pathogen variants of the future. The methods
of the invention enable the identification of individual chimeric
antigens that provide efficient protection against a variety of
existing pathogens, providing improved vaccines for troops and
civilian populations.
[0066] The methods of the invention provide an evolution-based
approach, such as DNA shuffling in particular, that is an optimal
approach to improve the immunogenicity of many types of antigens.
For example, the methods provide means of obtaining optimized
cancer antigens useful for preventing and treating malignant
diseases. Furthermore, an increasing number of self-antigens,
causing autoimmune diseases, and allergens, causing atopy, allergy
and asthma, have been characterized. The immunogenicity and
manufacturing of these antigens can likewise be improved with the
methods of this invention.
[0067] The antigen library immunization methods of the invention
provide a means by which one can obtain a recombinant antigen that
has improved ability to induce an immune response to a pathogenic
agent. A "pathogenic agent" refers to an organism or virus that is
capable of infecting a host cell. Pathogenic agents typically
include and/or encode a molecule, usually a polypeptide, that is
immunogenic in that an immune response is raised against the
immunogenic polypeptide. Often, the immune response raised against
an immunogenic polypeptide from one serotype of the pathogenic
agent is not capable of recognizing, and thus protecting against, a
different serotype of the pathogenic agent, or other related
pathogenic agents. In other situations, the polypeptide produced by
a pathogenic agent is not produced in sufficient amounts, or is not
sufficiently immunogenic, for the infected host to raise an
effective immune response against the pathogenic agent.
[0068] These problems are overcome by the methods of the invention,
which typically involve recombining two or more forms of a nucleic
acid that encode a polypeptide of the pathogenic agent, or antigen
involved in another disease or condition. These recombination
methods, referred to herein as "DNA shuffling", use as substrates
forms of the nucleic acid that differ from each other in two or
more nucleotides, so a library of recombinant nucleic acids
results. The library is then screened to identify at least one
optimized recombinant nucleic acid that encodes an optimized
recombinant antigen that has improved ability to induce an immune
response to the pathogenic agent or other condition. The resulting
recombinant antigens often are chimeric in that they are recognized
by antibodies (Abs) reacting against multiple pathogen strains, and
generally can also elicit broad spectrum immune responses. Specific
neutralizing antibodies are known to mediate protection against
several pathogens of interest, although additional mechanisms, such
as cytotoxic T lymphocytes, are likely to be involved. The concept
of chimeric, multivalent antigens inducing broadly reacting
antibody responses is further illustrated in FIG. 1.
[0069] In preferred embodiments, the different forms of the nucleic
acids that encode antigenic polypeptides are obtained from members
of a family of related pathogenic agents. This scheme of performing
DNA shuffling using nucleic acids from related organisms, known as
"family shuffling," is described in Crameri et al. ((1998) Nature
391: 288-291) and is shown schematically in FIG. 2. Polypeptides of
different strains and serotypes of pathogens generally vary between
60-98%, which will allow for efficient family DNA shuffling.
Therefore, family DNA shuffling provides an effective approach to
generate multivalent, crossprotective antigens. The methods are
useful for obtaining individual chimeras that effectively protect
against most or all pathogen variants (FIG. 3A). Moreover,
immunizations using entire libraries or pools of shuffled antigen
chimeras can also result in identification of chimeric antigens
that protect against pathogen variants that were not included in
the starting population of antigens (for example, protection
against strain C by shuffled library of chimeras/mutants of strains
A and B in FIG. 3B). Accordingly, the antigen library immunization
approach enables the development of immunogenic polypeptides that
can induce immune responses against poorly characterized, newly
emerging pathogen variants.
[0070] Sequence recombination can be achieved in many different
formats and permutations of formats, as described in further detail
below. These formats share some common principles. For example, the
targets for modification vary in different applications, as does
the property sought to be acquired or improved. Examples of
candidate targets for acquisition of a property or improvement in a
property include genes that encode proteins which have immunogenic
and/or toxigenic activity when introduced into a host organism.
[0071] The methods use at least two variant forms of a starting
target. The variant forms of candidate substrates can show
substantial sequence or secondary structural similarity with each
other, but they should also differ in at least one and preferably
at least two positions. The initial diversity between forms can be
the result of natural variation, e.g., the different variant forms
(homologs) are obtained from different individuals or strains of an
organism, or constitute related sequences from the same organism
(e.g., allelic variations), or constitute homologs from different
organisms (interspecific variants). Alternatively, initial
diversity can be induced, e.g., the variant forms can be generated
by error-prone transcription, such as an error-prone PCR or use of
a polymerase which lacks proof-reading activity (see, Liao (1990)
Gene 88:107-111), of the first variant form, or, by replication of
the first form in a mutator strain (mutator host cells are
discussed in further detail below, and are generally well known). A
mutator strain can include any mutants in any organism impaired in
the functions of mismatch repair. These include mutant gene
products of mutS, mutT, mutH, mutL, ovrD, dcm, vsr, umuC, umuD,
sbcB, recj, etc. The impairment is achieved by genetic mutation,
allelic replacement, selective inhibition by an added reagent such
as a small compound or an expressed antisense RNA, or other
techniques. Impairment can be of the genes noted, or of homologous
genes in any organism. Other methods of generating initial
diversity include methods well known to those of skill in the art,
including, for example, treatment of a nucleic acid with a chemical
or other mutagen, through spontaneous mutation, and by inducing an
error-prone repair system (e.g., SOS) in a cell that contains the
nucleic acid. The initial diversity between substrates is greatly
augmented in subsequent steps of recombination for library
generation.
[0072] Properties Involved in Immunogenicity
[0073] The effectiveness of an antigen in inducing an immune
response against a pathogen can depend upon several factors, many
of which are not well understood. Most previously available methods
for increasing the effectiveness of antigens are dependent upon
understanding the molecular basis for these factors. However, DNA
shuffling and antigen library immunization according to the methods
of the invention are effective even where the molecular bases are
unknown. The methods of the invention do not rely upon a priori
assumptions.
[0074] Polynucleotide sequences that can positively or negatively
affect the immunogenicity of an antigen encoded by the
polynucleotide are often scattered throughout the entire antigen
gene. Several of these factors are shown diagrammatically in FIG.
4. By recombining different forms of polynucleotide that encode the
antigen, using DNA shuffling, followed by selection for those
chimeric polynucleotides that encode an antigen that can induce an
improved immune response, one can obtain primarily sequences that
have a positive influence on antigen immunogenicity. Those
sequences that negatively affect antigen immunogenicity are
eliminated (FIG. 4). One need not know the particular sequences
involved.
[0075] DNA Shuffling Methods
[0076] Generally, the methods of the invention entail performing
DNA recombination ("shuffling") and screening or selection to
"evolve" individual genes, whole plasmids or viruses, multigene
clusters, or even whole genomes (Stemmer (1995) Bio/Technology
13:549-553). Reiterative cycles of recombination and
screening/selection can be performed to further evolve the nucleic
acids of interest. Such techniques do not require the extensive
analysis and computation required by conventional methods for
polypeptide engineering. Shuffling allows the recombination of
large numbers of mutations in a minimum number of selection cycles,
in contrast to natural pair-wise recombination events (e.g., as
occur during sexual replication). Thus, the sequence recombination
techniques described herein provide particular advantages in that
they provide recombination between mutations in any or all of
these, thereby providing a very fast way of exploring the manner in
which different combinations of mutations can affect a desired
result. In some instances, however, structural and/or functional
information is available which, although not required for sequence
recombination, provides opportunities for modification of the
technique.
[0077] The DNA shuffling methods of the invention can involve at
least one of at least four different approaches to improve
immunogenic activity as well as to broaden specificity. First, DNA
shuffling can be performed on a single gene. Secondly, several
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 recombinants with the
desired activity. The shuffled genes can be cloned into appropriate
host cells, such as E. coli, yeast, plants, fungi, animal cells,
and the like, and those that encode antigens having the desired
properties can be identified by the methods described below. Third,
whole genome shuffling can be performed to shuffle genes that
encode antigenic polypeptides (along with other genomic nucleic
acids). For whole genome shuffling approaches, it is not even
necessary to identify which genes are being shuffled. Instead,
e.g., bacterial cell or viral genomes are combined and shuffled to
acquire recombinant polypeptides that have enhanced ability to
induce an immune response, as measured in any of the assays
described below. Fourth, antigenic polypeptide-encoding genes can
be codon modified to access mutational diversity not present in any
naturally occurring gene. Details on each of these procedures can
be found below.
[0078] Exemplary formats and examples for sequence recombination,
sometimes referred to as DNA shuffling, evolution, or molecular
breeding, have been described by the present inventors and
co-workers in co-pending applications U.S. patent application Ser.
No. 08/198,431, filed Feb. 17, 1994, Serial No. PCT/US95/02126,
filed, Feb. 17, 1995, Ser. No. 08/425,684, filed Apr. 18, 1995,
Ser. No. 08/537,874, filed Oct. 30, 1995, Ser. No. 08/564,955,
filed Nov. 30, 1995, Ser. No. 08/621,859, filed Mar. 25, 1996, Ser.
No. 08/621,430, filed Mar. 25, 1996, Serial No. PCT/US96/05480,
filed Apr. 18, 1996, Ser. No. 08/650,400, filed May 20, 1996, Ser.
No. 08/675,502, filed Jul. 3, 1996, Ser. No. 08/721, 824, filed
Sep. 27, 1996, Serial No. PCT/US97/17300, filed Sep. 26, 1997, and
Serial No. PCT/US97/24239, filed Dec. 17, 1997; Stemmer, Science
270:1510 (1995); Stemmer et al., Gene 164:49-53 (1995); Stemmer,
Bio/Technology 13:549-553 (1995); Stemmer, Proc. Natl. Acad. Sci.
U.S.A. 91:10747-10751. (1994); Stemmer, Nature 370:389-391 (1994);
Crameri et al., Nature Medicine 2(1):1-3 (1996); Crameri et al.,
Nature Biotechnology 14:315-319 (1996), each of which is
incorporated by reference in its entirety for all purposes.
[0079] Other methods for obtaining recombinant polynucleotides
and/or for obtaining diversity in nucleic acids used as the
substrates for shuffling include, for example, homologous
recombination (PCT/US98/05223; Publ. No. WO98/42727);
oligonucleotide-directed mutagenesis (for review see, Smith, Ann.
Rev. Genet. 19: 423-462 (1985); Botstein and Shortle, Science 229:
1193-1201 (1985); Carter, Biochem. J. 237: 1-7 (1986); Kunkel, "The
efficiency of oligonucleotide directed mutagenesis" in Nucleic
acids & Molecular Biology, Eckstein and Lilley, eds., Springer
Verlag, Berlin (1987)). Included among these methods are
oligonucleotide-directed mutagenesis (Zoller and Smith, Nucl. Acids
Res. 10: 6487-6500 (1982), Methods in Enzymol. 100: 468-500 (1983),
and Methods in Enzymol. 154: 329-350 (1987))
phosphothioate-modified DNA mutagenesis (Taylor et al., Nucl. Acids
Res. 13: 8749-8764 (1985); Taylor et al., Nucl. Acids Res. 13:
8765-8787 (1985); Nakamaye and Eckstein, Nucl. Acids Res. 14:
9679-9698 (1986); Sayers et al., Nucl. Acids Res. 16: 791-802
(1988); Sayers et al., Nucl. Acids Res. 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
Enzymol. 154: 367-382)); mutagenesis using gapped duplex DNA
(Kramer et al., Nucl. Acids Res. 12: 9441-9456 (1984); Kramer and
Fritz, Methods in Enzymol. 154: 350-367 (1987); Kramer et al.,
Nucl. Acids Res. 16: 7207 (1988)); and Fritz et al., Nucl. Acids
Res. 16: 6987-6999 (1988)). Additional suitable methods include
point mismatch repair (Kramer et al., Cell 38: 879-887 (1984)),
mutagenesis using repair-deficient host strains (Carter et al.,
Nucl. Acids Res. 13: 4431-4443 (1985); Carter, Methods in Enzymol.
154: 382-403 (1987)), deletion mutagenesis (Eghtedarzadeh and
Henikoff, Nucl. Acids Res. 14: 5115 (1986)), restriction-selection
and restriction-purification (Wells et al., Phil. Trans. R. Soc.
Lond. A 317: 415-423 (1986)), mutagenesis by total gene synthesis
(Nambiar et al., Science 223: 1299-1301 (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
(e.g., Bio-Rad, Amersham International, Anglian Biotechnology).
[0080] The breeding procedure starts with at least two substrates
that generally show some degree of sequence identity to each other
(i.e., at least about 30%, 50%, 70%, 80% or 90% sequence identity),
but differ from each other at certain positions. The difference can
be any type of mutation, for example, substitutions, insertions and
deletions. Often, different segments differ from each other in
about 5-20 positions. For recombination to generate increased
diversity relative to the starting materials, the starting
materials must differ from each other in at least two nucleotide
positions. That is, if there are only two substrates, there should
be at least two divergent positions. If there are three substrates,
for example, one substrate can differ from the second at a single
position, and the second can differ from the third at a different
single position. The starting DNA segments can be natural variants
of each other, for example, allelic or species variants. The
segments can also be from nonallelic genes showing some degree of
structural and usually functional relatedness (e.g., different
genes within a superfamily, such as the family of Yersinia
V-antigens, for example). The starting DNA segments can also be
induced variants of each other. For example, one DNA segment can be
produced by error-prone PCR replication of the other, the nucleic
acid can be treated with a chemical or other mutagen, or by
substitution of a mutagenic cassette. Induced mutants can also be
prepared by propagating one (or both) of the segments in a
mutagenic strain, or by inducing an error-prone repair system in
the cells. In these situations, strictly speaking, the second DNA
segment is not a single segment but a large family of related
segments. The different segments forming the starting materials are
often the same length or substantially the same length. However,
this need not be the case; for example; one segment can be a
subsequence of another. The segments can be present as part of
larger molecules, such as vectors, or can be in isolated form.
[0081] The starting DNA segments are recombined by any of the
sequence recombination formats provided herein to generate a
diverse library of recombinant DNA segments. Such a library can
vary widely in size from having fewer than 10 to more than
10.sup.5, 10.sup.9, 10.sup.12 or more members. In some embodiments,
the starting segments and the recombinant libraries generated will
include full-length coding sequences and any essential regulatory
sequences, such as a promoter and polyadenylation sequence,
required for expression. In other embodiments, the recombinant DNA
segments in the library can be inserted into a common vector
providing sequences necessary for expression before performing
screening/selection.
[0082] Substrates for Evolution of Optimized Recombinant
Antigens
[0083] The invention provides methods of obtaining recombinant
polynucleotides that encode antigens that exhibit improved ability
to induce an immune response to a pathogenic agent. The methods are
applicable to a wide range of pathogenic agents, including
potential biological warfare agents 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.
[0084] 1. Bacterial Pathogens and Toxins
[0085] In some embodiments, the methods of the invention are
applied to bacterial pathogens, as well as to toxins produced by
bacteria and other organisms. One can use the methods to obtain
recombinant polypeptides that can induce an immune response against
the pathogen, as well as recombinant toxins that are less toxic
than native toxin polypeptides. Often, the polynucleotides of
interest encode polypeptides that are present on the surface of the
pathogenic organism.
[0086] Among the pathogens for which the methods of the invention
are useful for producing protective immunogenic recombinant
polypeptides are the Yersinia species. Yersinia pestis, the
causative agent of plague, is one of the most virulent bacteria
known with LD.sub.50 values in mouse of less than 10 bacteria. The
pneumonic form of the disease is readily spread between humans by
aerosol or infectious droplets and can be lethal within days. A
particularly preferred target for obtaining a recombinant
polypeptide that can protect against Yersinia infection is the V
antigen, which is a 37 kDa virulence factor, induces protective
immune responses and is currently being evaluated as a subunit
vaccine (Brubaker (1991) Current Investigations of the Microbiology
of Yersinae, 12: 127). The V-antigen alone is not toxic, but Y
pestis isolates that lack the V-antigen are a virulent. The
Yersinia V-antigen has been successfully produced in E. coli by
several groups (Leary et al. (1995) Infect. Immun. 3: 2854).
Antibodies that recognize the V-antigen can provide passive
protection against homologous strains, but not against heterologous
strains. Similarly, immunization with purified V antigen protects
against only homologous strains. To obtain cross-protective
recombinant V antigen, in a preferred embodiment, V antigen genes
from various Yersinia species are subjected to family shuffling.
The genes encoding the V antigen from Y. pestis, Y. enterocolitica,
and Y. pseudotuberculosis, for example, are 92-99% identical at the
DNA level, making them ideal for optimization using family
shuffling according to the methods of the invention. After
shuffling, the library of recombinant nucleic acids is screened
and/or selected for those that encode recombinant V antigen
polypeptides that can induce an improved immune response and/or
have greater cross-protectivity.
[0087] Bacillus anthracis, the causative agent of anthrax, is
another example of a bacterial target against which the methods of
the invention are useful. The anthrax protective antigen (PA)
provides protective immune responses in test animals, and
antibodies against PA also provide some protection. However, the
immunogenicity of PA is relatively poor, so multiple injections are
typically required when wild-type PA is used. Co-vaccination with
lethal factor (LF) can improve the efficacy of wild-type PA
vaccines, but toxicity is a limiting factor. Accordingly the DNA
shuffling and antigen library immunization methods of the invention
can be used to obtain nontoxic LF. Polynucleotides that encode LF
from various B. anthracis strains are subjected to family
shuffling. The resulting library of recombinant LF nucleic acids
can then be screened to identify those that encode recombinant LF
polypeptides that exhibit reduced toxicity. For example, one can
inoculate tissue culture cells with the recombinant LF polypeptides
in the presence of PA and select those clones for which the cells
survive. If desired, one can then backcross the nontoxic LF
polypeptides to retain the immunogenic epitopes of LF. Those that
are selected through the first screen can then be subjected to a
secondary screen. For example, one can test for the ability of the
recombinant nontoxic LF polypeptides to induce an immune response
(e.g., CTL or antibody response) in a test animal such as mice. In
preferred embodiments, the recombinant nontoxic LF polypeptides are
then tested for ability to induce protective immunity in test
animals against challenge by different strains of B. anthracis.
[0088] The protective antigen (PA) of B. anthracis is also a
suitable target for the methods of the invention. PA-encoding
nucleic acids from various strains of B. anthracis are subjected to
DNA shuffling. One can then screen for proper folding in, for
example, E. coli, using polyclonal antibodies. Screening for
ability to induce broad-spectrum antibodies in a test animal is
also typically used, either alone or in addition to a preliminary
screening method. In presently preferred embodiments, those
recombinant polynucleotides that exhibit the desired properties can
be backcrossed so that the immunogenic epitopes are maintained.
Finally, the selected recombinants are tested for ability to induce
protective immunity against different strains of B. anthracis in a
test animal.
[0089] The Staphylococcus aurees and Streptococcus toxins are
another example of a target polypeptide that can be altered using
the methods of the invention. Strains of Staphylococcus aureus and
group A Streptococci are involved in a range of diseases, including
food poisoning, toxic shock syndrome, scarlet fever and various
autoimmune disorders. They secrete a variety of toxins, which
include at least five cytolytic toxins, a coagulase, and a variety
of enterotoxins. The enterotoxins are classified as superantigens
in that they crosslink MHC class II molecules with T cell receptors
to cause a constitutive T cell activation (Fields et al. (1996)
Nature 384: 188). This results in the accumulation of pathogenic
levels of cytokines that can lead to multiple organ failure and
death. At least thirty related, yet distinct enterotoxins have been
sequenced and can be phylogenetically grouped into families.
Crystal structures have been obtained for several members alone and
in complex with MHC class II molecules. Certain mutations in the
MHC class 11-binding site of the toxins strongly reduce their
toxicity and can form the basis of attenuated vaccines (Woody et
al. (1997) Vaccine 15: 133). However, a successful immune response
to one type of toxin may provide protection against closely related
family members, whereas little protection against toxins from the
other families is observed. Family shuffling of enterotoxin genes
from various family members can be used to obtain recombinant toxin
molecules that have reduced toxicity and can induce a
cross-protective immune response. Shuffled antigens can also be
screened to identify antigens that elicit neutralizing antibodies
in an appropriate animal model such as mouse or monkey. Examples of
such assays can include ELISA formats in which the elicited
antibodies prevent binding of the enterotoxin to the MHC complex
and/or T cell receptors on cells or purified forms. These assays
can also include formats where the added antibodies would prevent T
cells from being cross-linked to appropriate antigen presenting
cells.
[0090] Cholera is an ancient, potentially lethal disease caused by
the bacterium Vibrio cholerae and an effective vaccine for its
prevention is still unavailable. Much of the pathogenesis of this
disease is caused by the cholera enterotoxin. Ingestion of
microgram quantities of cholera toxin can induce severe diarrhea
causing loss of tens of liters of fluid. Cholera toxin is a complex
of a single catalytic A subunit with a pentameric ring of identical
B subunits. Each subunit is inactive on its own. The B subunits
bind to specific ganglioside receptors on the surface of intestinal
epithelial cells and trigger the entry of the A subunit into the
cell. The A subunit ADP-ribosylates a regulatory G protein
initiating a cascade of events causing a massive, sustained flow of
electrolytes and water into the intestinal lumen resulting in
extreme diarrhea.
[0091] The B subunit of cholera toxin is an attractive vaccine
target for a number of reasons. It is a major target of protective
antibodies generated during cholera infection and contains the
epitopes for antitoxin neutralizing antibodies. It is nontoxic
without the A subunit, is orally effective, and stimulates
production of a strong IgA-dominated gut mucosal immune response,
which is essential in protection against cholera and cholera toxin.
The B subunit is also being investigated for use as an adjuvant in
other vaccine preparations, and therefore, evolved toxins may
provide general improvements for a variety of different vaccines.
The heat-labile enterotoxins (LT) from enterotoxigenic E. coli
strains are structurally related to cholera toxin and are 75%
identical at the DNA sequence level. To obtain optimized
recombinant toxin molecules that exhibit reduced toxicity and
increased ability to induce an immune response that is protective
against V. cholerae and E. coli, the genes that encode the related
toxins are subjected to DNA shuffling.
[0092] The recombinant toxins are then tested for one or more of a
several desirable traits. For example, one can screen for improved
cross-reactivity of antibodies raised against the recombinant toxin
polypeptides, for lack of toxicity in a cell culture assay, and for
ability to induce a protective immune response against the
pathogens and/or against the toxins themselves. The shuffled clones
can be selected by phage display and/or screened by phage ELISA and
ELISA assays for the presence of epitopes from the different
serotypes. Variant proteins with multiple epitopes can then be
purified and used to immunize mice or other test animal. The animal
serum is then assayed for antibodies to the different B chain
subtypes and variants that elicit a broad cross-reactive response
will be evaluated further in a virulent challenge model. The E.
coli and V. cholerae toxins can also act as adjuvants that are
capable of enhancing mucosal immunity and oral delivery of vaccines
and proteins. Accordingly, one can test the library of recombinant
toxins for enhancement of the adjuvant activity.
[0093] Shuffled antigens can also be screened for improved
expression levels and stability of the B chain pentamer, which may
be less stable than when in the presence of the A chain in the
hexameric complex. Addition of a heat treatment step or denaturing
agents such as salts, urea, and/or guanidine hydrochloride can be
included prior to ELISA assays to measure yields of correctly
folded molecules by appropriate antibodies. It is sometimes
desirable to screen for stable monomeric B chain molecules, in an
ELISA format, for example, using antibodies that bind monomeric,
but not pentameric B chains. Additionally, the ability of shuffled
antigens to elicit neutralizing antibodies in an appropriate animal
model such as mouse or monkey can be screened. For example,
antibodies that bind to the B chain and prevent its binding to its
specific ganglioside receptors on the surface of intestinal
epithelial cells may prevent disease. Similarly antibodies that
bind to the B chain and prevent its pentamerization or block A
chain binding may be useful in preventing disease.
[0094] The 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) Aliment.
Pharmacol. Ther. 1: 73-7; Blaser and Crabtree (1996) Am. J. Clin.
Pathol. 106: 565-7; Censini et al. (1996) Proc. Nat'l. Acad. Sci.
USA 93: 14648-14643). Other suitable H. pylon antigens include, for
example, four immunoreactive proteins of 45-65 kDa as reported by
Chatha et al. (1997) Indian J. Med. Res. 105: 170-175 and the H.
pylon GroES homologue (HspA) (Kansau et al. (1996) Mol. Microbiol.
22: 1013-1023. Other suitable bacterial antigens include, but are
not limited to, the 43-kDa and the fimbrilin (41 kDa) proteins of
P. gingivalis (Boutsl et al. (1996) Oral Microbiol. Immunol. 11:
236-241); pneumococcal surface protein A (Briles et al. (1996) Ann.
NY Acad. Sci. 797: 118-126); Chlamydia psittaci antigens, 80-90 kDa
protein and 110 kDa protein (Buendia et al. (1997) FEMS Microbiol.
Lett. 150: 113-9); the chlamydial exoglycolipid antigen (GLXA)
(Whittum-Hudson et al. (1996) Nature Med. 2: 1116-1121); Chlamydia
pneumoniae species-specific antigens in the molecular weight ranges
92-98, 51-55, 43-46 and 31.5-33 kDa and genus-specific antigens in
the ranges 12, 26 and 65-70 kDa (Halme et al. (1997) Scand. J
Immunol. 45: 378-84); Neisseria gonorrhoeae (GC) or Escherichia
coli phase-variable opacity (Opa) proteins (Chen and Gotschlich
(1996) Proc. Nat'l. Acad. Sci. USA 93: 14851-14856), any of the
twelve immunodominant proteins of Schistosoma mansoni (ranging in
molecular weight from 14 to 208 kDa) as described by Cutts and
Wilson (1997) Parasitology 114: 245-55; the 17-kDa protein antigen
of Brucella abortus (De Mot et al. (1996) Curr. Microbiol. 33:
26-30); a gene homolog of the 17-kDa protein antigen of the
Gram-negative pathogen Brucella abortus identified in the
nocardioform actinomycete Rhodococcus sp. NI86/21 (De Mot et al.
(1996) Curr. Microbiol. 33: 26-30); the staphylococcal enterotoxins
(SEs) (Wood et al. (1997) FEMS Immunol. Med. Microbiol. 17: 1-10),
a 42-kDa M. hyopneumoniae NrdF ribonucleotide reductase R2 protein
or 15-kDa subunit protein of M. hyopneumoniae (Fagan et al. (1997)
Infect. Immun. 65: 2502-2507), the meningococcal antigen PorA
protein (Feavers et al. (1997) Clin. Diagn. Lab. Immunol. 3:
444-50); pneumococcal surface protein A (PspA) (McDaniel et al.
(1997) Gene Ther. 4: 375-377); F. tularensis outer membrane protein
FopA (Fulop et al. (1996) FEMS Immunol. Med. Microbiol. 13:
245-247); the major outer membrane protein within strains of the
genus Actinobacillus (Hartmann et al. (1996) Zentralbl. Bakteriol.
284: 255-262); p60 or listeriolysin (Hly) antigen of Listeria
monocytogenes (Hess et al. (1996) Proc. Nat'l. Acad. Sci. USA 93:
1458-1463); flagellar (G) antigens observed on Salmonella
enteritidis and S. pullorum (Holt and Chaubal (1997) J. Clin.
Microbiol. 35: 1016-1020); Bacillus anthracis protective antigen
(PA) (Ivins et al. (1995) Vaccine 13: 1779-1784); Echinococcus
granulosus antigen 5 (Jones et al. (1996) Parasitology 113:
213-222); the rol genes of Shigella dysenteriae I and Escherichia
coli K-12 (Klee et al. (1997) J. Bacteriol. 179: 2421-2425); cell
surface proteins Rib and alpha of group B streptococcus (Larsson et
al. (1996) Infect. Immun. 64: 3518-3523); the 37 kDa secreted
polypeptide encoded on the 70 kb virulence plasmid of pathogenic
Yersinia spp. (Leary et al. (1995) Contrib. Microbiol. Immunol. 13:
216-217 and Roggenkamp et al. (1997) Infect. Immun. 65: 446-51);
the OspA (outer surface protein A) of the Lyme disease spirochete
Borrelia burgdorferi (Li et al. (1997) Proc. Nat'l. Acad. Sci. USA
94: 3584-3589, Padilla et al. (1996) J. Infect. Dis. 174: 739-746,
and Wallich et al. (1996) Infection 24: 396-397); the Brucella
melitensis group 3 antigen gene encoding Omp28 (Lindler et al.
(1996) Infect. Immun. 64: 2490-2499); the PAc antigen of
Streptococcus mutans (Murakami et al. (1997) Infect. Immun. 65:
794-797); pneumolysin, Pneumococcal neuraminidases, autolysin,
hyaluronidase, and the 37 kDa pneumococcal surface adhesin A (Paton
et al. (1997) Microb. Drug Resist. 3: 1-10); 29-32, 41-45,
63-71.times.10(3) MW antigens of Salmonella typhi (Perez et al.
(1996) Immunology 89: 262-267); K-antigen as a marker of Klebsiella
pneumoniae (Priamukhina and Morozova (1996) Klin. Lab. Diagn.
47-9); nocardial antigens of molecular mass approximately 60, 40,
20 and 15-10 kDa (Prokesova et al. (1996) Int. J. Immunopharmacol.
18: 661-668); Staphylococcus aureus antigen ORF-2 (Rieneck et al.
(1997) Biochim Biophys Acta 1350: 128-132); GlpQ antigen of
Borrelia hemmsii (Schwan et al. (1996) J. Clin. Microbiol. 34:
2483-2492); cholera protective antigen (CPA) (Sciortino (1996) J.
Diarrhoeal Dis. Res. 14: 16-26); a 190-kDa protein antigen of
Streptococcus mutans (Senpuku et al. (1996) Oral Microbiol.
Immunol. 11: 121-128); Anthrax toxin protective antigen (PA)
(Sharma et al. (1996) Protein Expr. Purif 7: 33-38); Clostridium
perfringens antigens and toxoid (Strom et al. (1995) Br. J.
Rheumatol. 34: 1095-1096); the SEF14 fimbrial antigen of Salmonella
enteritidis (Thorns et al. (1996) Microb. Pathog. 20: 235-246); the
Yersinia pestis capsular antigen (F1 antigen) (Titball et al.
(1997) Infect. Immun. 65: 1926-1930); a 35-kilodalton protein of
Mycobacterium leprae (Triccas et al. (1996) Infect. Immun. 64:
5171-5177); the major outer membrane protein, CD, extracted from
Moraxella (Branhamella) catarrhalis (Yang et al. (1997) FEMS
Immunol. Med. Microbiol. 17: 187-199); pH 6 antigen (PsaA protein)
of Yersinia pestis (Zav'yalov et al. (1996) FEMS Immunol. Med.
Microbiol. 14: 53-57); a major surface glycoprotein, gp63, of
Leishmania major (Xu and Liew (1994) Vaccine 12: 1534-1536; Xu and
Liew (1995) Immunology 84: 173-176); mycobacterial heat shock
protein 65, mycobacterial 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 tuberculosis antigen 85 (Ag85) (Huygen
et al. (1996) Nat. Med. 2: 893-898); the 45/47 kDa antigen
complex.(APA) of Mycobacterium tuberculosis, M. bovis and BCG (Hom
et al. (1996) J. Immunol. Methods 197: 151-159); the mycobacterial
antigen, 65-kDa heat shock protein, hsp65 (Tascon et al. (1996)
Nat. Med. 2: 888-892); the mycobacterial antigens MPB64, MPB70,
MPB57 and alpha antigen (Yamada et al. (1995) Kekkaku 70: 639-644);
the M. tuberculosis 38 kDa protein (Vordermeier et al. (1995)
Vaccine 13: 1576-1582); the MPT63, MPT64 and MPT-59 antigens from
Mycobacterium tuberculosis (Manca et al. (1997) Infect. Immun. 65:
16-23; Oettinger et al. (1997) Scand. J. Immunol. 45: 499-503;
Wilcke et al. (1996) Tuber. Lung Dis. 77: 250-256); the
35-kilodalton protein of Mycobacterium leprae (Triccas et al.
(1996) Infect. Immun. 64: 5171-5177); the ESAT-6 antigen of
virulent mycobacteria (Brandt et al. (1996) J. Immunol. 157:
3527-3533; Pollock and Andersen (1997) J. Infect. Dis. 175:
1251-1254); Mycobacterium tuberculosis 16-kDa antigen (Hsp16.3)
(Chang et al. (1996) J. Biol. Chem. 271: 7218-7223); and the
18-kilodalton protein of Mycobacterium leprae (Baumgart et al.
(1996) Infect. Immun. 64: 2274-2281).
[0095] 2. Viral Pathogens
[0096] The methods of the invention are also useful for obtaining
recombinant nucleic acids and polypeptides that have enhanced
ability to induce an immune response against viral pathogens. While
the bacterial recombinants described above are typically
administered in polypeptide form, recombinants that confer viral
protection are preferably administered in nucleic acid form, as
genetic vaccines.
[0097] One illustrative example is the Hantaan virus. Glycoproteins
of this virus typically accumulate at the membranes of the Golgi
apparatus of infected cells. This poor expression of the
glycoprotein prevents the development of efficient genetic vaccines
against these viruses. The methods of the invention solve this
problem by performing DNA shuffling on nucleic acids that encode
the glycoproteins and identifying those recombinants that exhibit
enhanced expression in a host cell, and/or for improved
immunogenicity when administered as a genetic vaccine. A convenient
screening method for these methods is to express the recombinant
polynucleotides as fusion proteins to PIG, which results in display
of the polypeptides on the surface of the host cell (Whitehom et
al. (1995) Biotechnology (N Y) 13:1215-9). Fluorescence-activated
cell sorting is then used to sort and recover those cells that
express an increased amount of the antigenic polypeptide on the
cell surface. This preliminary screen can be followed by
immunogenicity tests in mammals, such as mice. Finally, in
preferred embodiments, those recombinant nucleic acids are tested
as genetic vaccines for their ability to protect a test animal
against challenge by the virus.
[0098] The flaviviruses are another example of a viral pathogen for
which the methods of the invention are useful for obtaining a
recombinant polypeptide or genetic vaccine that is effective
against a viral pathogen. The flaviviruses consist of three
clusters of antigenically related viruses: Dengue 1-4 (62-77%
identity), Japanese, St. Louis and Murray Valley encephalitis
viruses (75-82% identity), and the tick-borne encephalitis viruses
(77-96% identity). Dengue virus can induce protective antibodies
against SLE and Yellow fever (40-50% identity), but few efficient
vaccines are available. To obtain genetic vaccines and recombinant
polypeptides that exhibit enhanced cross-reactivity and
immunogenicity, the polynucleotides that encode envelope proteins
of related viruses are subjected to DNA shuffling. The resulting
recombinant polynucleotides can be tested, either as genetic
vaccines or by using the expressed polypeptides, for ability to
induce a broadly reacting neutralizing antibody response. Finally,
those clones that are favorable in the preliminary screens can be
tested for ability to protect a test animal against viral
challenge.
[0099] 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) Am. J. Trop. Med. Hyg. 50: 322-328;
Putnak et al. (1996) Am. J Trop. Med. Hyg. 55: 504-10); HIV
antigens Gag, Pol, Vif and Nef (Vogt et al. (1995) Vaccine 13:
202-208); HIV antigens gp120 and gp160 (Achour et al. (1995) Cell.
Mol. Biol. 41: 395-400; Hone et al. (1994) Dev. Biol. Stand. 82:
159-162); gp41 epitope of human immunodeficiency virus (Eckhart et
al. (1996) J. Gen. Virol. 77: 2001-2008); rotavirus antigen VP4
(Mattion et al. (1995) J. Virol. 69: 5132-5137); the rotavirus
protein VP7 or VP7sc (Emslie et al. (1995) J. Virol. 69: 1747-1754;
Xu et al. (1995) J. Gen. Virol. 76: 1971-1980); herpes simplex
virus (HSV) glycoproteins gB, gC, gD, gE, gG, gH, and gI (Fleck et
al. (1994) Med. Microbiol. Immunol. (Berl) 183: 87-94 [Mattion,
1995]; Ghiasi et al. (1995) Invest. Ophthalmol. Vis. Sci. 36:
1352-1360; McLean et al. (1994) J. Infect. Dis. 170: 1100-1109);
immediate-early protein ICP47 of herpes simplex virus-type 1
(HSV-1) (Banks et al. (1994) Virology 200: 236-245);
immediate-early (IE) proteins ICP27, ICP0, and ICP4 of herpes
simplex virus (Manickan et al. (1995) J. Virol. 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 proteins VP1 (Kawase et al. (1995) Virology 211:
359-366) or VP2 (Brown et al. (1994) Virology 198: 477-488);
Hepatitis B virus core and e antigen (Schodel et al. (1996)
Intervirology 39: 104-106); hepatitis B surface antigen (Shiau and
Murray (1997) J. Med. Virol. 51: 159-166); hepatitis B surface
antigen fused to the core antigen of the virus (Id.); Hepatitis B
virus core-preS2 particles (Nemeckova et al. (1996) Acta Virol. 40:
273-279); HBV preS2-S protein (Kutinova et al. (1996) Vaccine 14:
1045-1052); VZV glycoprotein I (Kutinova et al. (1996) Vaccine 14:
1045-1052); rabies virus glycoproteins (Xiang et al. (1994)
Virology 199: 132-140; 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); the
hepatitis C virus (HCV) nucleocapsid protein in a secreted or a
nonsecreted form, or as a fusion-protein with the middle (pre-S2
and S) or major (S) surface antigens of hepatitis B virus (HBV)
(Inchauspe et al. (1997) DNA Cell Biol. 16: 185-195; Major et al.
(1995) J. Virol. 69: 5798-5805); the hepatitis C virus antigens:
the core protein (pC); E1 (pE1) and E2 (pE2) alone or as fusion
proteins (Saito et al. (1997) Gastroenterology 112: 1321-1330); the
gene encoding respiratory syncytial virus fusion protein (PFP-2)
(Falsey and Walsh (1996) Vaccine 14: 1214-1218; Piedra et al.
(1996) Pediatr. Infect. Dis. J. 15: 23-31); the VP6 and VP7 genes
of rotaviruses (Choi et al. (1997) Virology 232: 129-138; Jin et
al. (1996) Arch. Virol. 141: 2057-2076); the E1, E2, E3, E4, E5, E6
and E7 proteins of human papillomavirus (Brown et al. (1994)
Virology 201: 46-54; Dillner et al. (1995) Cancer Detect. Prev. 19:
381-393; Krul et al. (1996) Cancer Immunol. Immunother. 43: 44-48;
Nakagawa et al. (1997) J. Infect. Dis. 175: 927-931); a human
T-lymphotropic virus type I gag protein (Porter et al. (1995) J.
Med. Virol. 45: 469-474); Epstein-Barr virus (EBV) gp340 (Mackett
et al. (1996) J. Med. Virol. 50: 263-271); the 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 (1996) J. Virol. 70: 4849-4853; Khanna et al.
(1995) Virology 214: 633-637); the measles virus nucleoprotein (N)
(Fooks et al. (1995) Virology 210: 456-465); and cytomegalovirus
glycoprotein gB (Marshall et al. (1994) J. Med. Virol. 43: 77-83)
or glycoprotein gH (Rasmussen et al. (1994) J. Infect. Dis. 170:
673-677).
[0100] 3. Parasites
[0101] Antigens from parasites can also be optimized by the methods
of the invention. These include, but are not limited to, the
schistosome gut-associated antigens CAA (circulating anodic
antigen) and CCA (circulating cathodic antigen) in Schistosoma
mansoni, S. haematobium or S. japonicum (Deelder et al. (1996)
Parasitology 112: 21-35); a multiple antigen peptide (MAP) composed
of two distinct protective antigens derived from the parasite
Schistosoma mansoni (Ferru et al. (1997) Parasite Immunol. 19:
1-11); Leishimania parasite surface molecules (Lezama-Davila (1997)
Arch. Med. Res. 28: 47-53); third-stage larval (L3) antigens of L.
Ioa (Akue et al. (1997) J. Infect. Dis. 175: 158-63); the genes,
Tams1-1 and Tams1-2, encoding the 30-and 32-kDa major merozoite
surface antigens of Theileria annulata (Ta) (d'Oliveira et al.
(1996) Gene 172: 33-39); Plasmodium falciparum merozoite surface
antigen 1 or 2 (al-Yaman et al. (1995) Trans. R. Soc. Trop. Med.
Hyg. 89: 555-559; Beck et al. (1997) J. Infect. Dis. 175: 921-926;
Rzepczyk et al. (1997) Infect. Immun. 65: 1098-1100);
circumsporozoite (CS) protein-based B-epitopes from Plasmodium
berghei, (PPPPNPND)2 and Plasmodiumn yoelii, (QGPGAP)3QG, along
with a P. berghei T-helper epitope KQIRDSITEEWS. (Reed et al.
(1997) Vaccine 15: 482-488); NYVAC-Pf7 encoded Plasmodium
falciparum antigens derived from the sporozoite (circumsporozoite
protein and sporozoite surface protein 2), liver (liver stage
antigen 1), blood (merozoite surface protein 1, serine repeat
antigen, and apical membrane antigen 1), and sexual (25-kDa
sexual-stage antigen) stages of the parasite life cycle were
inserted into a single NYVAC genome to generate NYVAC-Pf7 (Tine et
al. (1996) Infect. Immun. 64: 3833-3844); Plasmodium falciparum
antigen Pfs230 (Williamson et al. (1996) Mol. Biochem. Parasitol.
78: 161-169); Plasmodium falciparum apical membrane antigen (AMA-1)
(Lal et al. (1996) Infect. Immun. 64: 1054-1059); Plasmodium
falciparum proteins Pfs28 and Pfs25 (Duffy and Kaslow (1997)
Infect. Immun. 65: 1109-1113); Plasmodium falciparum merozoite
surface protein, MSP1 (Hui et al. (1996) Infect. Immun. 64:
1502-1509); the malaria antigen Pf332 (Ahlborg et al. (1996)
Immunology 88: 630-635); Plasmodium falciparum erythrocyte membrane
protein 1 (Baruch et al. (1995) Proc. Nat'l. Acad. Sci. USA 93:
3497-3502; Baruch et al. (1995) Cell 82: 77-87); Plasmodium
falciparum merozoite surface antigen, PfMSP-1 (Egan et al. (1996)
J. Infect. Dis. 173: 765-769); Plasmodium falciparum antigens SERA,
EBA-175, RAPI and RAP2 (Riley (1997) J. Pharm. Pharmacol. 49:
21-27); Schistosoma japonicum paramyosin (Sj97) or fragments
thereof (Yang et al. (1995) Biochem. Biophys. Res. Commun. 212:
1029-1039); and Hsp70 in parasites (Maresca and Kobayashi (1994)
Experientia 50: 1067-1074).
[0102] 4. Allergy
[0103] The invention also provides methods of obtaining reagents
that are useful for treating allergy. In one embodiment, the
methods involve making a library of recombinant polynucleotides
that encode an allergen, and screening the library to identify
those recombinant polynucleotides that exhibit improved properties
when used as immunotherapeutic reagents for treating allergy. For
example, specific immunotherapy of allergy using natural antigens
carries a risk of inducing anaphylaxis, which can be initiated by
cross-linking of high-affinity IgE receptors on mast cells.
Therefore, allergens that are not recognized by pre-existing IgE
are desirable. The methods of the invention provide methods by
which one can obtain such allergen variants. Another improved
property of interest is induction of broader immune responses,
increased safety and efficacy.
[0104] Synthesis of polyclonal and allergen-specific IgE requires
multiple interactions between B cells, T cells and professional
antigen-presenting cells (APC). Activation of naive, unprimed B
cells is initiated when specific B cells recognize the allergen by
cell surface immunoglobulin (sIg). However, costimulatory molecules
expressed by activated T cells in both soluble and membrane-bound
forms are necessary for differentiation of B cells into
IgE-secreting plasma cells. Activation of T helper cells requires
recognition of an antigenic peptide in the context of MHC class II
molecules on the plasma membrane of APC; such as monocytes,
dendritic cells, Langerhans cells or primed B cells. Professional
APC can efficiently capture the antigen and the peptide-MHC class
II complexes are formed in a post-Golgi, proteolytic intracellular
compartment and subsequently exported to the plasma membrane, where
they are recognized by T cell receptor (TCR) (Whitton (1998) Curr.
Top. Microbiol. Immunol. 232: 1-13). In addition, activated B cells
express CD80 (B7-1) and CD86 (B7-2, B70), which are the counter
receptors for CD28 and which provide a costimulatory signal for T
cell activation resulting in T cell proliferation and cytokine
synthesis. Since allergen-specific T cells from atopic individuals
generally belong to the T.sub.H2 cell subset, activation of these
cells also leads to production of IL-4 and IL-13, which, together
with membrane-bound costimulatory molecules expressed by activated
T helper cells, direct B cell differentiation into IgE-secreting
plasma cells.
[0105] Mast cells and eosinophils are key cells in inducing
allergic symptoms in target organs. Recognition of specific antigen
by IgE bound to high-affinity IgE receptors on mast cells,
basophils or eosinophils results in crosslinking of the receptors
leading to degranulation of the cells and rapid release of mediator
molecules, such as histamine, prostaglandins and leukotrienes,
causing allergic symptoms.
[0106] Immunotherapy of allergic diseases currently includes
hyposensibilization treatments using increasing doses of allergen
injected to the patient. These treatments result skewing of immune
responses towards T.sub.H1 phenotype and increase the ratio of
IgG/IgE antibodies specific for allergens. Because these patients
have circulating IgE antibodies specific for the allergens, these
treatments include significant risk of anaphylactic reactions. In
these reactions, free circulating allergen is recognized by IgE
molecules bound to high-affinity IgE receptors on mast cells and
eosinophils. Recognition of the allergen results in crosslinking of
the receptors leading to release of mediators, such as histamine,
prostaglandins, and leukotrienes, which cause the allergic
symptoms, and occasionally anaphylactic reactions. Other problems
associated with hyposensibilization include low efficacy and
difficulties in producing allergen extracts reproducibly.
[0107] The methods of the invention provide a means to obtain
allergens that, when used in genetic vaccines, provide a means of
circumventing the problems that have limited the usefulness of
previously known hyposensibilization treatments. For example, by
expressing antigens on the surface of cells, such as muscle cells,
the risk of anaphylactic reactions is significantly reduced. This
can be conveniently achieved by using genetic vaccine vectors that
encode transmembrane forms of allergens. The allergens can also be
modified in such a way that they are efficiently expressed in
transmembrane forms, further reducing the risk of anaphylactic
reactions. Another advantage provided by the use of genetic
vaccines for hyposensibilization is that the genetic vaccines can
include cytokines and accessory molecules which further direct the
immune responses towards the T.sub.H1 phenotype, thus reducing the
amount of IgE antibodies produced and increasing the efficacy of
the treatments. To further reduce IgE production, one can
administer the shuffled allergens using vectors that have been
evolved to induce primarily IgG and IgM responses, with little or
no IgE response (see, e.g., U.S. patent application Ser. No.
09/021,769, filed Feb. 11, 1998).
[0108] In these methods, polynucleotides encoding known allergens,
or homologs or fragments thereof (e.g., immunogenic peptides) are
inserted into DNA vaccine vectors and used to immunize allergic and
asthmatic individuals. Alternatively, the shuffled allergens are
expressed in manufacturing cells, such as E. coli or yeast cells,
and subsequently purified and used to treat the patients or prevent
allergic disease. DNA shuffling or other recombination method can
be used to obtain allergens that activate T cells but cannot induce
anaphylactic reactions. For example, a library of recombinant
polynucleotides that encode allergen variants can be expressed in
cells, such as antigen presenting cells, which are than contacted
with PBMC or T cell clones from atopic patients. Those library
members that efficiently activate T.sub.H cells from the atopic
patients can be identified by assaying for T cell proliferation, or
by cytokine synthesis (e.g., synthesis of IL-2, IL-4, IFN-.gamma..
Those recombinant allergen variants that are positive in the in
vitro tests can then be subjected to in vivo testing.
[0109] Examples of allergies that can be treated include, but are
not limited to, allergies against house dust mite, grass pollen,
birch pollen, ragweed pollen, hazel pollen, cockroach, rice, olive
tree pollen, fungi, mustard, bee venom. Antigens of interest
include those of animals, including the mite (e.g.,
Dermzatophagoides pteronyssinus, Dermatophagoides farinae, Blomia
tropicalis), such as the allergens der pl (Scobie et al. (1994)
Biochem. Soc. Trans. 22: 448S; Yssel et al. (1992) J. Immunol. 148:
738-745), der p2 (Chua et al. (1996) Clin. Exp. Allergy 26:
829-837), der p3 (Smith and Thomas (1996) Clin. Exp. Allergy 26:
571-579), der p5, der p V (Lin et al. (1994) J. Allergy Clin.
Immunol. 94: 989-996), der p6 (Bennett and Thomas (1996) Clin. Exp.
Allergy 26: 1150-1154), der p 7 (Shen et al. (1995) Clin. Exp.
Allergy 25: 416-422), der f2 (Yuuki et al. (1997) Int. Arch.
Allergy Immunol. 112: 44-48), der f3 (Nishiyama et al. (1995) FEBS
Lett. 377: 62-66), der f7 (Shen et al. (1995) Clin. Exp. Allergy
25: 1000-1006); Mag 3 (Fujikawa et al. (1996) Mol. Immunol. 33:
311-319). Also of interest as antigens are the house dust mite
allergens Tyr p2 (Eriksson et al. (1998) Eur. J. Biochem. 251:
443-447), Lep d1 (Schmidt et al. (1995) FEBS Lett. 370: 11-14), and
glutathione S-transferase (O'Neill et al. (1995) Immunol Lett. 48:
103-107); the 25,589 Da, 219 amino acid polypeptide with homology
with glutathione S-transferases (O'Neill et al. (1994) Biochim.
Biophys. Acta. 1219: 521-528); Blo t 5 (Arruda et al. (1995) Int.
Arch. Allergy Immunol. 107: 456-457); bee venom phospholipase A2
(Carballido et al. (1994) J. Allergy Clin. Immunol. 93: 758-767;
Jutel et al. (1995) J. Immunol. 154: 4187-4194); bovine
dermal/dander antigens BDA 11 (Rautiainen et al. (1995) J. Invest.
Dermatol. 105: 660-663) and BDA20 (Mantyjarvi et al. (1996) J.
Allergy Clin. Immunol. 97: 1297-1303); the major horse allergen Equ
c1 (Gregoire et al. (1996) J. Biol. Chem. 271: 32951-32959); Jumper
ant M. pilosula allergen Myr p I and its homologous allergenic
polypeptides Myr p2 (Donovan et al. (1996) Biochem. Mol. Biol. Int.
39: 877-885); 1-13, 14, 16 kD allergens of the mite Blomia
tropicalis (Caraballo et al. (1996) J. Allergy Clin. Immunol. 98:
573-579); the cockroach allergens Bla g Bd90K (Helm et al. (1996)
J. Allergy Clin. Immunol. 98: 172-80) and Bla g 2 (Arruda et al.
(1995) J. Biol. Chem. 270: 19563-19568); the cockroach Cr-PI
allergens (Wu et al. (1996) J. Biol. Chem. 271: 17937-17943); fire
ant venom allergen, Sol i 2 (Schmidt et al. (1996) J. Allergy Clin.
Immunol. 98: 82-88); the insect Chironomus thummi major allergen
Chi t 1-9 (Kipp et al. (1996) Int. Arch. Allergy Immunol. 110:
348-353); dog allergen Can f 1 or cat allergen Fel d 1 (Ingram et
al. (1995) J. Allergy Clin. Immunol. 96: 449-456); albumin,
derived, for example, from horse, dog or cat (Goubran Botros et al.
(1996) Immunology 88: 340-347); deer allergens with the molecular
mass of 22 kD, 25 kD or 60 kD (Spitzauer et al. (1997) Clin. Exp.
Allergy 27: 196-200); and the 20 kd major allergen of cow (Ylonen
et al. (1994) J. Allergy Clin. Immunol. 93: 851-858).
[0110] Pollen and grass allergens are also useful in vaccines,
particularly after optimization of the antigen by the methods of
the invention. Such allergens include, for example, Hor v9 (Astwood
and Hill (1996) Gene 182: 53-62, Lig v1 (Batanero et al. (1996)
Clin. Exp. Allergy 26: 1401-1410); Lol p 1 (Muller et al. (1996)
Int. Arch. Allergy Immunol. 109: 352-355), Lol p II (Tamborini et
al. (1995) Mol. Immunol. 32: 505-513), Lol pVA, Lol pVB (Ong et al.
(1995) Mol. Immunol. 32: 295-302), Lol p 9 (Blaher et al. (1996) J.
Allergy Clin. Immunol. 98: 124-132); Par J I (Costa et al. (1994)
FEBS Lett. 341: 182-186; Sallusto et al. (1996) J. Allergy Clin.
Immunol. 97: 627-637), Par j 2.0101 (Duro et al. (1996) FEBS Lett.
399: 295-298); Bet v1 (Faber et al. (1996) J. Biol. Chem. 271:
19243-19250), Bet v2 (Rihs et al. (1994) Int. Arch. Allergy
Inmimunol. 105: 190-194); Dac g3 (Guerin-Marchand et al. (1996)
Mol. Immunol. 33: 797-806); Phl p 1 (Petersen et al. (1995) J.
Allergy Clin. Immunol. 95: 987-994), Phl p 5 (Muller et al. (1996)
Int. Arch. Allergy Immunol. 109: 352-355), Phl p 6 (Petersen et al.
(1995) Int. Arch. Allergy Immunol. 108: 55-59); Cryj I (Sone et al.
(1994) Biochem. Bioph1ys. Res. Commun. 199: 619-625), Cryj II
(Namba et al. (1994) FEBS 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. Immunol. 98: 331-343), cyn d7 (Suphioglu et
al. (1997) FEBS Lett. 402: 167-172); Pha a 1 and isoforms of Pha a
5 (Suphioglu and Singh (1995) Clin. Exp. Allergy 25: 853-865); Cha
o 1 (Suzuki et al. (1996) Mol. Immunol. 33: 451-460); profilin
derived, e.g, from timothy grass or birch pollen (Valenta et al.
(1994) Biochem. Biophys. Res. Commun. 199: 106-118); P0149 (Wu et
al. (1996) Plant Mol. Biol. 32: 1037-1042); Ory s1 (Xu et al.
(1995) Gene 164: 255-259); and Amb a V and Amb t 5 (Kim et al.
(1996) Mol. Immunol. 33: 873-880; Zhu et al. (1995) J. Immunol.
155: 5064-5073).
[0111] Vaccines against food allergens can also be developed using
the methods of the invention. Suitable antigens for shuffling
include, for example, profilin (Rihs et al. (1994) Int. Arch.
Allergy Immunol. 105: 190-194); rice allergenic cDNAs belonging to
the alpha-amylase/trypsin inhibitor gene family (Alvarez et al.
(1995) Biochim Biophys Acta 1251: 201-204); the main olive
allergen, Ole e I (Lombardero et al. (1994) Clin Exp Allergy 24:
765-770); Sin a 1, the major allergen from mustard (Gonzalez De La
Pena et al. (1996) Eur J. Biochem. 237: 827-832); parvalbumin, the
major allergen of salmon (Lindstrom et al. (1996) Scand. J.
Immunol. 44: 335-344); apple allergens, such as the major allergen
Mal d I (Vanek-Krebitz et al. (1995) Biochem. Biophys. Res. Commun.
214: 538-551); and peanut allergens, such as Ara h I (Burks et al.
(1995) J. Clin. Invest. 96: 1715-1721).
[0112] The methods of the invention can also be used to develop
recombinant antigens that are effective against allergies to fungi.
Fungal allergens useful in these vaccines include, but are not
limited to, the allergen, Cla h III, of Cladosporium herbarum
(Zhang et al. (1995) J. Immunol. 154: 710-717); the allergen Psi c
2, a fungal cyclophilin, from the basidiomycete Psilocybe cubensis
(Horner et al. (1995) Int. Arch. Allergy Immunol. 107: 298-300);
hsp 70 cloned from a cDNA library of Cladosporium herbarum (Zhang
et al. (1996) Clin Exp Allergy 26: 88-95); the 68 kD allergen of
Penicillium notatum (Shen et al. (1995) Clin. 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 ribosomal protein P2 (Id.).
[0113] Other allergens that can be used in the methods of the
invention include latex allergens, such as a major allergen (Hev b
5) from natural rubber latex (Akasawa et al. (1996) J. Biol. Chem.
271: 25389-25393; Slateret al. (1996) J. Biol. Chem. 271:
25394-25399).
[0114] The invention also provides a solution to another
shortcoming of vaccination as a treatment for allergy and asthma.
While genetic vaccination primarily induces CD8.sup.+ T cell
responses, induction of allergen-specific IgE responses is
dependent on CD4.sup.+ T cells and their help to B cells.
T.sub.H2-type cells are particularly efficient in inducing IgE
synthesis because they secrete high levels of IL-4, IL-5 and IL-13,
which direct Ig isotype switching to IgE synthesis. IL-5 also
induces eosinophilia. The methods of the invention can be used to
develop recombinant antigens that efficiently induce CD4.sup.+ T
cell responses, and direct differentiation of these cells towards
the T.sub.H1 phenotype.
[0115] 5. Inflammatory and Autoimmune Diseases
[0116] Autoimmune diseases are characterized by immune response
that attacks tissues or cells of ones own body, or
pathogen-specific immune responses that also are harmful for ones
own tissues or cells, or non-specific immune activation which is
harmful for ones 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 using antigens that
are optimized using the methods of the invention.
[0117] These conditions are often characterized by an accumulation
of inflammatory cells, such as lymphocytes, macrophages, and
neutrophils, at the sites of inflammation. Altered cytokine
production levels are often observed, with increased levels of
cytokine production. Several autoimmune diseases, including
diabetes and rheumatoid arthritis, are linked to certain MHC
haplotypes. Other autoimmune-type disorders, such as reactive
arthritis, have been shown to be triggered by bacteria such as
Yersinia and Shigella, and evidence suggests that several other
autoimmune diseases, such as diabetes, multiple sclerosis,
rheumatoid arthritis, may also be initiated by viral or bacterial
infections in genetically susceptible individuals.
[0118] Current strategies of treatment generally include
anti-inflammatory drugs, such as NSAID or cyclosporin, and
antiproliferative drugs, such as methotrexate. These therapies are
non-specific, so a need exists for therapies having greater
specificity, and for means to direct the immune responses towards
the direction that inhibits the autoimmune process.
[0119] The present invention provides several strategies by which
these needs can be fulfilled. First, the invention provides methods
of obtaining antigens having greater tolerogenicity and/or have
improved antigenicity. In a preferred embodiment, the antigens
prepared according to the invention exhibit improved induction of
tolerance by oral delivery. Oral tolerance is characterized by
induction of immunological tolerance after oral administration of
large quantities of antigen. In animal models, this approach has
proven to be a very promising approach to treat autoimmune
diseases, and clinical trials are in progress to address the
efficacy of this approach in the treatment of human autoimmune
diseases, such as rheumatoid arthritis and multiple sclerosis. It
has also been suggested that induction of oral tolerance against
viruses used in gene therapy might reduce the immunogenicity of
gene therapy vectors. However, the amounts of antigen required for
induction of oral tolerance are very high and the methods of the
invention provide a means for obtaining antigens that exhibit a
significant improvement in induction of oral tolerance.
[0120] Expression library immunization (Barry et al. (1995) Nature
377: 632) is a particularly useful method of screening for optimal
antigens for use in genetic vaccines. For example, to identify
autoantigens present in Yersinia, Shigella, and the like, one can
screen for induction of T cell responses in HLA-B27 positive
individuals. Complexes that include epitopes of bacterial antigens
and MHC molecules associated with autoimmune diseases, e.g.,
HLA-B27 in association with Yersinia antigens can be used in the
prevention of reactive arthritis and ankylosing spondylitis in
HLA-B27 positive individuals.
[0121] Screening of optimized antigens can be done in animal models
which are known to those of skill in the art. Examples of suitable
models for various conditions include collagen induced arthritis,
the NFS/sld mouse model of human Sjogren's syndrome; a 120 kD
organ-specific autoantigen recently identified as an analog of
human cytoskeletal protein (.alpha.-fodrin (Haneji et al. (1997)
Science 276: 604), the New Zealand Black/White F1 hybrid mouse
model of human SLE, NOD mice, a mouse model of human diabetes
mellitus, fas/fas ligand mutant mice, which spontaneously develop
autoimmune and lymphoproliferative disorders (Watanabe-Fukunaga et
al. (1992) Nature 356: 314), and experimental autoimmune
encephalomyelitis (EAE), in which myelin basic protein induces a
disease that resembles human multiple sclerosis.
[0122] Autoantigens that can be shuffled according to the methods
of the invention and used in vaccines for treating multiple
sclerosis include, but are not limited to, myelin basic protein
(Stinissen et al. (1996) J. IVeurosci. Res. 45: 500-511) or a
fusion protein of myelin basic protein and proteolipid protein
(Elliott et al. (1996) J. Clin. Invest. 98: 1602-1612), proteolipid
protein (PLP) (Rosener et al. (1997) J. Neuroimmunol. 75: 28-34),
2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) (Rosener et
al. (1997) J. Neuroimmunol. 75: 28-34), the Epstein Barr virus
nuclear antigen-1 (EBNA-1) (Vaughan et al. (1996) J. Neuroimmunol.
69: 95-102), HSP70 (Salvetti et al. (1996) J. Neuroimmunol. 65:
143-53; Feldmann et al. (1996) Cell 85: 307).
[0123] Target antigens that, after shuffling according to the
methods of the invention, can be used to treat scleroderma,
systemic sclerosis, and systemic lupus erythematosus include, for
example, (-2-GPI, 50 kDa glycoprotein (Blank et al. (1994) J.
Autoimmun. 7: 441-455), Ku (p70/p80) autoantigen, or its 80-kd
subunit protein (Hong et al. (1994) Invest. Ophthalmol. Vis. Sci.
35: 4023-4030; Wang et al. (1994) J. Cell Sci. 107: 3223-3233), the
nuclear autoantigens La (SS-B) and Ro (SS-A) (Huang et al. (1997)
J. Clin. Immunol. 17: 212-219; Igarashi et al. (1995) Autoimmunity
22: 33-42; Keech et al. (1996) Clin. Exp. Immunol. 104: 255-263;
Manoussakis et al. (1995) J. Autoimmun. 8: 959-969; Topfer et al.
(1995) Proc. Nat'l. Acad. Sci. USA 92: 875-879), proteasome (-type
subunit C9 (Feist et al. (1996) J. Exp. Med. 184: 1313-1318),
Scleroderma antigens Rpp 30, Rpp 38 or Scl-70 (Eder et al. (1997)
Proc. Nat'l. Acad. Sci. USA 94: 1101-1106; Hietarinta et al. (1994)
Br. J Rheumatol. 33: 323-326), the centrosome autoantigen PCM-1
(Bao et al. (1995) Autoimmunity 22: 219-228),
polymyositis-scleroderma autoantigen (PM-Scl) (Kho et al. (1997) J.
Biol. Chem. 272: 13426-13431), scleroderma (and other systemic
autoimmune disease) autoantigen CENP-A (Muro et al. (1996) Clin.
Immunol. Immunopathol. 78: 86-89), U5, a small nuclear
ribonucleoprotein (snRNP) (Okano et al. (1996) Clin. Immunol.
Immunopathol. 81: 41-47), the 100-kd protein of PM-Scl autoantigen
(Ge et al. (1996) Arthritis Rheum. 39: 1588-1595), the nucleolar
U3- and Th(7-2) ribonucleoproteins (Verheijen et al. (1994) J.
Immunol. Methods 169: 173-182), the ribosomal protein L7 (Neu et
al. (1995) Clin. Exp. Immunol. 100: 198-204), hPopl (Lygerou et al.
(1996) EMBO J. 15: 5936-5948), and a 36-kd protein from nuclear
matrix antigen (Deng et al. (1996) Arthritis Rheum. 39:
1300-1307).
[0124] Hepatic autoimmune disorders can also be treated using
improved recombinant antigens that are prepared according to the
methods described herein. Among the antigens that are useful in
such treatments are the cytochromes P450 and
UDP-glucuronosyl-transferases (Obermayer-Straub and Manns (1996)
Baillieres Clin. Gastroenterol. 10: 501-532), the cytochromes P450
2C9 and P450 1A2 (Bourdi et al. (1996) Chem. Res. Toxicol. 9:
1159-1166; Clemente et al. (1997) J. Clin. Endocrinol. Metab. 82:
1353-1361), LC-1 antigen (Klein et al. (1996) J. Pediatr.
Gastroenterol. Nutr. 23: 461-465), and a 230-kDa Golgi-associated
protein (Funaki et al. (1996) Cell Struct. Funct. 21: 63-72).
[0125] For treatment of autoimmune disorders of the skin, useful
antigens include, but are not limited to, the 450 kD human
epidermal autoantigen (Fujiwara et al. (1996) J. Invest. Dermatol.
106: 1125-1130), the 230 kD and 180 kD bullous pemphigoid antigens
(Hashimoto (1995) Keio J. Med. 44: 115-123; Murakami et al. (1996)
J. Dermatol. Sci. 13: 112-117), pemphigus foliaceus antigen
(desmoglein 1), pemphigus vulgaris antigen (desmoglein 3), BPAg2,
BPAg1, and type VII collagen (Batteux et al. (1997) J. Clin.
Immunol. 17: 228-233; Hashimoto et al. (1996) J. Dermatol. Sci. 12:
10-17), a 168-kDa mucosal antigen in a subset of patients with
cicatricial pemphigoid (Ghohestani et al. (1996) J. Invest.
Dermatol. 107: 136-139), and a 218-kd nuclear protein (218-kd Mi-2)
(Seelig et al. (1995) Arthritis Rheum. 38: 1389-1399).
[0126] The methods of the invention are also useful for obtaining
improved antigens for treating insulin dependent diabetes mellitus,
using one or more of antigens which include, but are 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; Schloot et
al. (1997) Diabetologia 40: 332-338), viral proteins homologous to
GAD65 (Jones and Crosby (1996) Diabetologia 39: 1318-1324), islet
cell antigen-related protein-tyrosine phosphatase (PTP) (Cui et al.
(1996) J. Biol. Chem. 271: 24817-24823), GM2-1 ganglioside (Cavallo
et al. (1996) J. Endocrinol. 0.150: 113-120; Dotta et al. (1996)
Diabetes 45: 1193-1196), glutamic acid decarboxylase (GAD) (Nepom
(1995) Curr. Opin. Immunol. 7: 825-830; Panina-Bordignon et al.
(1995) J. Exp. Med. 181: 1923-1927), an 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, the single T cell
epitope recognized by T cells from diabetes patients (Karges et al.
(1997) Biochim. Biophys. Acta 1360: 97-101), ICA 512, an
autoantigen of type I diabetes (Solimena et al. (1996) EMBO J. 15:
2102-2114), an islet-cell protein tyrosine phosphatase and the
37-kDa autoantigen derived from it in type I diabetes (including
IA-2, IA-2) (La Gasse et al. (1997) Mol. Med. 3: 163-173), the 64
kDa protein from In-111 cells or human thyroid follicular cells
that is immunoprecipitated with sera from patients with islet cell
surface antibodies (ICSA) (Igawa et al. (1996) Endocr. J. 43:
299-306), phogrin, a homologue of the human transmembrane protein
tyrosine phosphatase, an autoantigen of type I diabetes (Kawasaki
et al. (1996) Biochem. Biophys. Res. Commun. 227: 440-447), the 40
kDa and 37 kDa tryptic fragments and their precursors IA-2 and IA-2
in IDDM (Lampasona et al. (1996) J. Immunol. 157: 2707-2711;
Notkins et al. (1996) J. Autoimmun. 9: 677-682), insulin or a
cholera toxoid-insulin conjugate (Bergerot et al. (1997) Proc.
Nat'l. Acad. Sci. USA 94: 4610-4614), carboxypeptidase H, the human
homologue of gp330, which is a renal epithelial glycoprotein
involved in inducing Heymann nephritis in rats, and the 38-kD islet
mitochondrial autoantigen (Arden et al. (1996) J. Clin. Invest. 97:
551-561.
[0127] Rheumatoid arthritis is another condition that is treatable
using optimized antigens prepared according to the present
invention. Useful antigens for rheumatoid arthritis treatment
include, but are not limited to, the 45 kDa DEK nuclear antigen, in
particular onset juvenile rheumatoid arthritis and iridocyclitis
(Murray et al. (1997) J. Rheumatol. 24: 560-567), human cartilage
glycoprotein-39, an autoantigen in rheumatoid arthritis (Verheijden
et al. (1997) Arthritis Rheum. 40: 1115-1125), a 68k autoantigen in
rheumatoid arthritis (Blass et al. (1997) Ann. Rheum. Dis. 56:
317-322), collagen (Rosloniec et al. (1995) J. Immunol. 155:
4504-4511), collagen type II (Cook et al. (1996) Arthritis Rheum.
39: 1720-1727; Trentham (1996) Ann. N. Y Acad. Sci. 778: 306-314),
cartilage link protein (Guerassimov et al. (1997) J. Rheumatol. 24:
959-964), ezrin, radixin and moesin, which are auto-immune antigens
in rheumatoid arthritis (Wagatsuma et al. (1996) Mol. Immunol. 33:
1171-1176), and mycobacterial heat shock protein 65 (Ragno et al.
(1997) Arthritis Rheum. 40: 277-283).
[0128] Also among the conditions for which one can obtain an
improved antigen suitable for treatment are autoimmune thyroid
disorders. Antigens that are useful for these applications include,
for example, thyroid peroxidase and the thyroid stimulating hormone
receptor (Tandon and Weetman (1994) J. R. Coll. Physicians Lond.
28: 10-18), thyroid peroxidase from human Graves' thyroid tissue
(Gardas et al. (1997) Biochem. Biophys. Res. Commun. 234: 366-370;
Zimmer et al. (1997) Histochem. Cell. Biol. 107: 115-120), a 64-kDa
antigen associated with thyroid-associated ophthalmopathy (Zhang et
al. (1996) Clin. Immunol. Immunopathol. 80: 236-244), the human TSH
receptor (Nicholson et al. (1996) J. Mol. Endocrinol. 16: 159-170),
and the 64 kDa protein from In-111 cells or human thyroid
follicular cells that is immunoprecipitated with sera from patients
with islet cell surface antibodies (ICSA) (Igawa et al. (1996)
Endocr. J. 43: 299-306).
[0129] Other conditions and associated antigens include, but are
not limited to, Sjogren's syndrome (-fodrin; Haneji et al. (1997)
Science 276: 604-607), myastenia gravis (the human M2 acetylcholine
receptor or fragments thereof, specifically the second
extracellular loop of the human M2 acetylcholine receptor; Fu et
al. (1996) Clin. Immunol. Immunopathol. 78: 203-207), vitiligo
(tyrosinase; Fishman et al. (1997) Cancer 79: 1461-1464), a 450 kD
human epidermal autoantigen recognized by serum from individual
with blistering skin disease, and ulcerative colitis (chromosomal
proteins HMG1 and HMG2; Sobajima et al. (1997) Clin. Exp. Immunol.
107: 135-140).
[0130] 6. Cancer
[0131] Immunotherapy has great promise for the treatment of cancer
and prevention of metastasis. By inducing an immune response
against cancerous cells, the body's immune system can be enlisted
to reduce or eliminate cancer. Improved antigens obtained using the
methods of the invention provide cancer immunotherapies of
increased effectiveness compared to those that are presently
available.
[0132] One approach to cancer immunotherapy is vaccination using
vaccines that include or encode antigens that are specific for
tumor cells or by injecting the patients with purified recombinant
cancer antigens. The methods of the invention can be used for
obtaining antigens that exhibit an enhancement of immune responses
against known tumor-specific antigens, and also to search for novel
protective antigenic sequences. Antigens having optimized
expression, processing, and presentation can be obtained as
described herein. The approach used for each particular cancer can
vary. For treatment of hormone-sensitive cancers (for example,
breast cancer and prostate cancer), methods of the invention can be
used to obtain optimized hormone antagonists. For highly
immunogenic tumors, including melanoma, one can screen for
recombinant antigens that optimally boost the immune response
against the tumor. Breast cancer, in contrast, is of relatively low
immunogenicity and exhibits slow progression, so individual
treatments can be designed for each patient. Prevention of
metastasis is also a goal in design of cancer vaccines.
[0133] Among the tumor-specific antigens that can be used in the
antigen shuffling methods of the invention are: bullous pemphigoid
antigen 2, prostate mucin antigen (PMA) (Beckett and Wright (1995)
Int. J. Cancer 62: 703-710), tumor associated Thomsen-Friedenreich
antigen (Dahlenborg et al. (1997) Int. J Cancer 70: 63-71),
prostate-specific antigen (PSA) (Dannull and Belldegrun (1997) Br.
J. Urol. 1: 97-103), luminal epithelial antigen (LEA 135) of breast
carcinoma and bladder transitional cell carcinoma (TCC) (Jones et
al. (1997) Anticancer Res. 17: 685-687), cancer-associated serum
antigen (CASA) and cancer antigen 125 (CA 125) (Kierkegaard et al.
(1995) Gynecol. Oncol. 59: 251-254), the epithelial glycoprotein 40
(EGP40) (Kievit et al. (1997) Int. J. Cancer 71: 237-245), squamous
cell carcinoma antigen (SCC) (Lozza et al. (1997) 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
79: 1461-1464), cell nuclear antigen (PCNA) of cerebral cavernomas
(Notelet et al. (1997) Surg. Neurol. 47: 364-370), DF31MUC1 breast
cancer antigen (Apostolopoulos et al. (1996) Immunol. Cell. Biol.
74: 457-464; Pandey et al. (1995) Cancer Res. 55: 4000-4003),
carcinoembryonic antigen (Paone et al. (1996) J. Cancer Res. Clin.
Oncol. 122: 499-503; Schlom et al. (1996) Breast Cancer Res. Treat.
38: 27-39), tumor-associated antigen CA 19-9 (Tolliver and O'Brien
(1997) South Med. J. 90: 89-90; Tsuruta et al. (1997) Urol. Int.
58: 20-24), human melanoma antigens MART-1/Melan-A27-35 and gp 100
(Kawakami and Rosenberg (1997) Int. Rev. Immunol. 14: 173-192;
Zajac et al. (1997) Int. J Cancer 71: 491-496), the T and Tn
pancarcinoma (CA) glycopeptide epitopes (Springer (1995) Crit. Rev.
Oncog. 6: 57-85), a 35 kD tumor-associated autoantigen in papillary
thyroid carcinoma (Lucas et al. (1996) Anticancer Res. 16:
2493-2496), KH-1 adenocarcinoma antigen (Deshpande and Danishefsky
(1997) Nature 387: 164-166), the A60 mycobacterial antigen (Maes et
al. (1996) J. Cancer Res. Clin. Oncol. 122: 296-300), heat shock
proteins (HSPs) (Blachere and Srivastava (1995) Semin. Cancer Biol.
6: 349-355), and MAGE, tyrosinase, melan-A and gp75 and mutant
oncogene products (e.g., p53, ras, and HER-2/neu (Bueler and
Mulligan (1996) Mol. Med. 2: 545-555; Lewis and Houghton (1995)
Semin. Cancer Biol. 6: 321-327; Theobald et al. (1995) Proc. Nat'l.
Acad. Sci. USA 92: 11993-11997).
[0134] 7. Contraception
[0135] Genetic vaccines that contain optimized antigens obtained by
the methods of the invention are also useful for contraception. For
example, genetic vaccines can be obtained that encode sperm cell
specific antigens, and thus induce anti-sperm immune responses.
Vaccination can be achieved by, for example, administration of
recombinant bacterial strains, e.g. Salmonella and the like, which
express sperm antigen, as well as by induction of neutralizing
anti-hCG antibodies by vaccination by DNA vaccines encoding human
chorionic gonadotropin (hCG), or a fragment thereof.
[0136] Sperm antigens which can be used in the 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. Androl. 33:141-144),
the synthetic sperm peptide, P10G (O'Rand et al. (1993) J. Reprod.
Immunol. 25: 89-102), the 135 kD, 95 kD, 65 kD, 47 kD, 41 kD and 23
kD proteins of sperm, and the FA-1 antigen (Naz et al. (1995) Arch.
Androl. 35: 225-231), and the 35 kD fragment of cytokeratin 1
(Lucas et al. (1996) Anticancer Res. 16: 2493-2496).
[0137] The methods of the invention can also be used to obtain
genetic vaccines that are expressed specifically in testis. For
example, polynucleotide sequences that direct expression of genes
that are specific to testis can be used (e.g., fertilization
antigen-1 and the like). In addition to sperm antigens, antigens
expressed on oocytes or hormones regulating reproduction may be
useful targets of contraceptive vaccines. For example, genetic
vaccines can be used to generate antibodies against gonadotropin
releasing hormone (GnRH) or zona pellucida proteins (Miller et al.
(1997) Vaccine 15:1858-1862). Vaccinations using these molecules
have been shown to be efficacious 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).
[0138] Methods of Selecting and Identifying Optimized Recombinant
Antigens
[0139] Once one has performed DNA shuffling to obtain a library of
polynucleotides that encode recombinant antigens, the library is
subjected to selection and/or screening to identify those library
members that encode antigenic peptides that have improved ability
to induce an immune response to the pathogenic agent. Selection and
screening of recombinant polynucleotides that encode polypeptides
having an improved ability to induce an immune response can involve
either in vivo and in vitro methods, but most often involves a
combination of these methods. For example, in a typical embodiment
the members of a library of recombinant nucleic acids are picked,
either individually or as pools. The clones can be subjected to
analysis directly, or can be expressed to produce the corresponding
polypeptides. In a presently preferred embodiment, an in vitro
screen is performed to identify the best candidate sequences for
the in vivo studies. Alternatively, the library can be subjected to
in vivo challenge studies directly. The analyses can employ either
the nucleic acids themselves (e.g., as genetic vaccines), or the
polypeptides encoded by the nucleic acids. A schematic diagram of a
typical strategy is shown in FIG. 5. Both in vitro and in vivo
methods are described in more detail below.
[0140] If a recombination cycle is performed in vitro, the products
of recombination, i.e., recombinant segments, are sometimes
introduced into cells before the screening step. Recombinant
segments can also be linked to an appropriate vector or other
regulatory sequences before screening. Alternatively, products of
recombination generated in vitro are sometimes packaged in viruses
(e.g., bacteriophage) before screening. If recombination is
performed in vivo, recombination products can sometimes be screened
in the cells in which recombination occurred. In other
applications, recombinant segments are extracted from the cells,
and optionally packaged as viruses, before screening.
[0141] Often, improvements are achieved after one round of
recombination and selection. However, recursive sequence
recombination can also be employed to achieve still further
improvements in a desired property, or to bring about new (or
"distinct") properties. Recursive sequence recombination entails
successive cycles of recombination to generate molecular diversity.
That is, one creates a family of nucleic acid molecules showing
some sequence identity to each other but differing in the presence
of mutations. In any given cycle, recombination can occur in vivo
or in vitro, intracellularly or extracellularly. Furthermore,
diversity resulting from recombination can be augmented in any
cycle by applying prior methods of mutagenesis (e.g., error-prone
PCR or cassette mutagenesis) to either the substrates or products
for recombination.
[0142] In a presently preferred embodiment, polynucleotides that
encode optimized recombinant antigens are subjected to molecular
backcrossing, which provides a means to breed the shuffled
chimeras/mutants back to a parental or wild-type sequence, while
retaining the mutations that are critical to the phenotype that
provides the optimized immune responses. In addition to removing
the neutral mutations, molecular backcrossing can also be used to
characterize which of the many mutations in an improved variant
contribute most to the improved phenotype. This cannot be
accomplished in an efficient library fashion by any other method.
Backcrossing is performed by shuffling the improved sequence with a
large molar excess of the parental sequences.
[0143] The nature of screening or selection depends on what
property or characteristic is to be acquired or the property or
characteristic for which improvement is sought, and many examples
are discussed below. It is not usually necessary to understand the
molecular basis by which particular products of recombination
(recombinant segments) have acquired new or improved properties or
characteristics relative to the starting substrates. For example, a
gene that encodes an antigenic polypeptide can have many component
sequences each having a different intended role (see, e.g., FIG.
4). Each of these component sequences can be varied and recombined
simultaneously. Screening/selection can then be performed, for
example, for recombinant segments that have increased ability to
induce an immune response to a pathogenic agent without the need to
attribute such improvement to any of the individual component
sequences of the recombinant polynucleotide.
[0144] Depending on the particular screening protocol used for a
desired property, initial round(s) of screening can sometimes be
performed using bacterial cells due to high transfection
efficiencies and ease of culture. However, especially for testing
of immunogenic activity, test animals are used for library
expression and screening. Similarly other types of screening which
are not amenable to screening in bacterial or simple eukaryotic
library cells, are performed in cells selected for use in an
environment close to that of their intended use. Final rounds of
screening can be performed in cells or organisms that are as close
as possible to the precise cell type or organism of intended
use.
[0145] If further improvement in a property is desired, at least
one, and usually a collection, of recombinant segments surviving a
first round of screening/selection are subject to a further round
of recombination. These recombinant segments can be recombined with
each other or with exogenous segments representing the original
substrates or further variants thereof. Again, recombination can
proceed in vitro or in vivo. If the previous screening step
identifies desired recombinant segments as components of cells, the
components can be subjected to further recombination in vivo, or
can be subjected to further recombination in vitro, or can be
isolated before performing a round of in vitro recombination.
Conversely, if the previous screening step identifies desired
recombinant segments in naked form or as components of viruses,
these segments can be introduced into cells to perform a round of
in vivo recombination. The second round of recombination,
irrespective how performed, generates further recombinant segments
which encompass additional diversity than is present in recombinant
segments resulting from previous rounds.
[0146] The second round of recombination can be followed by a
further round of screening/selection according to the principles
discussed above for the first round. The stringency of
screening/selection can be increased between rounds. Also, the
nature of the screen and the property being screened for can vary
between rounds if improvement in more than one property is desired
or if acquiring more than one new property is desired. Additional
rounds of recombination and screening can then be performed until
the recombinant segments have sufficiently evolved to acquire the
desired new or improved property or function.
[0147] The practice of this invention involves the construction of
recombinant nucleic acids and the expression of genes in
transfected 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 such as expression vectors are well-known
to persons of skill. General texts which describe molecular
biological techniques useful herein, including mutagenesis, include
Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods
in Enzymology volume 152 Academic Press, Inc., San Diego, Calif.
(Berger); Sambrook et al., Molecular Cloning--A Laboratory Manual
(2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y., 1989 ("Sambrook") and Current Protocols in Molecular
Biology, F. M. Ausubel et al., eds., Current Protocols, a joint
venture between Greene Publishing Associates, Inc. and John Wiley
& Sons, Inc., (supplemented through 1998) ("Ausubel")).
Examples of techniques sufficient to direct persons of skill
through in vitro amplification methods, including the polymerase
chain reaction (PCR) the ligase chain reaction (LCR), Q-replicase
amplification and other RNA polymerase mediated techniques (e.g.,
NASBA) are found in Berger, Sambrook, and Ausubel, as well as
Mullis et al. (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide
to Methods and Applications (Innis et al. eds) Academic Press Inc.
San Diego, Calif. (1990) (Innis); Amnheim & Levinson (Oct. 1,
1990) C&EN 36-47; The Journal OF NIH Research (1991)3, 81-94;
(Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli
et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al.
(1989) J. Clin. Chem 35, 1826; Landegren et al. (1988) Science 241,
1077-1080; Van Brunt (1990) Biotechnology 8, 291-294; Wu and
Wallace (1989) Gene 4, 560; Barringer et al. (1990) Gene 89, 117,
and Sooknanan and Malek (1995) Biotechnology 13: 563-564. Improved
methods of cloning in vitro amplified nucleic acids are described
in Wallace et al., U.S. Pat. No. 5,426,039. Improved methods of
amplifying large nucleic acids by PCR are summarized in Cheng et
al. (1994) Nature 369: 684-685 and the references therein, in which
PCR amplicons of up to 40 kb are generated. One of skill will
appreciate that essentially any RNA can be converted into a double
stranded DNA suitable for restriction digestion, PCR expansion and
sequencing using reverse transcriptase and a polymerase. See,
Ausubel, Sambrook and Berger, all supra.
[0148] Oligonucleotides for use as probes, e.g., in in vitro
amplification methods, for use as gene probes, or as shuffling
targets (e.g., synthetic genes or gene segments) are typically
synthesized chemically according to the solid phase phosphoramidite
triester method described by Beaucage and Caruthers (1981)
Tetrahedron Letts., 22(20):1859-1862, e.g., using an automated
synthesizer, as described in Needham-VanDevanter et al. (1984)
Nucleic Acids Res., 12:6159-6168. Oligonucleotides can also be
custom made and ordered from a variety of commercial sources known
to persons of skill.
[0149] Indeed, essentially any nucleic acid with a known sequence
can be custom ordered from any of a variety of commercial sources,
such as The Midland Certified Reagent Company (mcrc@oligos.com),
The Great American Gene Company (http://www.genco.com), ExpressGen
Inc. (www.expressgen.com), Operon Technoloigies Inc. (Alameda,
Calif.) and many others. Similarly, peptides and antibodies can be
custom ordered from any of a variety of sources, such as
PeptidoGenic (pkim@ccnet.com), HTI Bio-products, Inc.
(http://www.htibio.com), BMA Biomedicals Ltd (U.K.), Bio-Synthesis,
Inc., and many others.
[0150] 1. Purification and In Vitro Analysis of Recombinant Nucleic
Acids and Polypeptides
[0151] Once DNA shuffling has been performed, the resulting library
of recombinant polynucleotides can be subjected to purification and
preliminary analysis in vitro, in order to identify the most
promising candidate recombinant nucleic acids. Advantageously, the
assays can be practiced in a high-throughput format. For example,
to purify individual shuffled recombinant antigens, clones can
robotically picked into 96-well formats, grown, and, if desired,
frozen for storage.
[0152] Whole cell lysates (V-antigen), periplasmic extracts, or
culture supernatants (toxins) can be assayed directly by ELISA as
described below, but high throughput purification is sometimes also
needed. Affinity chromatography using immobilized antibodies or
incorporation of a small nonimmunogenic affinity tag such as a
hexahistidine peptide with immobilized metal affinity
chromatography will allow rapid protein purification. High
binding-capacity reagents with 96-well filter bottom plates provide
a high throughput purification process. The scale of culture and
purification will depend on protein yield, but initial studies will
require less than 50 micrograms of protein. Antigens showing
improved properties can be purified in larger scale by FPLC for
re-assay and animal challenge studies.
[0153] In some embodiments, the shuffled antigen-encoding
polynucleotides are assayed as genetic vaccines. Genetic vaccine
vectors containing the shuffled antigen sequences can be prepared
using robotic colony picking and subsequent robotic plasmid
purification. Robotic plasmid purification protocols are available
that allow purification of 600-800 plasmids per day. The quantity
and purity of the DNA can also be analyzed in 96-well plates, for
example. In a presently preferred embodiment, the amount of DNA in
each sample is robotically normalized, which can significantly
reduce the variation between different batches of vectors.
[0154] Once the proteins and/or nucleic acids are picked and
purified as desired, they can be subjected to any of a number of in
vitro analysis methods. Such screenings include, for example, phage
display, flow cytometry, and ELISA assays to identify antigens that
are efficiently expressed and have multiple epitopes and a proper
folding pattern. In the case of bacterial toxins, the libraries may
also be screened for reduced toxicity in mammalian cells.
[0155] As one example, to identify recombinant antigens that are
cross-reactive, one can use a panel of monoclonal antibodies for
screening. A humoral immune response generally targets multiple
regions of antigenic proteins. Accordingly, monoclonal antibodies
can be raised against various regions of immunogenic proteins
(Alving et al. (1995) Immunol. Rev. 145: 5). In addition, there are
several examples of monoclonal antibodies that only recognize one
strain of a given pathogen, and by definition, different serotypes
of pathogens are recognized by different sets of antibodies. For
example, a panel of monoclonal antibodies have been raised against
VEE envelope proteins, thus providing a means to recognize
different subtypes of the virus (Roehrig and Bolin (1997) J. Clin.
Microbiol. 35: 1887). Such antibodies, combined with phage display
and ELISA screening, can be used to enrich recombinant antigens
that have epitopes from multiple pathogen strains. Flow cytometry
based cell sorting will further allow for the selection of variants
that are most efficiently expressed.
[0156] Phage display provides a powerful method for selecting
proteins of interest from large libraries (Bass et al. (1990)
Proteins: Struct. Funct. Genet. 8: 309; Lowman and Wells (1991)
Methods: A Companion to Methods Enz. 3(3);205-216. Lowman and Wells
(1993) J. Mol. Biol. 234;564-578). Some recent reviews on the phage
display technique include, for example, McGregor (1996) Mol
Biotechnol. 6(2):155-62; Dunn (1996) Curr. Opin. Biotechnol.
7(5):547-53; Hill et al. (1996) Mol Microbiol 20(4):685-92; Phage
Display of Peptides and Proteins: A Laboratory Manual. B K. Kay, J.
Winter, J, McCafferty eds., Academic Press 1996; O'Neil et al.
(1995) Curr. Opin. Struct. Biol. 5(4):443-9; Phizicky et al. (1995)
Microbiol Rev. 59(1):94-123; Clackson et al. (1994) Trends
Biotechnol. 12(5):173-84; Felici et al. (1995) Biotechnol. Annu.
Rev. 1:149-83; Burton (1995) Immunotechnology 1(2):87-94.) See,
also, Cwirla et al., Proc. Natl. Acad. Sci. USA 87: 6378-6382
(1990); Devlin et al., Science 249: 404-406 (1990), Scott &
Smith, Science 249: 386-388 (1990); Ladner et al., U.S. Pat. No.
5,571,698. Each phage particle displays a unique variant protein on
its surface and packages the gene encoding that particular variant.
The shuffled genes for the antigens are fused to a protein that is
expressed on the phage surface, e.g., gene III of phage M13, and
cloned into phagemid vectors. In a presently preferred embodiment,
a suppressible stop codon (e.g., an amber stop codon) separates the
genes so that in a suppressing strain of E. coli, the antigen-gillp
fusion is produced and becomes incorporated into phage particles
upon infection with M13 helper phage. The same vector can direct
production of the unfused antigen alone in a nonsuppressing E. coli
for protein purification.
[0157] The genetic packages most frequently used for display
libraries are bacteriophage, particularly filamentous phage, and
especially phage M13, Fd and Fl. Most work has involved inserting
libraries encoding polypeptides to be displayed into either gIII or
gVIII of these phage forming a fusion protein. See, e.g., Dower, WO
91/19818; Devlin, WO 91/18989; MacCafferty, WO 92/01047 (gene III);
Huse, WO 92/06204; Kang, WO 92/18619 (gene VIII). Such a fusion
protein comprises a signal sequence, usually but not necessarily,
from the phage coat protein, a polypeptide to be displayed and
either the gene III or gene VIII protein or a fragment thereof.
Exogenous coding sequences are often inserted at or near the
N-terminus of gene III or gene VIII although other insertion sites
are possible.
[0158] Eukaryotic viruses can be used to display polypeptides in an
analogous manner. For example, display of human heregulin fused to
gp70 of Moloney murine leukemia virus has been reported by Han et
al., Proc. Natl. Acad. Sci. USA 92: 9747-9751 (1995). Spores can
also be used as replicable genetic packages. In this case,
polypeptides are displayed from the outer surface of the spore. For
example, spores from B. subtilis have been reported to be suitable.
Sequences of coat proteins of these spores are provided by Donovan
et al., J. Mol. Biol. 196, 1-10 (1987). Cells can also be used as
replicable genetic packages. Polypeptides to be displayed are
inserted into a gene encoding a cell protein that is expressed on
the cells surface. Bacterial cells including Salmonella
typhimurium, Bacillus subtilis, Pseudomonas aeruaginosa, Vibrio
cholerae, Klebsiella pneumonia, Neisseria gonorrhoeae, Neisseria
meningitidis, Bacteroides nodosus, Moraxella bovis, and especially
Escherichia coli are preferred. Details of outer surface proteins
are discussed by Ladner et al., U.S. Pat. No. 5,571,698 and
references cited therein. For example, the lamB protein of E. coli
is suitable.
[0159] A basic concept of display methods that use phage or other
replicable genetic package is the establishment of a physical
association between DNA encoding a polypeptide to be screened and
the polypeptide. This physical association is provided by the
replicable genetic package, which displays a polypeptide as part of
a capsid enclosing the genome of the phage or other package,
wherein the polypeptide is encoded by the genome. The establishment
of a physical association between polypeptides and their genetic
material allows simultaneous mass screening of very large numbers
of phage bearing different polypeptides. Phage displaying a
polypeptide with affinity to a target, e.g., a receptor, bind to
the target and these phage are enriched by affinity screening
to-the target. The identity of polypeptides displayed from these
phage can be determined from their respective genomes. Using these
methods a polypeptide identified as having a binding affinity for a
desired target can then be synthesized in bulk by conventional
means, or the polynucleotide that encodes the peptide or
polypeptide can be used as part of a genetic vaccine.
[0160] Variants with specific binding properties, in this case
binding to family-specific antibodies, are easily enriched by
panning with immobilized antibodies. Antibodies specific for a
single family are used in each round of panning to rapidly select
variants that have multiple epitopes from the antigen families. For
example, A-family specific antibodies can be used to select those
shuffled clones that display A-specific epitopes in the first round
of panning. A second round of panning with B-specific antibodies
will select from the "A" clones those that display both A- and
B-specific epitopes. A third round of panning with C-specific
antibodies will select for variants with A, B, and C epitopes. A
continual selection exists during this process for clones that
express well in E. coli and that are stable throughout the
selection. Improvements in factors such as transcription,
translation, secretion, folding and stability are often observed
and will enhance the utility of selected clones for use in vaccine
production.
[0161] Phage ELISA methods can be used to rapidly characterize
individual variants. These assays provide a rapid method for
quantitation of variants without requiring purification of each
protein. Individual clones are arrayed into 96-well plates, grown,
and frozen for storage. Cells in duplicate plates are infected with
helper phage, grown overnight and pelleted by centrifugation. The
supernatants containing phage displaying particular variants are
incubated with immobilized antibodies and bound clones are detected
by anti-M13 antibody conjugates. Titration series of phage
particles, immobilized antigen, and/or soluble antigen competition
binding studies are all highly effective means to quantitate
protein binding. Variant antigens displaying multiple epitopes will
be further studied in appropriate animal challenge models.
[0162] Several groups have reported an in vitro ribosome display
system for the screening and selection of mutant proteins with
desired properties from large libraries. This technique can be used
similarly to phage display to select or enrich for variant antigens
with improved properties such as broad cross reactivity to
antibodies and improved folding (see, e.g., Hanes et al. (1997)
Proc. Nat'l. Acad. Sci. USA 94(10):4937-42; Mattheakis et al.
(1994) Proc. Nat'l. Acad. Sci. USA 91(19):9022-6; He et al. (1997)
Nucl. Acids Res. 25(24):5132-4; Nemoto et al. (1997) FEBS Lett.
414(2):405-8).
[0163] Other display methods exist to screen antigens for improved
properties such as increased expression levels, broad cross
reactivity, enhanced folding and stability. These include, but are
not limited to display of proteins on intact E. coli or other cells
(e.g., Francisco et al. (1993) Proc. Nat'l. Acad. Sci. USA 90:
1044-10448; Lu et al. (1995) Bio/Technology 13: 366-372). Fusions
of shuffled antigens to DNA-binding proteins can link the antigen
protein to its gene in an expression vector (Schatz et al. (1996)
Methods Enzymol. 267: 171-91; Gates et al. (1996) J. Mol. Biol.
255: 373-86.)
[0164] The various display methods and ELISA assays can be used to
screen for shuffled antigens with improved properties such as
presentation of multiple epitopes, improved immunogenicity,
increased expression levels, increased folding rates and
efficiency, increased stability to factors such as temperature,
buffers, solvents, improved purification properties, etc. Selection
of shuffled antigens with improved expression, folding, stability
and purification profile under a variety of chromatographic
conditions can be very important improvements to incorporate for
the vaccine manufacturing process.
[0165] To identify recombinant antigenic polypeptides that exhibit
improved expression in a host cell, flow cytometry is a useful
technique. Flow cytometry provides a method to efficiently analyze
the functional properties of millions of individual cells. One can
analyze the expression levels of several genes simultaneously, and
flow cytometry-based cell sorting allows for the selection of cells
that display properly expressed antigen variants on the cell
surface or in the cytoplasm. Very large numbers (>10.sup.7) of
cells can be evaluated in a single vial experiment, and the pool of
the best individual sequences can be recovered from the sorted
cells. These methods are particularly useful in the case of, for
example, Hantaan virus glycoproteins, which are generally very
poorly expressed in mammalian cells. This approach provides a
general solution to improve expression levels of pathogen antigens
in mammalian cells, a phenomenon that is critical for the function
of genetic vaccines.
[0166] To use flow cytometry to analyze polypeptides that are not
expressed on the cell surface, one can engineer the recombinant
polynucleotides in the library such that the polynucleotide is
expressed as a fusion protein that has a region of amino acids
which is targeted to the cell membrane. For example, the region can
encode a hydrophobic stretch of C-terminal amino acids which
signals the attachment of a phosphoinositol-glycan (PIG) terminus
on the expressed protein and directs the protein to be expressed on
the surface of the transfected cell (Whitehom et al. (1995)
Biotechnology (N Y) 13:1215-9). With an antigen that is naturally a
soluble protein, this method will likely not affect the three
dimensional folding of the protein in this engineered fusion with a
new C-terminus. With an antigen that is naturally a transmembrane
protein (e.g., a surface membrane protein on pathogenic viruses,
bacteria, protozoa or tumor cells) there are at least two
possibilities. First, the extracellular domain can be engineered to
be in fusion with the C-terminal sequence for signaling
PIG-linkage. Second, the protein can be expressed in toto relying
on the signalling of the host cell to direct it efficiently to the
cell surface. In a minority of cases, the antigen for expression
will have an endogenous PIG terminal linkage (e.g., some antigens
of pathogenic protozoa).
[0167] Those cells expressing the antigen can be identified with a
fluorescent monoclonal antibody specific for the C-terminal
sequence on PIG-linked forms of the surface antigen. FACS analysis
allows quantitative assessment of the level of expression of the
correct form of the antigen on the cell population. Cells
expressing the maximal level of antigen are sorted and standard
molecular biology methods are used to recover the plasmid DNA
vaccine vector that conferred this reactivity. An alternative
procedure that allows purification of all those cells expressing
the antigen (and that may be useful prior to loading onto a cell
sorter since antigen expressing cells may be a very small minority
population), is to rosette or pan-purify the cells expressing
surface antigen. Rosettes can be formed between antigen expressing
cells and erythrocytes bearing covalently coupled antibody to the
relevant antigen. These are readily purified by unit gravity
sedimentation. Panning of the cell population over petri dishes
bearing immobilized monoclonal antibody specific for the relevant
antigen can also be used to remove unwanted cells.
[0168] In the high throughput assays of the invention, it is
possible to screen up to several thousand different shuffled
variants in a single day. For example, each well of a microtiter
plate can be used to run a separate assay, or, if concentration or
incubation time effects are to be observed, every 5-10 wells can
test a single variant. Thus, a single standard microtiter plate can
assay about 100 (e.g., 96) reactions. If 1536 well plates are used,
then a single plate can easily assay from about 100 to about 1500
different reactions. It is possible to assay several different
plates per day; assay screens for up to about 6,000-20,000
different assays (i.e., involving different nucleic acids, encoded
proteins, concentrations, etc.) is possible using the integrated
systems of the invention. More recently, microfluidic approaches to
reagent manipulation have been developed, e.g., by Caliper
Technologies (Palo Alto, Calif.).
[0169] In one aspect, library members, e.g., cells, viral plaques,
or the like, are separated on solid media to produce individual
colonies (or plaques). Using an automated colony picker (e.g., the
Q-bot, Genetix, U.K.), colonies or plaques are identified, picked,
and up to 10,000 different mutants inoculated into 96 well
microtiter dishes, optionally containing glass balls in the wells
to prevent aggregation. The Q-bot does not pick an entire colony
but rather inserts a pin through the center of the colony and exits
with a small sampling of cells (or viruses in plaque applications).
The time the pin is in the colony, the number of dips to inoculate
the culture medium, and the time the pin is in that medium each
effect inoculum size, and each can be controlled and optimized. The
uniform process of the Q-bot decreases human handling error and
increases the rate of establishing cultures (roughly 10,000/4
hours). These cultures are then shaken in a temperature and
humidity controlled incubator. The glass balls in the microtiter
plates act to promote uniform aeration of cells dispersal of cells,
or the like, similar to the blades of a fermentor. Clones from
cultures of interest can be cloned by limiting dilution. Plaques or
cells constituting libraries can also be screened directly for
production of proteins, either by detecting hybridization, protein
activity, protein binding to antibodies, or the like.
[0170] The ability to detect a subtle increase in the performance
of a shuffled library member over that of a parent strain relies on
the sensitivity of the assay. The chance of finding the organisms
having an improvement in ability to induce an immune response is
increased by the number of individual mutants that can be screened
by the assay. To increase the chances of identifying a pool of
sufficient size, a prescreen that increases the number of mutants
processed by 10-fold can be used. The goal of the prescreen will be
to quickly identify mutants having equal or better product titers
than the parent strain(s) and to move only these mutants forward to
liquid cell culture for subsequent analysis.
[0171] A number of well known robotic systems have also been
developed for solution phase chemistries useful in assay systems.
These systems include automated workstations like the automated
synthesis apparatus developed by Takeda Chemical Industries, LTD.
(Osaka, Japan) and many robotic systems utilizing robotic arms
(Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca,
Hewlett-Packard, Palo Alto, Calif) which mimic the manual synthetic
operations performed by a scientist. Any of the above devices are
suitable for use with the present invention, e.g., for
high-throughput screening of molecules encoded by codon-altered
nucleic acids. The nature and implementation of modifications to
these devices (if any) so that they can operate as discussed herein
with reference to the integrated system will be apparent to persons
skilled in the relevant art.
[0172] High throughput screening systems are commercially available
(see, e.g., Zymark Corp., Hopkinton, Mass.; Air Technical
Industries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton,
Calif.; Precision Systems, Inc., Natick, Mass., etc.). These
systems typically automate entire procedures including all sample
and reagent pipetting, liquid dispensing, timed incubations, and
final readings of the microplate in detector(s) appropriate for the
assay. These configurable systems provide high throughput and rapid
start up as well as a high degree of flexibility and
customization.
[0173] The manufacturers of such systems provide detailed protocols
the various high throughput. Thus, for example, Zymark Corp.
provides technical bulletins describing screening systems for
detecting the modulation of gene transcription, ligand binding, and
the like. Microfluidic approaches to reagent manipulation have also
been developed, e.g., by Caliper Technologies (Palo Alto,
Calif.).
[0174] Optical images viewed (and, optionally, recorded) by a
camera or other recording device (e.g., a photodiode and data
storage device) are optionally further processed in any of the
embodiments herein, e.g., by digitizing the image and/or storing
and analyzing the image on a computer. As noted above, in some
applications, the signals resulting from assays are florescent,
making optical detection approaches appropriate in these instances.
A variety of commercially available peripheral equipment and
software is available for digitizing, storing and analyzing a
digitized video or digitized optical image, e.g., using PC (Intel
x86 or Pentium chip-compatible DOS, OS2 WINDOWS, WINDOWS NT or
WINDOWS95 based machines), MACINTOSH, or UNIX based (e.g., SUN work
station) computers.
[0175] One conventional system carries light from the assay device
to a cooled charge-coupled device (CCD) camera, in common use in
the art. A CCD camera includes an array of picture elements
(pixels). The light from the specimen is imaged on the CCD.
Particular pixels corresponding to regions of the specimen (e.g.,
individual hybridization sites on an array of biological polymers)
are sampled to obtain light intensity readings for each position.
Multiple pixels are processed in parallel to increase speed. The
apparatus and methods of the invention are easily used for viewing
any sample, e.g., by fluorescent or dark field microscopic
techniques.
[0176] Integrated systems for analysis in the present invention
typically include a digital computer with high-throughput liquid
control software, image analysis software, data interpretation
software, a robotic liquid control armature for transferring
solutions from a source to a destination operably linked to the
digital computer, an input device (e.g., a computer keyboard) for
entering data to the digital computer to control high throughput
liquid transfer by the robotic liquid control armature and,
optionally, an image scanner for digitizing label signals from
labeled assay component. The image scanner interfaces with the
image analysis software to provide a measurement of optical
intensity. Typically, the intensity measurement is interpreted by
the data interpretation software to show whether the optimized
recombinant antigenic polypeptide products are produced.
[0177] 2. Antigen Library Immunization
[0178] In a presently preferred embodiment, antigen library
immunization (ALI) is used to identify optimized recombinant
antigens that have improved immunogenicity. ALI involves
introduction of the library of recombinant antigen-encoding nucleic
acids, or the recombinant antigens encoded by the shuffled nucleic
acids, into a test animal. The animals are then subjected to in
vivo challenge using live pathogens. Neutralizing antibodies and
cross-protective immune responses are studied after immunization
with the entire libraries, pools and/or individual antigen
variants.
[0179] Methods of immunizing test animals are well known to those
of skill in the art. In presently preferred embodiments, test
animals are immunized twice or three times at two week intervals.
One week after the last immunization, the animals are challenged
with live pathogens (or mixtures of pathogens), and the survival
and symptoms of the animals is followed. Immunizations using test
animal challenge are described in, for example, Roggenkamp et al.
(1997) Infect. Immun. 65: 446; Woody et al. (1997) Vaccine 2: 133;
Agren et al. (1997) J. Immunol. 158: 3936; Konishi et al. (1992)
Virology 190: 454; Kinney et al. (1988) J. Virol. 62: 4697;
Iacono-Connors et al. (1996) Virus Res. 43: 125; Kochel et al.
(1997) Vaccine 15: 547; and Chu et al. (1995) J. Virol. 69:
6417.
[0180] The immunizations can be performed by injecting either the
recombinant polynucleotides themselves, i.e., as a genetic vaccine,
or by immunizing the animals with polypeptides encoded by the
recombinant polynucleotides. Bacterial antigens are typically
screened primarily as recombinant proteins, whereas viral antigens
are preferably analyzed using genetic vaccinations.
[0181] To dramatically reduce the number of experiments required to
identify individual antigens having improved immunogenic
properties, one can use pooling and deconvolution, as diagrammed in
FIG. 6. Pools of recombinant nucleic acids, or polypeptides encoded
by the recombinant nucleic acids, are used to immunize test
animals. Those pools that result in protection against pathogen
challenge are then subdivided and subjected to additional analysis.
The high throughput in vitro approaches described above can be used
to identify the best candidate sequences for the in vivo
studies.
[0182] The challenge models that can be used to screen for
protective antigens include pathogen and toxin models, such as
Yersinia bacteria, bacterial toxins (such as Staphylococcal and
Streptococcal enterotoxins, E. coli/V. cholerae enterotoxins),
Venezuelan equine encephalitis virus (VEE), Flaviviruses (Japanese
encephalitis virus, Tick-borne encephalitis virus, Dengue virus),
Hantaan virus, Herpes simplex, influenza virus (e.g., Influenza A
virus), Vesicular Stomatitis Virus, Pseudomonas aeruginosa,
Salmonella typhimurium, Escherichia coli, Klebsiella pneumoniae,
Toxoplasma gondii, Plasmodium yoedii, Herpes simplex, influenza
virus (e.g., Influenza A virus), and Vesicular Stomatitis Virus.
However, the test animals can also be challenged with tumor cells
to enable screening of antigens that efficiently protect against
malignancies. Individual shuffled antigens or pools of antigens are
introduced into the animals intradermally, intramuscularly,
intravenously, intratracheally, anally, vaginally, orally, or
intraperitoneally and antigens that can prevent the disease are
chosen, when desired, for further rounds of shuffling and
selection. Eventually, the most potent antigens, based on in vivo
data in test animals and comparative in vitro studies in animals
and man, are chosen for human trials, and their capacity to prevent
and treat human diseases is investigated.
[0183] In some embodiments, antigen library immunization and
pooling of individual clones is used to immunize against a pathogen
strain that was not included in the sequences that were used to
generate the library. The level of crossprotection provided by
different strains of a given pathogen can significantly. However,
homologous titer is always higher than heterologous titer. Pooling
and deconvolution is especially efficient in models where minimal
protection is provided by the wild-type antigens used as starting
material for shuffling (for example minimal protection by antigens
A and B against strain C in FIG. 3B). This approach can be taken,
for example, when evolving the V-antigen of Yersinae or Hantaan
virus glycoproteins.
[0184] In some embodiments, the desired screening involves analysis
of the immune response based on immunological assays known to those
skilled in the art. Typically, the test animals are first immunized
and blood or tissue samples are collected for example one to two
weeks after the last immunization. These studies enable one to one
can measure immune parameters that correlate to protective
immunity, such as induction of specific antibodies (particularly
IgG) and induction of specific T lymphocyte responses, in addition
to determining whether an antigen or pools of antigens provides
protective immunity. Spleen cells or peripheral blood mononuclear
cells can be isolated from immunized test animals and measured for
the presence of antigen-specific T cells and induction of cytokine
synthesis. ELISA, ELISPOT and cytoplasmic cytokine staining,
combined with flow cytometry, can provide such information on a
single-cell level.
[0185] Common immunological tests that can be used to identify the
efficacy of immunization include antibody measurements,
neutralization assays and analysis of activation levels or
frequencies of antigen presenting cells or lymphocytes that are
specific for the antigen or pathogen. The test animals that can be
used in such studies include, but are not limited to, mice, rats,
guinea pigs, hamsters, rabbits, cats, dogs, pigs and monkeys.
Monkey is a particularly useful test animal because the MHC
molecules of monkeys and humans are very similar.
[0186] Virus neutralization assays are useful for 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 detection of antibodies in the sera of immunized
animal and analysis of these antibodies for their capacity 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 described by Dolin R (J. Infect. Dis. 1995, 172:1175-8,).
Virus neutralization assays provide means to screen for antigens
that also provide protective immunity.
[0187] In some embodiments, shuffled antigens are screened for
their capacity to induce T cell activation in vivo. More
specifically, peripheral blood mononuclear cells or spleen cells
from injected mice can be isolated and the capacity of cytotoxic T
lymphocytes to lyse infected, autologous target cells is studied.
The spleen cells can be reactivated with the specific antigen in
vitro. In addition, T helper cell activation and differentiation is
analyzed by measuring cell proliferation or production of T.sub.H1
(IL-2 and IFN-.gamma.) and T.sub.H2 (IL-4 and IL-5) cytokines by
ELISA and directly in CD4.sup.+ T cells by cytoplasmic cytokine
staining and flow cytometry. Based on the cytokine production
profile, one can also screen for alterations in the capacity of the
antigens to direct T.sub.H1/T.sub.H2 differentiation (as evidenced,
for example, by changes in ratios of IL-4/IFN-.gamma., IL-4/IL-2,
IL-5/IFN-.gamma., IL-5/IL-2, IL-13/IFN-.gamma., IL-13/IL-2). The
analysis of the T cell activation induced by the antigen variants
is a very useful screening method, because potent activation of
specific T cells in vivo correlates to induction of protective
immunity.
[0188] The frequency of antigen-specific CD8.sup.+ T cells in vivo
can also be directly analyzed using tetramers of MHC class I
molecules expressing specific peptides derived from the
corresponding pathogen antigens (Ogg and McMichael, Curr. Opin.
Immunol. 1998, 10:393-6; Altman et al., Science 1996, 274:94-6).
The binding of the tetramers can be detected using flow cytometry,
and will provide information about the efficacy of the shuffled
antigens to induce activation of specific T cells. For example,
flow cytometry and tetramer stainings provide an efficient method
of identifying T cells that are specific to a given antigen or
peptide. Another method involves panning using plates coated with
tetramers with the specific peptides. This method allows large
numbers of cells to be handled in a short time, but the method only
selects for highest expression levels. The higher the frequency of
antigen-specific T cells in vivo is, the more efficient the
immunization has been, enabling identification of the antigen
variants that have the most potent capacity to induce protective
immune responses. These studies are particularly useful when
conducted in monkeys, or other primates, because the MHC class I
molecules of humans mimic those of other primates more closely than
those of mice.
[0189] Measurement of the activation of antigen presenting cells
(APC) in response to immunization by antigen variants is another
useful screening method. Induction of APC activation can be
detected based on changes in surface expression levels of
activation antigens, such as B7-1 (CD80), B7-2 (CD86), MHC class I
and II, CD14, CD23, and Fc receptors, and the like.
[0190] Shuffled cancer antigens that induce cytotoxic T cells that
have the capacity to kill cancer cells can be identified by
measuring the capacity of T cells derived from immunized animals to
kill cancer cells in vitro. Typically the cancer cells are first
labeled with radioactive isotopes and the release of radioactivity
is an indication of tumor cell killing after incubation in the
presence of T cells from immunized animals. Such cytotoxicity
assays are known in the art.
[0191] An indication of the efficacy of an antigen to activate T
cells specific for, for example, cancer antigens, allergens or
autoantigens, is also the degree of skin inflammation when the
antigen is injected into the skin of a patient or test animal.
Strong inflammation is correlated with strong activation of
antigen-specific T cells. Improved activation of tumor-specific T
cells may lead to enhanced killing of the tumors. In case of
autoantigens, one can add immunomodulators that skew the responses
towards T.sub.H2, whereas in the case of allergens a T.sub.H1
response is desired. Skin biopsies can be taken, enabling detailed
studies of the type of immune response that occurs at the sites of
each injection (in mice and monkeys large numbers of
injections/antigens can be analyzed). Such studies include
detection of changes in expression of cytokines, chemokines,
accessory molecules, and the like, by cells upon injection of the
antigen into the skin.
[0192] To screen for antigens that have optimal capacity to
activate antigen-specific T cells, peripheral blood mononuclear
cells from previously infected or immunized humans individuals can
be used. This is a particularly useful method, because the MHC
molecules that will present the antigenic peptides are human MHC
molecules. 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. Because these
individuals have increased frequencies of pathogen-specific T cells
in circulation, antigens expressed in PBMCs or purified APCs of
these individuals will induce proliferation and cytokine production
by antigen-specific CD4.sup.+ and CD8.sup.+ T cells. Thus, antigens
that simultaneously harbor epitopes from several antigens can be
recognized by their capacity to stimulate T cells from various
patients infected or immunized with different pathogen antigens,
cancer antigens, autoantigens or allergens. One buffy coat derived
from a blood donor contains lymphocytes from 0.5 liters of blood,
and up to 10.sup.4 PBMC can be obtained, enabling very large
screening experiments using T cells from one donor.
[0193] When healthy vaccinated individuals (lab volunteers) are
studied, one can make EBV-transformed B cell lines from these
individuals. These cell lines can be used as antigen presenting
cells in subsequent experiments using blood from the same donor;
this reduces interassay and donor-to-donor variation. In addition,
one can make antigen-specific T cell clones, after which antigen
variants are introduced to EBV transformed B cells. The efficiency
with which the transformed B cells induce proliferation of the
specific T cell clones is then studied. When working with specific
T cell clones, the proliferation and cytokine synthesis responses
are significantly higher than when using total PBMCs, because the
frequency of antigen-specific T cells among PBMC is very low.
[0194] CTL epitopes can be presented by most cells types since the
class I major histocompatibility complex (MHC) surface
glycoproteins are widely expressed. Therefore, transfection of
cells in culture by libraries of shuffled antigen sequences in
appropriate expression vectors can lead to class I epitope
presentation. If specific CTLs directed to a given epitope have
been isolated from an individual, then the co-culture of the
transfected presenting cells and the CTLs can lead to release by
the CTLs of cytokines, such as IL-2, IFN-.gamma., or TNF, if the
epitope is presented. Higher amounts of released TNF will
correspond to more efficient processing and presentation of the
class I epitope from the shuffled, evolved sequence. Shuffled
antigens that induce cytotoxic T cells that have the capacity to
kill infected cells can also be identified by measuring the
capacity of T cells derived from immunized animals to kill infected
cells in vitro. Typically the target cells are first labeled with
radioactive isotopes and the release of radioactivity is an
indication of target cell killing after incubation in the presence
of T cells from immunized animals. Such cytotoxicity assays are
known in the art.
[0195] A second method for identifying optimized CTL epitopes does
not require the isolation of CTLs reacting with the epitope. In
this approach, cells expressing class I MHC surface glycoproteins
are transfected with the library of evolved sequences as above.
After suitable incubation to allow for processing and presentation,
a detergent soluble extract is prepared from each cell culture and
after a partial purification of the MHC-epitope complex (perhaps
optional) the products are submitted to mass spectrometry
(Henderson et al. (1993) Proc. Nat'l. Acad. Sci. USA 90:
10275-10279). Since the sequence is known of the epitope whose
presentation to be increased, one can calibrate the mass
spectrogram to identify this peptide. In addition, a cellular
protein can be used for internal calibration to obtain a
quantitative result; the cellular protein used for internal
calibration could be the MHC molecule itself. Thus one can measure
the amount of peptide epitope bound as a proportion of the MHC
molecules.
[0196] Use of Recombinant Multivalent Antigens
[0197] The multivalent antigens of the invention are useful for
treating and/or preventing the various diseases and conditions with
which the respective antigens are associated. For example, the
multivalent antigens can be expressed in a suitable host cell and
are administered in polypeptide form. Suitable formulations and
dosage regimes for vaccine delivery are well known to those of
skill in the art.
[0198] In presently preferred embodiments, the optimized
recombinant polynucleotides that encode improved allergens are used
in conjunction with a genetic vaccine vector. The choice of vector
and components can also be optimized for the particular purpose of
treating allergy, for example, or other conditions. For example,
the polynucleotide that encodes the recombinant antigenic
polypeptide can be placed under the control of a promoter, e.g., a
high activity or tissue-specific promoter. The promoter used to
express the antigenic polypeptide can itself be optimized using
recombination and selection methods analogous to those described
herein. The vector can contain immunostimulatory sequences such as
are described in copending, commonly assigned U.S. patent
application Ser. No. ______, entitled "Optimization of
Immunomodulatory Molecules," filed as TTC Attorney Docket No.
18097-030300US on Feb. 10, 1999. A vector engineered to direct a
T.sub.H1 response is preferred for many of the immune responses
mediated by the antigens described herein (see, e.g., copending,
commonly assigned U.S. patent application Ser. No. ______, entitled
"Genetic Vaccine Vector Engineering," filed on Feb. 10, 1999 as TTC
Attorney Docket No. 18097-030100US). It is sometimes advantageous
to employ a genetic vaccine that is targeted for a particular
target cell type (e.g., an antigen presenting cell or an antigen
processing cell); suitable targeting methods are described in
copending, commonly assigned U.S. patent application Ser. No.
______, entitled "Targeting of Genetic Vaccine Vectors," filed on
Feb. 10, 1999 as TTC Attorney Docket No. 18097-030200US.
[0199] Genetic vaccines that encode the multivalent antigens
described herein can be delivered to a mammal (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 by systemic administration (e.g.,
intravenous, intraperitoneal, intramuscular, subdermal,
intracranial, anal, vaginal, oral, buccal route or they can be
inhaled) or they can be administered by topical application.
Alternatively, vectors can be delivered to cells ex vivo, such as
cells explanted from an individual patient (e.g., lymphocytes, bone
marrow aspirates, tissue biopsy) or universal donor hematopoietic
stem cells, followed by reimplantation of the cells into a patient,
usually after selection for cells which have incorporated the
vector.
[0200] A large number of delivery methods are well known to those
of skill in the art. Such methods include, for example
liposome-based gene delivery (Debs and Zhu (1993) WO 93/24640;
Mannino and Gould-Fogerite (1988) BioTechniques 6(7): 682-691; Rose
U.S. Pat. No. 5,279,833; Brigham (1991) WO 91/06309; and Felgner et
al. (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7414), as well as
use of viral vectors (e.g., adenoviral (see, e.g., Bems 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 (Pt 3): 297-306 for review), papillomaviral,
retroviral (see, e.g., Buchscher et al. (1992) J. Virol. 66(5)
2731-2739; Johann et al. (1992) J. Virol. 66 (5):1635-1640 (1992);
Sommerfelt et al., (1990) Virol. 176:58-59; Wilson et al. (1989) J.
Virol. 63:2374-2378; Miller et al., J. Virol. 65:2220-2224 (1991);
Wong-Staal et al., PCT/US94/05700, and Rosenburg and Fauci (1993)
in Fundamental Immunology, Third Edition Paul (ed) Raven Press,
Ltd., New York and the references therein, and Yu et al., Gene
Therapy (1994) supra.), and adeno-associated viral vectors (see,
West et al. (1987) Virology 160:38-47; Carter et al. (1989) U.S.
Pat. No. 4,797,368; Carter et al. WO 93/24641 (1993); Kotin (1994)
Human Gene Therapy 5:793-801; Muzyczka (1994) J. Clin. Invst.
94:1351 and Samulski (supra) for an overview of AAV vectors; see
also, Lebkowski, U.S. Pat. No. 5,173,414; Tratschin et al. (1985)
Mol. Cell. Biol. 5(11):3251-3260; Tratschin, et al. (1984) Mol.
Cell. Biol., 4:2072-2081, Hermonat and Muzyczka (1984) Proc. Natl.
Acad. Sci. USA, 81:6466-6470; McLaughlin et al. (1988) and Samulski
et al. (1989) J. Virol., 63:03822-3828), and the like.
[0201] "Naked" DNA and/or RNA that comprises a genetic vaccine can
be introduced directly into a tissue, such as muscle. See, e.g.,
U.S. Pat. No. 5,580,859. Other methods such as "biolistic" or
particle-mediated transformation (see, e.g., Sanford et al., U.S.
Pat. No. 4,945,050; U.S. Pat. No. 5,036,006) are also suitable for
introduction of genetic vaccines into cells of a mammal according
to the invention. These methods are useful not only for in vivo
introduction of DNA into a mammal, but also for ex vivo
modification of cells for reintroduction into a mammal. As for
other methods of delivering genetic vaccines, if necessary, vaccine
administration is repeated in order to maintain the desired level
of immunomodulation.
[0202] Genetic vaccine vectors (e.g., adenoviruses, liposomes,
papillomaviruses, retroviruses, etc.) can be administered directly
to the mammal for transduction of cells in vivo. The genetic
vaccines obtained using the methods of the invention can be
formulated as pharmaceutical compositions for administration in any
suitable manner, including parenteral (e.g., subcutaneous,
intramuscular, intradermal, or intravenous), topical, oral, rectal,
intrathecal, buccal (e.g., sublingual), or local administration,
such as by aerosol or transdermally, for prophylactic and/or
therapeutic treatment. Pretreatment of skin, for example, by use of
hair-removing agents, may be useful in transdermal delivery.
Suitable methods of administering such packaged nucleic acids are
available and well known to those of skill in the art, and,
although more than one route can be used to administer a particular
composition, a particular route can often provide a more immediate
and more effective reaction than another route.
[0203] 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 pharmaceutical
compositions of the present invention. A variety of aqueous
carriers can be used, e.g., 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 such as pH adjusting and
buffering agents, toxicity adjusting agents and the like, for
example, sodium acetate, sodium chloride, potassium chloride,
calcium chloride, sodium lactate and the like. The concentration of
genetic vaccine vector in these formulations can vary widely, and
will be selected primarily based on fluid volumes, viscosities,
body weight and the like in accordance with the particular mode of
administration selected and the patient's needs.
[0204] Formulations suitable for oral administration can consist of
(a) liquid solutions, such as an effective amount of the packaged
nucleic acid suspended in diluents, such as water, saline or PEG
400; (b) capsules, sachets or tablets, each containing a
predetermined amount of the active ingredient, as liquids, solids,
granules or gelatin; (c) suspensions in an appropriate liquid; and
(d) suitable emulsions. Tablet forms can include one or more of
lactose, sucrose, mannitol, sorbitol, calcium phosphates, 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, buffering agents, moistening
agents, preservatives, flavoring agents, dyes, disintegrating
agents, and pharmaceutically compatible carriers. Lozenge forms can
comprise the active ingredient in a flavor, usually sucrose and
acacia or tragacanth, as well as pastilles comprising the active
ingredient in an inert base, such as gelatin and glycerin or
sucrose and acacia emulsions, gels, and the like containing, in
addition to the active ingredient, carriers known in the art. It is
recognized that the genetic vaccines, when administered orally,
must be protected from digestion. This is typically accomplished
either by complexing the vaccine vector with a composition to
render it resistant to acidic and enzymatic hydrolysis or by
packaging the vector in an appropriately resistant carrier such as
a liposome. Means of protecting vectors from digestion are well
known in the art. The pharmaceutical compositions can be
encapsulated, e.g., in liposomes, or in a formulation that provides
for slow release of the active ingredient.
[0205] The packaged nucleic acids, alone or in combination with
other suitable components, can be made into aerosol formulations
(e.g., they can be "nebulized") to be administered via inhalation.
Aerosol formulations can be placed into pressurized acceptable
propellants, such as dichlorodifluoromethane, propane, nitrogen,
and the like.
[0206] Suitable formulations for rectal administration include, for
example, suppositories, which consist of the packaged nucleic acid
with a suppository base. Suitable suppository bases include natural
or synthetic triglycerides or paraffin hydrocarbons. In addition,
it is also possible to use gelatin rectal capsules which consist of
a combination of the packaged nucleic acid with a base, including,
for example, liquid triglycerides, polyethylene glycols, and
paraffin hydrocarbons.
[0207] Formulations suitable for parenteral administration, such
as, for example, by intraarticular (in the joints), intravenous,
intramuscular, intradermal, intraperitoneal, and subcutaneous
routes, include aqueous and non-aqueous, isotonic sterile injection
solutions, which can contain antioxidants, buffers, bacteriostats,
and solutes that render the formulation isotonic with the blood of
the intended recipient, and aqueous and non-aqueous sterile
suspensions that can include suspending agents, solubilizers,
thickening agents, stabilizers, and preservatives. In the practice
of this invention, compositions can be administered, for example,
by intravenous infusion, orally, topically, intraperitoneally,
intravesically or intrathecally. Parenteral administration and
intravenous administration are the preferred methods of
administration. The formulations of packaged nucleic acid can be
presented in unit-dose or multi-dose sealed containers, such as
ampoules and vials.
[0208] Injection solutions and suspensions can be prepared from
sterile powders, granules, and tablets of the kind previously
described. Cells transduced by the packaged nucleic acid can also
be administered intravenously or parenterally.
[0209] 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 employed and
the condition of the patient, as well as the body weight or
vascular surface area of the patient to be treated. The size of the
dose also will be determined by the existence, nature, and extent
of any adverse side-effects that accompany the administration of a
particular vector, or transduced cell type in a particular
patient.
[0210] In determining the effective amount of the vector to be
administered in the treatment or prophylaxis of an infection or
other condition, the physician evaluates vector toxicities,
progression of the disease, and the production of anti-vector
antibodies, if any. In general, the dose equivalent of a naked
nucleic acid from a vector is from about 1 pg to 1 Mg for a typical
70 kilogram patient, and doses of vectors used to deliver the
nucleic acid are calculated to yield an equivalent amount of
therapeutic nucleic acid. Administration can be accomplished via
single or divided doses.
[0211] In therapeutic applications, compositions are administered
to a patient suffering from a disease (e.g., an infectious disease
or autoimmune disorder) in an amount sufficient to cure or at least
partially arrest the disease and its complications. An amount
adequate to accomplish this is defined as a "therapeutically
effective dose." Amounts effective for this use will depend upon
the severity of the disease and the general state of the patient's
health. Single or multiple administrations of the compositions may
be administered depending on the dosage and frequency as required
and tolerated by the patient. In any event, the composition should
provide a sufficient quantity of the proteins of this invention to
effectively treat the patient.
[0212] In prophylactic applications, compositions are administered
to a human or other mammal to induce an immune response that can
help protect against the establishment of an infectious disease or
other condition.
[0213] The toxicity and therapeutic efficacy of the genetic vaccine
vectors provided by the invention are determined using standard
pharmaceutical procedures in cell cultures or experimental animals.
One can determine the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population) using procedures presented herein and
those otherwise known to those of skill in the art.
[0214] A typical pharmaceutical composition for intravenous
administration would be about 0.1 to 10 mg per patient per day.
Dosages from 0.1 up to about 100 mg per patient per day may be
used, particularly when the drug is administered to a secluded site
and not into the blood stream, such as into a body cavity or into a
lumen of an organ. Substantially higher dosages are possible in
topical administration. Actual methods for preparing parenterally
administrable compositions will be known or apparent to those
skilled in the art and are described in more detail in such
publications as Remington's Pharmaceutical Science, 15th ed., Mack
Publishing Company, Easton, Pa. (1980).
[0215] The multivalent antigenic polypeptides of the invention, and
genetic vaccines that express the polypeptides, can be packaged in
packs, dispenser devices, and kits for administering genetic
vaccines to a mammal. For example, packs or dispenser devices that
contain one or more unit dosage forms are provided. Typically,
instructions for administration of the compounds will be provided
with the packaging, along with a suitable indication on the label
that the compound is suitable for treatment of an indicated
condition. For example, the label may state that the active
compound within the packaging is useful for treating a particular
infectious disease, autoimmune disorder, tumor, or for preventing
or treating other diseases or conditions that are mediated by, or
potentially susceptible to, a mammalian immune response.
EXAMPLES
[0216] The following examples are offered to illustrate, but not to
limit the present invention.
Example 1
Development of Broad-Spectrum Vaccines Against Bacterial Pathogens
and Toxins
[0217] A. Evolution of Yersinia V-Antigens
[0218] This Example describes the use of DNA shuffling to develop
immunogens that produce strong cross-protective immune responses
against a variety of Yersinia strains. Passive immunization with
anti-V-antigen antibodies or active immunization with purified
V-antigen can provide protection from challenge with a virulent
autologous Yersinia species. However, protection against
heterologous species is limited (Motin et al. (1994) Infect. Immun.
62: 4192).
[0219] V-antigen genes from a variety of Yersinia strains,
including serotypes of Y. pestis, Y. enterocolitica, and Y.
pseudotuberculosis are subjected to DNA shuffling as described
herein. The Yersinia pestis V antigen coding sequence, for example,
is used as a query in a database search to identify homologous
genes that can be used in a family shuffling format to obtain
improved antigens. Results for a BLAST search of GenBank and EMBL
databases are shown in Table 1, in which each line represents a
unique sequence entry listing the database, accession number, locus
name, bit score and E value. See, Altschul et al. (1997) Nucleic
Acids Res. 25:3389-3402, for a description of the search
algorithm). Homologous antigens have been cloned and sequenced from
a number of related yet distinct Yersinia strains and additional
natural diversity is obtained by cloning antigen genes from other
strains. These genes and others or fragments thereof are cloned by
methods such as PCR, shuffled and screened for improved
antigens.
1TABLE 1 Sequences producing significant alignments Score E
Database/Accession No. Gene (bits) Value
gb.vertline.M26405.vertline.YEPLCR Yersinia pestis lcrG, lcrV, and
lcrH genes, 1945 0.0 co gb.vertline.AF053946.vertline.AF053946
Yersinia pestis plasmid pCD1, complete pla 1945 0.0
emb.vertline.X96802.vertline.YPTPIVANT Y. pseudotuberculosis V
antigen gene 1834 0.0 gb.vertline.M57893.vertline.YEPLCRGVHP
Yersinia pseudotuberculosis V-antigen 1818 0.0
gb.vertline.AF080155.vertlin- e.AF080155 Yersinia enterocolitica
pYV LcrV (lcrV) 1723 0.0 antigen
emb.vertline.X96801.vertline.YE96PVANT Y. enterocolitica V antigen
gene, strain Y- . . . 1667 0.0 emb.vertline.X96799.vertlin-
e.YE108VANT Y. enterocolitica V antigen gene, strain Y- . . . 1659
0.0 emb.vertline.X96800.vertline.YE527VANT Y. enterocolitica V
antigen gene, serotype . . . 1651 0.0
emb.vertline.X96798.vertline.YE808VA- NT Y. enterocolitica V
antigen gene, strain 1643 0.0 8081
emb.vertline.X96796.vertline.YE314VANT Y. enterocolitica V antigen
gene, strain WA. 1237 0.0 emb.vertline.X96797.vertline.YENCTVANT Y.
enterocolitica V antigen gene, strain 1221 0.0 NCTC
gb.vertline.S38727.vertline.S38727 lcrGVH operon: lcrV = V-antigen
[Yersinia 365 9e-99 pseudo.]
[0220] Shuffled clones are selected by phage display and/or
screened by ELISA to identify those recombinant nucleic acids that
encode polypeptides that have multiple epitopes corresponding to
the different serotypes. The shuffled antigen genes are cloned into
a filamentous phage genome for polyvalent phage display or a
suitable phagemid vector for monovalent phage display. A typical
protocol for panning antigens by phage display is as follows.
[0221] Coat an appropriate surface (e.g., Nunc Maxisorp tube or
multiwell plate) at 4.degree. C. overnight with the target
antibody, usually at a concentration of 1-10 .mu.g/ml in PBS or
other suitable buffer
[0222] Rinse and Block with PBSM (PBS+3% nonfat dry milk) at
37.degree. C. for 1-2 hr
[0223] Pre-block phage if needed (PBSM, RT 1 hr)
[0224] Rinse tube and allow phage to bind (usually 1 hr @37.degree.
C.)
[0225] Can vary time, temp, buffer, add a competitive inhibitor,
etc.
[0226] Wash extensively (15.times.) with PBST (PBS+0.1% TWEEN20),
then PBS
[0227] Elute bound phage with low pH (e.g., 10 mM glycine), 100 mM
triethylamine, competitive ligand, protease, etc. and then
neutralize pH if needed.
[0228] Infect E. coli with eluted phage to transduce expression
phagemid into new host. Titer and plate for colonies on drug
plates
[0229] Pool colonies into media, grow cells and infect with helper
phage to produce phage for next round
[0230] Phage ELISA assays are a useful method to rapidly evaluate
single clones after panning of libraries. Single colonies are
picked in individual wells of a multiwell plate containing 2YT
media and grown as a master plate. A replicate plate is infected
with helper phage and grown so that phage from a single well will
display a single antigen variant. A suitable protocol for phage
ELISA assays is as follows.
[0231] Coat microtiter plate with 50 .mu.l of 1 .mu.g/ml target
antibody 4.degree. C. overnight
[0232] Rinse and block with PBSM for 2 hrs @ 37.degree. C.
[0233] Rinse, add preblocked phage and allow to bind 1 hr @
37.degree. C.
[0234] Wash plates with PBST 3.times., then PBS 3.times.with 2 min
soaks
[0235] Add HRP(or AP)-conjugated anti-M13 antibodies for 1 hr @
37.degree. C.
[0236] Add substrate and measure absorbance
[0237] Identify positive clones for further evaluation
[0238] ELISA assays can also be used to screen for individual
antigens with multiple epitopes or increased expression levels.
Single colonies are picked in individual wells of a multiwell plate
containing appropriate media and grown as a master plate so that
antigens produced from a single well are a single antigen variant.
A replicate plate is grown and induced for protein production,
e.g., by addition of 0.5 mM IPTG for Lac repressor-based systems
and grown for an appropriate time for the antigen to be produced.
At this point a crude antigen preparation is made which depends on
the antigen and where it is produced. Secreted proteins can be
evaluated by assaying the cell supernatants after centrifugation.
Periplasmic proteins are often readily released from cells by
simple extraction into hyper- or hypo-tonic buffers.
Intracellularly produced proteins will require some form of cell
lysis such as detergent treatment to release them. A suitable
protocol for ELISA assays is as follows.
[0239] Coat microtiter plate with 50 .mu.l of 1 .mu.g/ml target
antibody 4.degree. C. overnight
[0240] Rinse and block with PBSM for 2 hrs @ 37.degree. C.
[0241] Rinse, add antigen prep and allow to bind 1 hr @ 37.degree.
C.
[0242] Wash plates with PBST 3.times.
[0243] Add HRP(or AP)-conjugated secondary antibody and incubate
for 1 hr @ 37.degree. C.
[0244] Add appropriate substrate and measure absorbance
[0245] Identify positive clones for further evaluation
[0246] Antibodies specific for many of the various antigens are
commercially available (e.g., Toxin Technology, Inc, Sarasota,
Fla.) or can be generated by immunizing suitable animals with
purified antigens. Protein A or Protein G Sepharose (Pharmacia) can
be used to purify immunoglobulins from the serum. Various affinity
purification schemes can be used to further purify family-specific
antibodies if needed such as immobilization of specific antigens to
NHS-, CNBr-, or epoxy-activated sepharose beads. Other related
antigens may be included soluble form to prevent binding and
immobilization of cross-reactive antibodies.
[0247] The multivalent polypeptides that are identified by the
initial screening protocol are purified and subjected to in vivo
screening. For example, the shuffled antigens selected by a
combination of any or none of these methods are purified and used
to immunize animals, initially mice, which are then evaluated for
improved immune responses. Typically 10 micrograms of protein is
injected to a suitable location with or without appropriate
adjuvant, e.g., Alhydrogel (EM Seargent Pulp and Chemical, Inc.)
and the animals are boosted with an additional dose after 2-4
weeks. At this point serum samples is drawn and evaluated by ELISA
assay for the presence of antibodies that cross-react against
multiple parental antigens. In this ELISA assay format the antigens
are coated onto multiwell plates, then serial dilutions of each
sera is allowed to bind. After washing unbound antibodies, a
secondary HRP- or AP-conjugated antibody directed against the
appropriate test antibody constant region, e.g., goat anti-mouse
IgG Fe (Sigma) is bound. After another washing, the appropriate
substrate is added, e.g., O-phenylenediamine (Sigma). The
absorbance of each well is read by a plate reader at the
appropriate wavelength (e.g. 490 rum for OPD) and those producing
high antibody titers to multiple antigens are selected for further
evaluation.
[0248] Additionally, the ability of antigens to generate
neutralizing antibodies can be evaluated in an appropriate system.
Antigen variants that elicit a broad cross-reactive response are
evaluated further in a virulent challenge model with the
appropriate pathogenic organism. For example, the multivalent
polypeptides are used to immunize mice, which are then challenged
with live Yersinia bacteria. Those multivalent polypeptides that
protect against the challenge are identified and purified.
[0249] B. Evolution of Broad-Spectrum Vaccines Against Bacterial
Toxins
[0250] This Example describes the use of DNA shuffling to obtain
multivalent polypeptides that are effective in inducing an immune
response against a broad spectrum of bacterial toxins.
[0251] 1. Staphzylococcus
[0252] The Group A Streptococci, which can cause diseases such as
food poisoning, toxic shock syndrome, and autoimmune disorders, are
highly toxic by inhalation. The family of Group A Streptococcus
toxins numbers about 30 related members, making this group a
suitable target for family shuffling. Accordingly, this Example
describes the use of family DNA shuffling to create chimeric
proteins that are capable of eliciting broad spectrum
protection.
[0253] Nucleic acids that encode many diverse attenuated toxins are
subjected to DNA shuffling as described herein. Table 2 shows the
output of a BLAST search of GenBank, PDL, EMBL, and Swissprot using
the S. aureus enterotoxin B protein to identify homologous genes
that may be used in a family shuffling format to obtain improved
antigens.
2TABLE 2 Sequences producing significant alignments Score E
Database/Accession No. Gene (bits) Value
sp.vertline.P01552.vertline.ETXB_STAAU ENTEROTOXIN TYPE B PRECURSOR
(SEB) >g 554 e-157 pdb.vertline.ISE3.vertline. Stapylococcal
Enterotoxin B Complex 504 e-142 Tri. . .
pdb.vertline.ISEB.vertline.D Staphylococcus aureus
>gi.vertline.1633348.vertline.pdb 406 e-113 Staphyl. . .
sp.vertline.P23313.vertline.ETC3_STAAU ENTEROTOXIN TYPE C-3
PRECURSOR (SEC3) 376 e-103 sp.vertline.P01553.vertline.ETC1_STAAU
ENTEROTOXIN TYPE C-1 PRECURSOR (SEC1) 368 e-101
sp.vertline.P34071.vertline.ETC2_STAAU ENTEROTOXIN TYPE C-2
PRECURSOR (SEC2) 361 2e-99 gi.vertline.295145 (L13376) enterotoxin
[Staphylococcus 338 2e-92 gi.vertline.295151 (L13379) enterotoxin
[Staphylococcus 332 1e-90 gi.vertline.295143 (L13375) enterotoxin
[Staphylococcus 330 4e-90 gi.vertline.295149 (L13378) enterotoxin
[Staphylococcus 329 1e-89 pdb.vertline.IJCK.vertline.B Chain B,
T-Cell Receptor Beta Chain 328 2e-89 With S. . . gi.vertline.295141
(L13374) enterotoxin [Staphylococcus 328 3e-89
pdb.vertline.ISE2.vertline. Staphylococcal Enterotoxin C2, Monoc.
327 4e-89 Ente. . . gi.vertline.1906052 (U91526) type C enterotoxin
[Staphylo 326 8e-89 intermed. . . gi.vertline.295147 (L13337)
enterotoxin [Staphylococcus 323 7e-88 bbs.vertline.155101
enterotoxin=pyrogenic toxin [Staphylo 319 1e-86 4446, P. . .
gi.vertline.476764 (L29565) superantigen [Streptococcus 311 3e-84
gi.vertline.1245172 (U48792) Superantigen SSA [Streptococ 310 4e-84
pyogenes] > . . . gi.vertline.1245174 (U48793) superantigen SSA
[Streptococ 309 1e-83 pyogenes]
sp.vertline.P08095.vertline.SPEA_STRPY EXOTOXIN TYPE A PRECURSOR
(SCARLET FP 225 2e-58 gi.vertline.47288 (X61560) type A exotoxin
[Streptococc 211 3e-54 pyogenes] >gi.vertline. . . .
pir.vertline..vertline.S18783 exotoxin type A precursor (allele 3)
211 4e-54 Streptococcu. . . pir.vertline..vertline.S18786 exotoxin
type A precursor (allele 2) 209 2e-53 Streptococcu. . .
pir.vertline..vertline.S18789 exotoxin A precursor (allele 4) - Str
206 8e-53 pyo. . . gi.vertline.47328 (X61554) type A exotoxin
[Streptococc 196 9e-50 pyogenes] pir.vertline..vertline.A26152
Streptococcal pyrogenic exotoxin type 185 2e-46 precursor -. . .
sp.vertline.P20723.vertline.EXTD_ST- AAU ENTEROTOXIN TYPE D
PRECURSOR (SED) >g 131 3e-30
sp.vertline.P13163.vertline.ETXA_STAAU ENTEROTOXIN TYPE A PRECURSOR
(SEA) >g 129 2e-29 prf.vertline.1704203A enterotoxin A
[Staphylococcus aureus] 128 3e-29 pdb.vertline.IESF.vertline.A
Staphylococcus aureus >gi.vertline.1633233.vertline.pdb 125
2e-28 Staphyl. . . pir.vertline..vertline.A29566 enterotoxin A -
Staphylococcus aureus 125 2e-28 sp.vertline.P12993.vertline.EXTE_S-
TAAU ENTEROTOXIN TYPE E PRECURSOR (SEE) >g 118 3e-26
gi.vertline.510692 (011702) enterotoxin H [Staphylococcu 98 7e-20
>gi.vertline.10. . . gi.vertline.149047 (M94872) enterotoxin D
[Plasmid p1B48 89 2e-17 gi.vertline.2689563 (U93688) enterotoxin
[Staphylococcus 76 2e-13 gi.vertline.153785 (M97156) pyrogenic
exotoxin C [STrept 57 8e-08 pyogenes. . .
sp.vertline.P13380.vertline.SPEC_STRPY EXOTOXIN TYPE C PRECURSOR
(SPE C) 57 8e-08 gi.vertline.529754 (U02559) speC [Streptococcus
pyogenes 56 2e-07 pir.vertline..vertline.A30509 exotoxin C
precursor - Streptococcus 56 2e-07 >gi.vertline.1. . .
gi.vertline.529755 (U02560) speC [Streptococcus pyogenes 55 4e-07
pir.vertline..vertline.S27240 enterotoxin B - Staphylococcus 53
1e-06 aureus (fragments)
[0254] Shuffled recombinant clones are initially selected by phage
display and/or screened by ELISA for the presence of multiple
epitopes from the different families. Variant proteins with
multiple epitopes are purified and used to for in vivo screening as
described above. The mouse sera are analyzed for antibodies
specific for different toxin subtypes and variants that elicit
broadly cross-reactive responses will be evaluated further in
challenge models.
[0255] 2. Escherichia coli and Vibrio cholerae
[0256] This Example describes the use of DNA shuffling to obtain
cross-reactive multivalent polypeptides that induce an immune
response against the E. coli heat-labile toxin (LT), cholera toxin
(CT), and verotoxin (VT). Nucleic acids that encode cholera and LT
toxin B-chains are subjected to DNA shuffling. Table 3 shows the
results of a BLAST search using the V. cholerae toxin B-chain to
identify homologous genes that can be used in a family shuffling
format to obtain improved antigens. Homologous antigens have been
cloned and sequenced from a number of related yet distinct Vibrio
and E. coli strains, and additional natural diversity can be
obtained by cloning antigen genes from other strains. These genes
and others or fragments thereof can be cloned by methods such as
PCR, shuffled and screened for improved antigens.
3TABLE 3 Sequences producing significant alignments Score E
Database/Accession No. Gene (bits) Value
sp.vertline.P01556.vertline.CHTB_VIBCH CHOLERA ENTEROTOXIN, BETA
CHAIN PRECU 252 5e-67 gi.vertline.48890 (X58785) cholera toxin B
protein (CTH 248 8e-66 cholera. . . gi.vertline.758351 (X00171) ctx
B [Vibrio cholerea] 246 3e-65 prf.vertline..vertline.1001196A
toxin,cholera [Vibrio cholerae] 246 3e-65 gnl.vertline.PID.vertlin-
e.d1006853 (D30052) cholera toxin [Vibrio choler 244 1e-64
pir.vertline..vertline.XVVCB cholera enterotoxin chain B precursor
241 1e-63 cholerae gi.vertline.209556 (M23050) cholera toxin
subunit B prec 228 7e-60 [Artificia. . . bbs.vertline.168005
cholera-like enterotoxin B subunit [.backslash. 211 1e-54 cholrea,
. . . sp.vertline.P13811.vertline.ELBH_ECOL- I HEAT-LABILE
ENTEROTOXIN B CHAIN PRECU 209 5e-54 pdb.vertline.1XTC.vertline.D
Vibrio cholerae >gi.vertline.1827851.vert-
line.pdb.vertline.1XTC 207 2e-53 choler. . .
pdb.vertline.1FGB.vertline.D Vibrio cholerae
>gi.vertline.1942839.vert- line.pdb.vertline.1FGB 207 2e-53
choler. . . pdb.vertline.2CHB.vertline.D Chain D, Cholera Toxin
B-Pentamer Con 207 2e-53 With Gml. . . pdb.vertline.1CHP.vertline.D
Vibrio cholerae >gi.vertline.1421512.vertline.pdb.vertline.1CHP
205 1e-52 choler. . . pdb.vertline.1CHQ.vertline.D Vibrio cholerae
>gi.vertline.1421526.vertline.pdb.vertline.1CHQ 205 1e-52
choler. . . pdb.vertline.1CT1.vertline.D Chain D, Cholera Toxin
B-Pentamer Mut 204 1e-52 Bound . . .
sp.vertline.P32890.vertline.ELBP_ECOLI HEAT-LABILE ENTEROTOXIN B
CHAIN PRECU 204 2e-52 prf.vertline..vertline.0701264A toxin LTB
Cistron,heat labile [Escher 201 9e-52 coli]
pir.vertline..vertline.QLECB heat-labile enterotoxin chain B precu
201 2e-51 Escheric. . . bbs.vertline.131495 (S60731) heat-labile
enterotoxin B su 200 2e-51 B su. . . prf.vertline..vertline.770190A
toxin [Vibrio cholerae] 199 6e-51 pdb.vertline.1LTA.vertline.D
Escherichia coli >gi.vertline.494266.vert-
line.pdb.vertline.1LTA 179 4e-45 Escherichia c. . .
pdb.vertline.1TET.vertline.P Vibrio cholerae 34 0.31
[0257] Those chimeric toxins that elicit high levels of
neutralizing antibodies against both toxins and have improved
adjuvant properties are identified. For example, shuffled clones
are selected by phage display and/or screened by ELISA assays for
the presence of epitopes from the different parental B-chains.
Variants with multiple epitopes are purified and further studied
for their capacity to act as adjuvants and to elicit
cross-protective immune responses in challenge models.
Example 2
Evolution of Broad-Spectrum Vaccines Against Borrelia
burgdorferi
[0258] Lyme disease is currently one of the fastest-growing
infectious diseases in the United States. It is caused by infection
of the spirochete bacterium Borrelia burgdorferi, which is carried
and spread by the bite of infected ticks. Early signs of infection
include skin rash and flu-like symptoms. If left untreated Lyme
disease can cause arthritis, heart abnormalities, and facial
paralysis. Treatment of early Lyme disease with antibiotics can
stop the infection, but a lasting immunity may not develop making
reinfection possible. A current vaccine requires three
immunizations over a 1-year period to acquire immunity.
[0259] Both passive and active immunization with the purified B.
burgdorferi outer surface protein A (OspA) protein has been
successful in protecting against infection with B. burgdorferi, but
has no effect against ongoing infections, since this antigen is not
expressed in vertebrate hosts. OspA is normally anchored on the
outside of the cell by a covalently attached lipid moiety through
an amino terminal cysteine residue. In contrast, the outer surface
protein C (OspC) is highly expressed by the spirochete in
vertebrate hosts and vaccination of infected individuals with OspC
may be an effective therapeutic in curing the infection (Zhong et
al. (1997) Proc. Nat'l. Acad. Sci. USA 94 12533-12538.
[0260] A recent BLAST search (Altschul, et al., (1997) Nucleic
Acids Res. 25:3389-3402) of the non-redundant GenBank, PDB,
SwissProt, Spupdate, and PIR databases was used to identify
homologues of the OspA outer surface protein gene. This resulted in
the identification of over 200 entries related to OspA. One hundred
entries are shown in Table 4 below from different strains of B.
burgdorferi, B. garinii, B. afzelii, B. tanukii, and B. turdi that
share at least 83% DNA sequence identity to 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 the prevention of Lyme disease. These
genes are cloned by methods such as PCR, shuffled and screened for
improved antigens.
4TABLE 4 Sequences producing significant alignments Score E
Database/Accession No. Gene (bits) Value
dbj.vertline.AB016977.vertline.AB016977 Borrelia sp. gene for outer
surface 1629 0.0 prote . . . dbj.vertline.AB016978.vertlin-
e.AB016978 Borrelia sp. 10MT gene for outer 1614 0.0 surface . . .
dbj.vertline.AB016975.vertline.AB016975 Borrelia turdi gene for
outer surface 1526 0.0 pro . . . dbj.vertline.AB016976.ver-
tline.AB016976 Borrelia sp. gene for outer surface 1187 0.0 prote .
. . gb.vertline.S48323.vertline.S48323 ospA = outer surface protein
A [Borrelia 948 0.0 burgdor . . .
gb.vertline.L38657.vertline.BORFRA Borrelia burgdorferi (clone N3)
ospA 948 0.0 gene frag . . . emb.vertline.X80186.vertline.BBPTR-
OOPS B. burgdorferi PTro ospA gene 938 0.0
emb.vertline.X65598.vert- line.BBOSPA1 B. burgdorferi Osp A gene
(TRO) 938 0.0 gb.vertline.U20357.vertline.BBU20357 Borrelia
burgdorferi C-1-11 outer 876 0.0 surface pr . . .
gb.vertline.S88693.vertline.S8869- 3 outer surface protein A
[Borrelia 858 0.0 burgdorferi, . . .
emb.vertline.X66065.vertline.BBOSPROA B. burgdorferi OspA gene for
outer 839 0.0 surface p . . . emb.vertline.X85440.vertline-
.BGTISOSPA B. garinii ospA gene (TIS1 substrain) 839 0.0
emb.vertline.X85438.vertline.BAPLJOSPA B. afzelii ospA gene (PLj7
substrain) 837 0.0 >gi.vertline.9 . . .
emb.vertline.X80183.vertline.BBPBOOSPA B. burgdorferi PBo ospA gene
829 0.0 emb.vertline.Z29087.vertline.BBOSPAY B. burgdorferi (VS461)
OspA gene for 821 0.0 outer su . . .
emb.vertline.X85982.vertline.BADNAOSPA B. afzelii ospA gene 821 0.0
emb.vertline.X62161.vertline.BBOSPAG B. burgdorferi plasmid ospA
gene for 821 0.0 outer su . . . gb.vertline.U78301.vertline.BBU-
78301 Borrelia afzelii major outer membrane 821 0.0 surfac . . .
emb.vertline.X65599.vertline.BBOSPA2 B. burgdorferi Osp A gene
(PKO) 819 0.0 emb.vertline.X70365.vertline.BBOPSAA B. burgdorferi
OspA gene 819 0.0 emb.vertline.X85439.vertline.BAPLUOSPA B. afzelii
ospA gene (PLud substrain) 813 0.0 emb.vertline.X81047.vertline.BB-
OPSA B. burgdorferi plasmid OspA gene 813 0.0
gb.vertline.U20356.vertline.BAU20356 Borrelia afzelii BV1 outer
surface 813 0.0 protein A . . . emb.vertline.X85437.vertline.BAPHO-
OSPA B. afzelii ospA gene (PHo substrain) 797 0.0
emb.vertline.X80253.vertline.BBPWUDLL B. burgdorferi Wudll OspA
gene 791 0.0 emb.vertline.Z29086.vertline.BBOSPAX B. burgdorferi
(G25) OspA gene for outer 791 0.0 surf . . .
gb.vertline.L19702.vertl- ine.BORMAJOSPR Borrelia burgdorferi outer
surface 791 0.0 protein . . . emb.vertline.X62387.vertline.BBSPA B.
burgdorferi ospA gene for outer 789 0.0 surface prot . . .
emb.vertline.X60300.vertline.BBASPA B. burgdorferi gene for OspA
outer 785 0.0 surface pro . . . emb.vertline.X62624.vertline.BBK-
48OSPA B. burgdorferi ospA gene 767 0.0
gb.vertline.M88764.vertline- .BOROSPABA Borreila burgdorferi operon
major outer 759 0.0 mem . . . emb.vertline.X63412.vertline.BBPOSPA
B. burgdorferi plasmid ospA gene for 759 0.0 outer su . . .
gb.vertline.L36036.vertlin- e.BOROSPAL Borrelia burgdorferi outer
surface 743 0.0 protein A . . .
gb.vertline.U20358.vertline.BGU20358 Borrelia garinii LV4 outer
surface 743 0.0 protein A . . .
emb.vertline.X85442.vertline.BB297OSPA B. burgdorferi ospA gene
(297 substrain) 714 0.0 gb.vertline.L19701.vertline.BOROPSAB
Borrelia burgdorferi major outer 714 0.0 surface pro . . .
emb.vertline.X14407.vertline.BBOSPAB Borrelia burgdorferi ospA and
ospB 706 0.0 genes for . . . gb.vertline.AE00790.vertline.AE007- 90
Borrelia burgdorferi plasmid 1p54, 706 0.0 complet . . .
gb.vertline.U20360.vertline.BBU20360 Borrelia burgdorferi S-1-10
outer 706 0.0 surface pr . . . emb.vertline.X69606.vertline.BBKA-
OSPA B. burgdorferi OspA gene 706 0.0 >gi.vertline.1819262.vert-
line.gb.vertline.I284 . . . dbj.vertline.AB007100.vertline.AB007100
Borrelia garinii gene for outer surface 702 0.0 p . . .
gb.vertline.M57248.vertline.BOROSPA B. burgdorferei outer surface
protein A 698 0.0 (OspA) . . . emb.vertline.X80182.vertline.BBPKAO-
PSA B. burgdorferi PKa ospA gene 698 0.0
dbj.vertline.AB007101.vert- line.AB007101 Borrelia garinii gene for
outer surface 694 0.0 p . . .
emb.vertline.X85443.vertline.BBT25OSPA B. burgdorferi ospA gene
(T255 692 0.0 substrain) emb.vertline.X16467.vertline- .BBOSPA
Borrelia burgdorferi OspA gene for 690 0.0 outer surf . . .
gb.vertline.U20359.vertline.BBU20359 Borrelia sp. LV5 outer surface
protein 690 0.0 A pre . . . gb.vertline.AF026059.vertline.AF026059
Borrelia burgdorferi 50 kDa plasmid 682 0.0 lipopr . . .
emb.vertline.X85739.vertline.- BBDNAOSPA B. burgdorferi ospA gene
682 0.0 emb.vertline.X85441.vert- line.BGWABOSPA B. garinii ospA
gene (WABSou substrain) 676 0.0 gb.vertline.U93709.vertline.U93709
Borrelia garinii outer surface protein 674 0.0 A (ospA . . .
dbj.vertline.AB007099.vertline.AB007- 099 Borrelia garinii gene for
outer surface 662 0.0 p . . .
emb.vertline.X80251.vertline.BBPHEIOSP B. burgdorferi PHei ospA
gene 660 0.0 gb.vertline.U49190.vertline.BGU49190 Borrelia garinii
major outer membrane 652 0.0 surfac . . .
emb.vertline.X65600.vertline.BBOSPA3 B. burgdorferi Osp A gene (HE)
652 0.0 dbj.vertline.D29660.vertline.D29660 Borrelia burgdorferi
gene for 652 0.0 outersurface pr . . . dbj.vertline.AB016979.ve-
rtline.AB016979 Borrelia valaisiana gene for outer 648 0.0 surfac .
. . dbj.vertline.AB007109.vertline.AB007109 Borrelia garinii gene
for outer surface 646 0.0 p . . .
gb.vertline.U93707.vertline.U93707 Borrelia garinii outer surface
protein 644 0.0 A (ospA . . . dbj.vertline.AB007102.vertline.AB007-
102 Borrelia garinii gene for outer surface 642 0.0 p . . .
dbj.vertline.AB001041.vertline.AB001041 Borrelia garinii DNA for
outer surface 636 e-180 pr . . . dbj.vertline.AB007114.vertline.-
AB007114 Borrelia garinii gene for outer surface 632 e-179 p . . .
dbj.vertline.AB007105.vertline.AB007105 Borrelia garinii gene for
outer surface 632 e-179 p . . . gb.vertline.U93710.vertlin-
e.U93710 Borrelia garinii outer surface protein 628 e-178 A (ospA .
. . dbj.vertline.AB007106.vertline.AB007106 Borrelia garinii gene
for outer surface 624 e-177 p . . .
dbj.vertline.AB007104.vertline.AB007104 Borrelia garinii gene for
outer surface 624 e-177 p . . . gb.vertline.U93706.vertline.U937-
06 Borrelia garinii outer surface protein 620 e-176 A (ospA . . .
emb.vertline.X80256.vertline.BGPBROSPA B. garnii PBr ospA gene 613
e-173 dbj.vertline.AB007108.vertline.AB007108 Borrelia garinii gene
for outer surface 607 e-171 p . . .
gb.vertline.L81129.vertline.BOROSPAY Borrelia burgdorferi (isolate
2-1498 605 e-171 297) ou . . . gb.vertline.U93711.vertline.U93711
Borrelia garinii outer surface protein 605 e-171 A (ospA . . .
dbj.vertline.AB007103.vertline.AB007103 Borrelia garinii gene for
outer surface 587 e-165 p . . . gb.vertline.L81128.vertlin-
e.BOROSPAZ Borrelia burgdorferi (isolate 2-1498 581 e-164 Son 188 .
. . gb.vertline.L23137.vertline.BOROSPAC Borrelia burgdorferi
(27985CT2) OspA 577 e-162 gene, 3 . . .
gb.vertline.L23139.vertline.BOROSPAE Borrelia burgdorferi
(42373NY3) OspA 577 e-162 gene, 3 . . .
gb.vertline.L23142.vertline.BOROSP- AI Borrelia burgdorferi (CA3)
OspA gene, 577 e-162 3' end . . .
gb.vertline.L23136.vertline.BOROSPAA Borrelia burgdorferi (B19CT1)
OspA 577 e-162 gene, 3' e . . . gb.vertline.U93705.vertlin-
e.U93705 Borrelia garini outer surface protein 569 e-160 A (ospA .
. . gb.vertline.L23140.vertline.BOROSPAF Borreila burgdorferi
(41552MA) OspA 569 e-160 gene, 3' . . .
dbj.vertline.AB007110.vertline.AB007110 Borreila garinii gene for
outer surface 565 e-159 p . . . gb.vertline.L23143.vertline.BORO-
SPAJ Borreila burgdorferi (CA7) OspA gene, 561 e-158 3' end . . .
dbj.vertline.AB007112.vertline.AB007112 Borreila garinii gene for
outer surface 557 e-156 p . . . emb.vertline.X80254.vertli-
ne.BGT25OSPA B. garnii T25 ospA gene 557 e-156
gb.vertline.U93708.vertline.U93708 Borreila garinii outer surface
protein 553 e-155 A (ospA . . . dbj.vertline.AB007107.vertline.AB0-
07107 Borrelia garinii gene for outer surface 549 e-154 p . . .
gb.vertline.L23141.vertline.BOROSPAH Borrelia burgdorferi (2134W1)
OspA 545 e-153 gene, 3' . . . dbj.vertline.AB007111.vertli-
ne.AB007111 Borrelia garinii gene for outer surface 541 e-152 p . .
. dbj.vertline.AB007113.vertline.AB007113 Borrelia garinii gene for
outer surface 537 e-151 p . . .
gb.vertline.L23144.vertline.BOROSPAK Borrelia burgdorferi (CA8)
OspA gene, 529 e-148 3' end . . . gb.vertline.U78549.vertline.B-
AU78549 Borrelia afzelii major outer membrane 525 e-147 surfac . .
. dbj.vertline.AB009863 .vertline.AB009863 Borrelia garinii gene
for outer surface 519 e-145 p . . .
emb.vertline.X68059.vertline.BBOSPAGE B. burgdorferi OspA gene for
outer 498 e-139 surface p . . . dbj.vertline.AB009862.vertline.A-
B009862 Borrelia garinii gene for outer surface 466 e-129 p . . .
emb.vertline.X95360.vertline.BBOSPPFRA B. burgdorferi ospA gene
(strain PFra) 460 e-127 emb.vertline.X68541.vertline.BBPHEI B.
burgdorferi (PHEI) plasmid OspA gene 446 e-123 for ou . . .
emb.vertline.X68540.vertline.BBPWUDI B. burgdorferi (PWudI) plasmid
OspA gene 414 e-114 for . . . emb.vertline.X95358.vertline.BGOS-
PPLI B. garinii ospA gene (strain PLi) 414 e-114
dbj.vertline.AB009860.vertline.AB009860 Borrelia garinii gene for
outer surface 393 e-107 p . . . dbj.vertline.AB009858.vertline.A-
B009858 Borreila garinii gene for outer surface 365 8e-99 p . . .
dbj.vertline.AB009861.vertline.AB009861 Borrelia garinii gene for
outer surface 283 2e-74 p . . .
[0261] A BLAST search with the B. burgdorferi OspC protein gene
revealed over 200 related entries. Entries for one hundred
sequences sharing at least 82% DNA sequence identity are shown in
Table 5 below that provide a source of diversity for family
shuffling to obtain improved therapeutics in the treatment of Lyrne
disease. These genes are cloned by methods such as PCR, shuffled
and screened for improved antigens.
5TABLE 5 Sequences producing significant alignments Score E
Database/Accession No. Gene (bits) Value
gb.vertline.U04282.vertline.BBU04282 Borrelia burgdorferi GMP
synthetase 1261 0.0 (guaA) g . . .
gb.vertline.L42898.vertline.BOR31O- SPC Borrelia burgdorferi
(strain 25015) 1124 0.0 outer s . . .
gb.vertline.U01894.vertline.BBU01894 Borrelia burgdorferi B31 outer
surface 622 e-176 prote . . . dbj.vertline.D49497.vertline-
.BOROSPCA Borreila burgdorferi gene for outer 622 e-176 surface . .
. gb.vertline.AE000792.vertline.AE000792 Borrelia burgdorferi
plasmid cp26, 622 e-176 complet . . .
emb.vertline.X69596.vertline.BB31OSPC B. burgdorferi ospC gene for
outer 615 e-174 surface . . . gb.vertline.AF029860.vertline.AF02-
9860 Borrelia burgdorferi OC1 outer surface 523 e-146 pro . . .
gb.vertline.U91798.vertline.BBU91798 Borrelia burgdorferi strain L5
outer 519 e-145 surface . . . gb.vertline.L42887.vertline.-
BOR20OSPC Borrelia burgdorferi (strain Ip2) outer 517 e-145 sur . .
. gb.vertline.L81131.vertline.BOROSPCY Borrelia burgdorferi;
suostrain sensu 509 e-142 strict . . .
gb.vertline.U91792.vertline.BBU91792 Borrelia burgdorferi strain
HI1 outer 478 e-133 surfac . . . gb.vertline.U91797.vertline.-
BBU91797 Borrelia burgdorferi strain IP3 outer 462 e-128 surfac . .
. gb.vertline.U91801.vertline.BBU91801 Borrelia burgdorferi strain
PIF outer 444 e-123 surfac . . .
dbj.vertline.AB001377.vertline.AB001377 Borrelia japanica strain
NO67 DNA for 430 e-118 Out . . .
dbj.vertline.AB001378.vertline.AB0- 01378 Borrelia japonica strain
OvKK7 DNA for 430 e-118 Ou . . .
emb.vertline.X84783.vertline.BBOSPCTXW B. burgdorferi ospC gene
(strain TXGW) 418 e-115 dbj.vertline.AB000355.vertline.AB000355
Borrelia tanukii DNA for Outer surface 418 e-115 pr . . .
gb.vertline.U91799.vertline.BBU91799 Borrelia burgdorferi strain
IP1 outer 418 e-115 surfac . . . dbj.vertline.AB001376.vertlin-
e.AB001376 Borrelia japonica strain Fi3lo DNA for 414 e-114 Ou . .
. emb.vertline.X73624.vertline.BBOSPCC B. burgdorferi (DK26) OspC
gene 404 e-111 emb.vertline.X62162.vertline.BBPCG B. burgdorferi
gene for pC protein 398 e-109 emb.vertline.X69590.vertline.BBWUDOS-
PC B. burgdorferi OspC gene, 3' end 398 e-109
emb.vertline.X81521.vertline.BAOSPC1 B. afzelii (strain PBo) ospC
gene 377 e-102 dbj.vertline.AB000345.vertline.AB000345 Borrelia
afzelii DNA for Outer surface 365 6e-99 pr . . .
dbj.vertline.AB009900.vertline.AB009900 Borrelia afzelii gene for
outer surface 361 9e-98 p . . . dbj.vertline.AB009899.vertline.A-
B009899 Borrelia afzelii gene for outer surface 357 1e-96 p . . .
emb.vertline.X81523.vertline.BAOSPC2 B. afzelii (strain PLj7) ospC
gene 339 3e-91 gb.vertline.AF029871.vertline.AF029871 Borrelia
burgdorferi OCT2 outer surface 337 1e-90 pr . . .
dbj.vertline.AB009897.vertline.AB009897 Borrelia afzelii gene for
outer surface 337 1e-90 p . . . dbj.vertline.AB000354.vertline.A-
B000354 Borrelia tanukii DNA for Outer surface 337 1e-90 pr . . .
gb.vertline.L25413.vertline.BOROSPC Borrelia burgdorferi membrane
protein 335 5e-90 (ospC) . . . dbj.vertline.D49502.vertlin-
e.BOROSPCF Borrelia afzelii gene for outer surface 335 5e-90 pro .
. . emb.vertline.X83555.vertline.BSDNAOSPC B. burgdorferi (B.
paciticus strain) 333 2e-89 ospC gene
gb.vertline.L42874.vertline.BOR10OSPC Borrelia burgdorferi (strain
Ortn) 331 8e-89 outer su . . . dbj.vertline.D49503.vertline.BORO-
SPCG Borrelia afzelii gene for outer surface 331 8e-89 pro . . .
dbj.vertline.AB009894.vertline.AB009894 Borrelia afzelii gene for
outer surface 329 3e-88 p . . . gb.vertline.L42890.vertlin-
e.BOR23OSPC Borrelia burgdorferi (strain E61) outer 329 3e-88 sur .
. . gb.vertline.L42892.vertline.BOR25OSPC Borrelia burgdorferi
(strain aca1) 329 3e-88 outer su . . .
dbj.vertline.D49501.vertline.BOROSPCE Borrelia afzelii gene for
outer surface 327 1e-87 pro . . . gb.vertline.U04240.vertline.BB-
U04240 Borrelia burgdorferi GMP synthetase 317 1e-84 (guaA) a . . .
gb.vertline.U04280.vertline.BBU04280 Borrelia burgdorferi GMP
synthetase 317 1e-84 (guaA) g . . .
dbj.vertline.AB009901.vertline.AB009901 Borrelia afzelii gene for
outer surface 309 3e-82 p . . . dbj.vertline.AB009893.vertline.A-
B009893 Borrelia afzelii gene for outer surface 309 3e-82 p . . .
gb.vertline.L42883.vertline.BOR17OSPC Borrelia burgdorferi (strain
JSB) outer 307 1e-81 sur . . . gb.vertline.U04281.vertline-
.BBU04281 Borrelia burgdorferi HB19 outer surface 305 5e-81 prot .
. . dbj.vertline.AB009896.vertline.AB009896 Borrelia afzelii gene
for outer surface 305 5e-81 p . . .
emb.vertline.X81522.vertline.BBOSPC1 B. burgdorferi (strain PBre)
ospC gene 297 1e-78 dbj.vertline.AB000349.vertline.AB000349
Borrelia afzelii DNA for Outer surface 297 1e-78 pr . . .
emb.vertline.X73625.vertline.BBOSPCD B. burgdorferi (DK7) OspC gene
297 1e-78 dbj.vertline.D49509.vertline.BOROSPCM Borrelia garinii
gene for outer surface 295 4e-78 pro . . .
dbj.vertline.AB000346.vertline.AB000346 Borrelia afzelii DNA for
Outer surface 293 2e-77 pr . . . gb.vertline.AF029870.vertline.A-
F029870 Borrelia burgdorferi OC11 outer surface 289 3e-76 pr . . .
gb.vertline.L42895.vertline.BOR2BOSPC Borrelia burgdorferi (strain
28354) 289 3e-76 outer s . . . emb.vertline.X81524.vertlin-
e.BBOSPC2 B. burgdorferi (strain T255) ospC gene 289 3e-76
dbj.vertline.D88296.vertline.D88296 Borrelia afzelii 26kb circular
plasmid 289 3e-76 DNA fo . . . emb.vertline.X81526.vertlin-
e.BGOSPC2 B. garinii (strain WABSou) ospC gene 285 4e-75
>gi.vertline.8720 . . . emb.vertline.X84772.vertline.BBOSPCD32
B. garinii ospC gene (strain DK32) 285 4e-75
dbj.vertline.AB009902.vertline.AB009902 Borrelia afzelii gene for
outer surface 285 4e-75 p . . . dbj.vertline.D49505.vertline.BOR-
OSPCI Borrelia garinii gene for outer surface 281 7e-74 pro . . .
gb.vertline.U01892.vertline.BBU01892 Borrelia burgdorferi 2591
outer surface 281 7e-74 prot . . . emb.vertline.X83552.ver-
tline.BADNAOSPC B. afzelii (PLud strain) oSpC gene 280 3e-73
dbj.vertline.D49378.vertline.BOKOSPC64 Borrelia garinii (strain
HT64) ospC 278 1e-72 gene f . . . dbj.vertline.D49379.vertline.B-
OROSPCVS Borrelia afzelii (strain VS461) ospC 274 2e-71 gene . . .
gb.vertline.AF029864.vertline.AF029864 Borrelia burgdorferi OCS
outer surface 266 4e-69 pro . . . dbj.vertline.AB000350.ve-
rtline.AB000350 Borrelia afzelii DNA for Outer surface 266 4e-69 pr
. . . gb.vertline.U08284.vertline.BBU08284 Borrelia burgdorferi 297
outer surface 264 2e-68 prote . . .
dbj.vertline.AB000343.vertline.AB000343 Borrelia afzelii DNA for
Outer surface 260 2e-67 pr . . . dbj.vertline.AB000353.vertline.-
AB000353 Borrelia tanukii DNA for Outer surface 258 1e-66 pr . . .
emb.vertline.X84779.vertline.BBOSCMUL B. burgdorferi ospC gene
(strain MUL) 256 4e-66 emb.vertline.X84768.vertline.BBOSPCD15 B.
afzelii ospC gene (strain DK15) 256 4e-66 dbj.vertline.D88292.vert-
line.D88292 Borrelia garinii 26kb circular plasmid 254 2e-65 DNA fo
. . . dbj.vertline.D49507.vertline.BOROSPCK Borrelia garinii gene
for outer surface 254 2e-65 pro . . .
emb.vertline.X83556.vertline.BGOSPCN34 B. garinii (N34 strain) ospC
gene 250 2e-64 gb.vertline.AF029866.vertline.AF029866 Borrelia
burgdorferi OC7 outer surface 250 2e-64 pro . . .
dbj.vertline.D88294.vertline.D88294 Borrelia garinii 2bkb circular
plasmid 250 2e-64 DNA fo . . . gb.vertline.L42888.vertline-
.BOR21OSPC Borrelia burgdorferi (strain H9) outer 248 9e-64 surf .
. . gb.vertline.AF029862.vertline.AF029862 Borrelia burgdorferi OC3
outer surface 246 4e-63 pro . . .
dbj.vertline.AB009891.vertline.AB009891 Borrelia afzelii gene for
outer surface 246 4e-63 p . . . dbj.vertline.AB009898.vertline.A-
B009898 Borrelia afzelii gene for outer surface 246 4e-63 p . . .
gb.vertline.AF029861.vertline.AF029861 Borrelia burgdorferi OC2
outer surface 246 4e-63 pro . . . emb.vertline.X69593.vert-
line.BSTNOSPC B. burgdorferi OspC gene, 3' end 246 4e-63
dbj.vertline.D49377.vertline.BOROSPC57 Borrelia garinii (strain
HT57), ospC 244 1e-62 gene f . . . dbj.vertline.D49500.vertline.B-
OROSPCD Borrelia garinii gene for outer surface 244 1e-62 pro . . .
gb.vertline.L42896.vertline.BOR29OSPC Borrelia burgdorferi (strain
27579) 242 6e-62 outer s . . .
dbj.vertline.D49376.vertline.BOROSPCTC Borrelia garinii (strain
TCLSK) ospC 240 2e-61 gene . . . gb.vertline.L42873.vertline.BOR9-
OSPC Borrelia burgdorferi (strain SIMON) 238 9e-61 outer su . . .
dbj.vertline.D49381.vertline.BOROSPC37 Borrelia garinii (strain
HT37) ospC 238 9e-61 gene f . . . dbj.vertline.D49498.vert-
line.BOROSPCB Borrelia garinii gene for outer surface 328 9e-61 pro
. . . emb.vertline.X69592.vertline.BBT25OSPC B. burgdorferi OspC
gene, 3' end 232 6e-59 emb.vertline.X69594.vertline.BBPBROSPC B.
burgdorferi OspC gene, 3' end 228 9e-58 dbj.vertline.D49506.ver-
tline.BOROSPCJ Borrelia garinii gene for outer surface 220 2e-55
pro . . . emb.vertline.X69595.vertline.BBPBIOSPC B. burgdorferi
ospC gene for outer 220 2e-55 surface . . .
emb.vertline.X83554.vertline.BGOSPPTRO B. garinii (PTrob strain)
opsC gene 220 2e-55 emb.vertline.X73626.vertline.BBOSPPCE B.
burgdorferi (Dk6) OspC gene 220 2e-55 gb.vertline.L42870.vertline.-
BOR6OSPC Borrelia burgdorferi (strain VSDA) 220 2e-55 outer sur . .
. gb.vertline.L42894.vertline.BOR27OSPC Borrelia burgdorferi
(strain 28691) 218 8e-55 outer s . . .
dbj.vertline.D49504.vertline.BOROSPCH Borrelia garinii gene for
outer surface 208 8e-52 pro . . gb.vertline.L42868.vertline.BOR-
4OSPC Borrelia burgdorferi (strain ZS7) outer 206 3e-51 surf . . .
dbj.vertline.AB000358.vertline.AB000358 Borrelia japonica DNA for
Outer surface 204 1e-50 p . . . dbj.vertline.AB000351.vert-
line.AB000351 Borrelia japonica DNA for Outer surface 204 1e-50 p .
. .
Example 3
Evolution of Broad-Spectrum Vaccines Against Mycobacterium
[0262] Tuberculosis is an ancient bacterial disease caused by
Mycobacterium tuberculosis that continues to be an important public
health problem worldwide and calls are being made for an improved
effort in eradication (Morb. Mortal Wkly Rep (Aug. 21, 1998;
47(RR-13): 1-6). It infects over 50 million people and over 3
million people will die from tuberculosis this year. The currently
available vaccine, Bacille Calmette-Guenrn (BCG) is found to be
less effective in developing countries and an increasing number of
multidrug-resistant (MDR) strains are being isolated. The major
immunodominant antigen of M. tuberculosis is the 30-35 kDa (a.k.a.
antigen 85, alpha-antigen) which is normally a lipoglycoprotein on
the cell surface. Other protective antigens include a 65-kDa heat
shock protein, and a 36-kDa proline-rich antigen (Tascon et al.
(1996) Nat. Med. 2: 888-92).
[0263] Table 6 shows the output of a BLAST search using the 30-35
kDa major M. Tuberculosis antigen (a.k.a. antigen 85,
alpha-antigen) coding sequence to identify homologous genes that
may be used in a family shuffling format to obtain improved
antigens. Many homologous antigens have been cloned and sequenced
from a large number of related yet distinct mycobactenral strains.
These genes are cloned by methods such as PCR, shuffled and
screened for improved antigens.
6TABLE 6 Sequences producing significant alignments Score E
Database/Accession No. Gene (bits) Value
gb.vertline.U38939.vertline.MTU38939 Mycobacterium tuberculosis 30
KDa 1939 0.0 extracellu . . . emb.vertline.X62398.vertline.MT8- 5B
M. tuberculosis (strain Erdman) gene for 1939 0.0 85-B a . . .
emb.vertline.Z97193.vertline.MTCY180 Mycobacterium tuberculosis
H37Rv 1939 0.0 complete ge . . . emb.vertline.X62397.vertl-
ine.MB85B M. bovis (strain 1173P2) gene for 85-B 1931 0.0 antigen
gb.vertline.M21839.vertline.MSGBCGA M. bovis BCG gene encoding
alpha- 1869 0.0 antigen, comp . . .
emb.vertline.X53897.vertline.MKAANTIG Mycobacterium kansasii gene
for alpha 819 0.0 antigen dbj.vertline.D26187.vertline.MSGAA
Mycobacterium scrofuraceum DNA for 706 0.0 alpha-antig . . .
dbj.vertline.D16546.vertline.MSGAAG Mycobacterium intracellulare
gene for 581 e-164 alpha-a . . . emb.vertline.X63437.vertline.M-
AALANT M. avium gene for alpha-antigen 569 e-160
dbj.vertline.D14253.vertline.MSGATCC139 Mycobacterium
intracellulare DNA for 533 e-149 alph . . .
gb.vertline.L01095.vertline.MSGB3- 8COS M. leprae genomic DNA
sequence, cosmid 371 e-100 B38 . . .
emb.vertline.X60934.vertline.ML85BA M. leprae gene for 85-B antigen
363 4e-98 emb.vertline.Z11666.vertline.MLFBPAPR M. leprae
fibronectin-binding protein 347 2e-93 antige . . .
gb.vertline.M27016.vertline.MSG32KDA Mycobacterium tuberculosis 32
KDa 317 2e-84 antigen gene. emb.vertline.X53034.vertline.MB32P- G
Mycobacterium bovis gene for 32kDa 317 2e-84 protein
dbj.vertline.D26486.vertline.MSG32KDAP Mycobacterium bovis genes
for 32kDa 317 2e-84 protei . . . emb.vertline.AL022076.vertlin-
e.MTV026 Mycobacterium tuberculosis H37Rv 317 2e-84 complete g . .
. gb.vertline.U47335.vertline.MTU47335 Mycobacterium tuberculosis
317 2e-84 extracellular 32 . . . dbj.vertline.D78142.vertl-
ine.MSGBCGA85B Mycobacterium bovis gene for alpha 309 5e-82 antige
. . . emb.vertline.Y10378.vertline.MG85AANT M. gordonae gene
encoding 85-A antigen 303 3e-80 dbj.vertline.D78144.vertline.D7814-
4 Mycobacterium avium gene for MPT51, 258 2e-66 antigen 8 . . .
gb.vertline.M90648.vertline.MSG85AA Mycobacterium leprae 85-A
antigen gene, 236 6e-60 compl . . . dbj.vertline.D43841.vertline.M-
SGA85CA Mycobacterium leprae DNA for antigen 85 222 8e-56 com . . .
emb.vertline.X92567.vertline.MMARI147 M marinum gene for 32 kDa
protein 218 1e-54 (partial) emb.vertline.Z33658.vertline.M- A32KP16
M. avium (ATCC 19075) gene for 32kDa 192 7e-47 protein . . .
dbj.vertline.D87323.vertline.D87323 Mycobacterium avium gene for
antigen 186 5e-45 85C and . . . emb.vertline.Z33657.vertli-
ne.MA32KP15 M. avium (ATCC 15769) gene for 32KDa 184 2e-44 protein
. . . emb.vertline.Z33662.vertline.MI32KPI10 M. intracellulare
(ATCC 13950) gene for 168 1e-39 32k . . .
emb.vertline.X92566.vertline.MASIAI22 M. aslaticum gene for 32 kDa
protein 168 1e-39 (partial) emb.vertline.Y07715.vertline.M-
A32KPRO1 M. aslaticum gene segment of 32-KDa 168 1e-39 protein
emb.vertline.Z50760.vertline.MA32K511 M. avium complex gene for 32
KDa protein 168 1e-39 (pa . . . emb.vertline.Z50759.vertline.M-
A32K1112 M. avium complex gene for 32 kDa protein 167 4e-39 (p . .
. emb.vertline.Z50767.vertline.MA32K769 M. avium complex gene for
32 kDa protein 161 3e-37 (pa . . . emb.vertline.Z50774.ver-
tline.MA32K966 M. avium complex gene for 32 kDa protein 161 3e-37
(pa . . . emb.vertline.Z33659.vertline.MA32KP17 M. avium (ATCC
19074) gene for 32kDa 161 3e-37 protein . . .
emb.vertline.Z33661.vertline.MI32KP19 M. intracellulare (ATCC
35762) gene for 161 3e-37 32kD . . .
emb.vertline.Z50763.vertline.MA32- K559 M. avium complex gene for
32 kDa protein 161 3e-37 (pa . . .
emb.vertline.Z50772.vertline.MA32K961 M. avium complex gene for 32
kDa protein 157 4e-36 (pa . . . emb.vertline.Z50765.vertli-
ne.MA32K576 M. avium complex gene for 32 kDa protein 153 6e-35 (pa
. . . emb.vertline.Z50770.vertline.MA32K904 M. avium complex gene
for 32 kDa protein 153 6e-35 (pa . . .
emb.vertline.Z33667.vertline.MM32KPI15 M. malmoense gene for 32kDa
protein 153 6e-35 (partial) emb.vertline.Z50768.vertline.M- A32K814
M. avium complex gene for 32 kDa protein 153 6e-35 (pa . . .
emb.vertline.Z50764.vertline.MA32K575 M. avium complex gene for 32
kDa protein 149 1e-33 (pa . . . emb.vertline.Z50762.ver-
tline.MA32K558 M. avium complex gene for 32 kDa protein 145 2e-32
(pa . . . emb.vertline.X57229.vertline.MT85CG Mycobacteriun
tuberculosis gene for 145 2e-32 antigen 8 . . .
emb.vertline.Z50761.vertline.MA32K554 M. avium complex gene for 32
kDa protein 145 2e-32 (pa . . . emb.vertline.Z92770.vertline.M-
TC15 Mycobacterium tuberculosis H37Rv 145 2e-32 complete geno . . .
emb.vertline.X92570.vertline.MSZUL8 M. szulgai gene for 32 kDa
protein 141 2e-31 (partial) emb.vertline.Z50758.vertline.M-
A32K1076 M. avium complex gene for 32 kDa protein 127 4e-27 (p . .
. emb.vertline.Z50766.vertline.MA32K577 M. avium complex gene for
32 kDa protein 123 6e-26 (pa . . . emb.vertline.Z33654.ver-
tline.MB32KP12 M. bovis (BCG) gene for 32KDa protein 119 9e-25
(parti . . . emb.vertline.X92573.vertline.MTRIV151 M. triviale gene
for 32 kDa protein 109 9e-22 (partial)
emb.vertline.X92583.vertline.MCEL1236 M. celatum gene for 32 KDa
protein 100 8e-19 (partial) emb.vertline.Z21950.vertline.ML85APRA
M. leprae of 85A protein gene 90 8e-16 >gi.vertline.287923.ver-
tline.emb . . . gb.vertline.L78816.vertline.MSGB26CS Mycobacterium
leprae cosmid B26 DNA 88 3e-15 sequence.
emb.vertline.Z21951.vertline.ML85CPRA M. leprae or 85C protein gene
88 3e-15 gb.vertline.M90649.vertline.MSG85CA Mycobacterium leprae
85-C antigen gene, 88 3e-15 compl . . .
emb.vertline.X92582.vertline.MCEL1235 M. celatum gene for 32 kDa
protein 86 1e-14 (partial) emb.vertline.X92577.vertline.MPHLE89 M.
phlei gene for 32 KDa protein 82 2e-13 (partial)
emb.vertline.X92581.vertline.MBRA1077 M. branderi gene for 32 kDa
protein 82 2e-13 (partial) emb.vertline.Y07718.vertline.MF32KPRO4
M. flavescens gene segment or 32-kDa 82 2e-13 protein
emb.vertline.X92575.vertline.MFORT131 M. tortultum gene for 32 kDa
protein 80 8e-13 (partial) emb.vertline.Z33663.vertline.MA- 32KP111
M. avium-intracellulare complex gene for 76 1e-11 32 . . .
emb.vertline.X92576.vertline.MPERE132 M. peregrinum gene for 32 KDa
protein 74 5e-11 (partial) emb.vertline.Z50776.vertlin- e.MAH04894
M. avium complex gene for 32 kDa protein 68 3e-09 (pa . . .
emb.vertline.X92571.vertline.MXENO201 M. xenopi gene for 32 kDa
protein 68 3e-09 (partial) emb.vertline.Y07717.vertlin- e.MS32KPRO3
M. smegmatis gene segment of 32-KDa 66 1e-08 protein
emb.vertline.Z50769.vertline.MA32K822 M. avium complex gene for 32
kDa protein 60 7e-07 (pa . . . emb.vertline.Z50775.vertlin-
e.MAH03994 M. avium complex gene for 32 kDa protein 60 7e-07 (pa .
. . emb.vertline.Y07719.vertline.MV32KPRO5 M. vaccae gene segment
of 32-kDa protein 54 4e-05 emb.vertline.X92580.vertline.MVAC91 M.
vaccae gene for 32 kDa protein 54 4e-05 (partial)
emb.vertline.X92574.vertline.MNONC45 M. nonchromagenicum gene for
32 kDa 48 0.003 protein ( . . . emb.vertline.X92569.vertline.MSIM-
195 M. simiae gene for 32 kDa protein 48 0.003 (partial)
emb.vertline.AJ002150.vertline.MTAJ2150 Mycobacterium tuberculosis
H37Rv, MPT51 46 0.011 gene emb.vertline.Z79700.vertline.MTCY10D7
Mycobacterium tuberculosis H37Rv 44 0.043 complete g . . .
gb.vertline.M58472.vertline.ATUCAT A. tumefaciens chloramphenicol
42 0.17 acetyltransferas . . . emb.vertline.X92578.vertline.MSMEG9-
0 M. smegmatis gene for 32 KDa protein 40 0.66 (partial)
emb.vertline.X92572.vertline.MTER260 M. terrae gene for 32 kDa
protein 40 0.66 (partial) emb.vertline.X92568.vertline.MSCRO149 M.
scrotulaceum gene for 32 KDa protein 40 0.66 (par . . .
emb.vertline.Z33666.vertline.MG32KPI14 M. gordonae (ATCC 14470)
gene for 32kDa 40 0.66 pro . . .
gb.vertline.M17700.vertline.FLCNPC- A Infuenza C/California/78
nucleoprotein 38 2.6 RNA ( . . .
Example 4
Evolution of Broad-Spectrum Vaccines Against Helicobacter
pylori
[0264] Chronic infection of the gastroduodenal mucosae by
Helicobacter pylori bacteria is responsible for chronic active
gastritis, peptic ulcers, and gastric cancers such as
adenocarcinoma and low-grade B-cell lymphoma. An increasing
occurrence of antibiotic-resistant strains is limiting this
therapy. The use of vaccines to both prevent and treat ongoing
infections is being actively pursued (Crabtree JE (1998) Gut 43:
7-8; Axon AT (1998) Gut 43 Suppl 1: S70-3; Dubois et al. (1998)
Infect. Immun. 66: 4340-6; Tytgat GN (1998) Aliment. Pharmacol.
Ther. 12 Suppl 1: 123-8; Blaser MJ (1998) 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) Pharm. World
Sci. 20: 1-17.
[0265] Identification of appropriate Helicobacter antigens for use
in preventive and therapeutic vaccines can include two-dimensional
gel electrophoresis, sequence analysis, and serum profiling (McAtee
et al. (1998) Clin. Diagn. Lab. Immunol. 5:537-42; McAtee et al.
(1998) Helicobacter 3: 163-9). Antigenic differences between
related Helicobacter species and strains can limit the use of
vaccines for prevention and treatment of infections (Keenan et al.
(1998) FEMS Microbiol Lett. 161: 21-7).
[0266] In this Example, DNA family shuffling of related yet
immunologically distinct antigens allows for the isolation of
complex chimeric antigens that can provide a broad cross-reactive
protection against many related strains and species of
Helicobacter. Mouse models of persistent infection by mouse-adapted
H. pylori strains that have been used to evaluate therapeutic use
of vaccines against infection are used to evaluate shuffled
antigens (Crabtree JE (1998) Gut 43: 7-8; Axon AT (1998) Gut 43
Suppl 1:S70-3).
[0267] The vacuolating cytotoxin (VacA) and cytotoxin associated
gene products (CagA) have been evaluated as a vaccine against H.
pylori infection in animal models which supports the application of
this approach in humans.
[0268] Table 7 shows the results of a BLAST search using the H.
pylori VacA gene to identify homologous genes that can be used in a
family shuffling format to obtain improved antigens. Homologous
antigens have been cloned and sequenced from a number of related
yet distinct H. pylori strains and additional natural diversity can
be obtained by cloning antigen genes from other strains. These
genes and others or fragments thereof are cloned by methods such as
PCR, shuffled and screened for improved antigens.
7TABLE 7 Sequences producing significant alignments Score E
Database/Accession No. Gene (bits) Value
gb.vertline.U95971.vertline.HPU95971 Helicobacter pylori 95-54
(J128) 7874 0.0 inactive cy . . . gb.vertline.AE000598.vertline.H-
PAE000598 Helicobacter pylori section 76 of 2468 0.0 134 of . . .
gb.vertline.AF001358.vertline.HPAF001358 Helicobacter pylori
vacuolating 2405 0.0 cytotoxi . . .
emb.vertline.Z26883.vertline.HPCYTTOX H. pylori gene for cytotoxin.
2389 0.0 gb.vertline.U05677.vertline.HPU05677 Helicobacter pylori
87-203 2389 0.0 vacuolating cytot . . .
gb.vertline.U05676.vertline.HPU05676 Helicobacter pylori 60190 2355
0.0 cysteinyl-tRNA syn . . . gb.vertline.U29401.vertline.HPU2940- 1
Helicobacter pylori vacuolating 2345 0.0 cytotoxin ho . . .
emb.vertline.AJ006969.vertline.HPY6969 Helicobacter pylori vacA
gene, 2103 0.0 strain Mz28 . . . gb.vertline.S72494.vertline.AS7-
2494 140 kda cytotoxin [Helicobacter 2050 0.0 pylori, Genomi . . .
gb.vertline.U07145.vertline.HPU07145 Helicobacter pylori NCTC 11638
2050 0.0 cysteinyl tRN . . . emb.vertline.AJ006968.v-
ertline.HPY6968 Helicobacter pylori vacA gene, 2032 0.0 strain Mz26
. . . emb.vertline.AJ006970.vertline.HPY6970 Helicbacter pylori
vacA gene, 1992 0.0 strain Mz29 . . .
gb.vertline.AF077939.vertline.AF077939 Helicobacter pylori strain
166 1834 0.0 vacuolating . . . gb.vertline.AF077940.vertline.A-
F077940 Helicobacter pylori strain 539 1814 0.0 vacuolating . . .
gb.vertline.AF077941.vertline.AF077941 Helicobacter pylori strain
549 1778 0.0 vacuolating . . . gb.vertline.AF077938.vertli-
ne.AF077938 Helicobacter pylori strain 50 1746 0.0 vacuolating . .
. gb.vertline.U63255.vertline.HPU63255 Helicobacter pylori
vacuolating 835 0.0 cytotoxin ge . . .
gb.vertline.U63270.vertline.HPU63270 Helicobacter pylori
vacuolating 819 0.0 cytotoxin ge . . .
gb.vertline.U63272.vertline.HPU6327- 2 Helicobacter pylori
vacuolating 819 0.0 cytotoxin ge . . .
gb.vertline.U63283.vertline.HPU63283 Helicobacter pylori
vacuolating 819 0.0 cytotoxin ge . . .
gb.vertline.U63284.vertline.HPU6328- 4 Helicobacter pylori
vacuolating 819 0.0 cytatoxin ge . . .
gb.vertline.U63262.vertline.HPU63262 Helicobacter pylori
vacuolating 803 0.0 cytotoxin ge . . .
gb.vertline.U63268.vertline.HPU6326- 8 Helicobacter pylori
vacuolating 803 0.0 cytotoxin ge . . .
gb.vertline.U63273.vertline.HPU63273 Helicobacter pylori
vacuolating 803 0.0 cytotoxin ge . . .
gb.vertline.U63282.vertline.HPU6328- 2 Helicobacter pylori
vacuolating 803 0.0 cytotoxin ge . . .
gb.vertline.U63259.vertline.HPU63259 Helicobacter pylori
vacuolating 795 0.0 cytotoxin ge . . .
gb.vertline.U63276.vertline.HPU6327- 6 Helicobacter pylori
vacuolating 779 0.0 cytotoxin ge . . .
gb.vertline.U63287.vertline.HPU63287 Helicobacter pylori
vacuolating 779 0.0 cytotoxin ge . . .
gb.vertline.U63263.vertline.HPU6326- 3 Helicobacter pylori
vacuolating 771 0.0 cytotoxin ge . . .
gb.vertline.Ub3269.vertline.HPU63269 Helicobacter pylori
vacuolating 771 0.0 cytotaxin ge . . .
gb.vertline.U63286.vertline.HPU6328- 6 Helicobacter pylori
vacuolating 771 0.0 cytotoxin ge . . .
gb.vertline.U63275.vertline.HPU63275 Helicobacter pylori
vacuolating 763 0.0 cytotoxin ge . . .
gb.vertline.U63279.vertline.HPU6327- 9 Helicobacter pylori
vacuolating 763 0.0 cytotoxin ge . . .
gb.vertline.U63277.vertline.HPU63277 Helicobacter pylori
vacuolating 755 0.0 cytotoxin ge . . .
gb.vertline.U63280.vertline.HPU6328- 0 Helicobacter pylori
vacuolating 755 0.0 cytotoxin ge . . .
gb.vertline.U63265.vertline.HPU63265 Helicobacter pylori
vacuolating 747 0.0 cytotoxin ge . . .
gb.vertline.U63267.vertline.HPU6326- 7 Helicobacter pylori
vacuolating 747 0.0 cytotoxin ge . . .
gb.vertline.U63281.vertline.HPU63281 Helicobacter pylori
vacuolating 741 0.0 cytotoxin ge . . .
gb.vertline.U63261.vertline.HPU6326- 1 Helicobacter pylori
vacuolating 739 0.0 cytotoxin ge . . .
gb.vertline.U63274.vertline.HPU63274 Helicobacter pylori
vacuolating 739 0.0 cytotoxin ge . . .
gb.vertline.U63285.vertline.HPU6328- 5 Helicobacter pylori
vacuolating 737 0.0 cytotoxin ge . . .
emb.vertline.AJ009430.vertline.HPAJ9430 Helicobacter pylori vacA
gene 737 0.0 (partial), . . . emb.vertline.AJ009435.vertline.HP-
AJ9435 Helicobacter pylori vacA gene 730 0.0 (partial), . . .
emb.vertline.AJ009439.vertline.HPAJ9439 Helicobacter pylori vacA
gene 730 0.0 (partial) , . . . gb.vertline.U63271.vertline.HPU6-
3271 Helicobacter pylori vacuolating 724 0.0 cytotoxin ge . . .
emb.vertline.AJ009418.vertline.HPAJ9418 Helicobacter pylori vacA
gene 722 0.0 (partial), . . . emb.vertline.AJ009422.vertline.HP-
AJ9422 Helicobacter pylori vacA gene 722 0.0 (partial), . . .
gb.vertline.U63256.vertline.HPU63256 Helicobacter pylori
vacuolating 716 0.0 cytotoxin ge . . .
gb.vertline.U63266.vertline.HPU- 63266 Helicobacter pylori
vacuolating 716 0.0 cytotoxin ge . . .
emb.vertline.AJ009420.vertline.HPAJ9420 Helicobacter pylori vacA
gene 714 0.0 (partial), . . . emb.vertline.AJ009424.vertli-
ne.HPAJ9424 Helicobacter pylori vacA gene 714 0.0 (partial), . . .
emb.vertline.AJ009431.vertline.HPAJ9431 Helicobacter pylori VacA
gene 714 0.0 (partial), . . . gb.vertline.U63260.vertline.-
HPU63260 Helicobacter pylori vacuolating 708 0.0 cytotoxin ge . . .
gb.vertline.U63278.vertline.HPU63278 Helicobacter pylori
vacuolating 708 0.0 cytotoxin ge . . .
emb.vertline.AJ009419.vertline.HPAJ9419 Helicobacter pylori vacA
gene 706 0.0 (partial), . . .
emb.vertline.AJ009428.vertline.HPAJ94- 28 Helicobacter pylori vacA
gene 706 0.0 (partial), . . .
emb.vertline.AJ009437.vertline.HPAJ9437 Helicobacter pylori vacA
gene 706 0.0 (partial), . . .
emb.vertline.AJ009427.vertline.HPAJ94- 27 Helicobacter pylori vacA
gene 704 0.0 (partial), . . . gb.vertline.U63257.vertline.HPU63257
Helicobacter pylori vacuolating 700 0.0 cytotoxin ge . . .
emb.vertline.AJ009423.vertline.HPAJ- 9423 Helicobacter pylori vacA
gene 698 0.0 (partial), . . .
emb.vertline.AJ009432.vertline.HPAJ9432 Helicobacter pylori vacA
gene 692 0.0 (partial), . . .
emb.vertline.AJ009417.vertline.HPAJ94- 17 Helicobacter pylori vacA
gene 688 0.0 (partial), . . .
emb.vertline.AJ009421.vertline.HPAJ9421 Helicobacter pylori vacA
gene 688 0.0 (partial), . . .
emb.vertline.AJ009426.vertline.HPAJ94- 26 Helicobacter pylori vacA
gene 688 0.0 (partial), . . .
emb.vertline.AJ009438.vertline.HPAJ9438 Helicobacter pylori vacA
gene 688 0.0 (partial), . . . gb.vertline.U63264.vertline.HPU63264
Helicobacter pylori vacuolating 676 0.0 cytotoxin ge . . .
emb.vertline.AJ009433.vertline.HPAJ9433 Helicobacter pylori vacA
gene 666 0.0 (partial), . . .
emb.vertline.AJ009425.vertline.HPAJ94- 25 Helicobacter pylori vacA
gene 658 0.0 (partial), . . . gb.vertline.U63258.vertline.HPU63258
Helicobacter pylori vacuolating 652 0.0 cytotoxin ge . . .
emb.vertline.AJ009442.vertline.HPAJ- 9442 Helicobacter pylori vacA
gene 626 e-177 (partial), . . .
emb.vertline.AJ009444.vertline.HPAJ9444 Helcobacter pylori vacA
gene 626 e-177 (partial), . . . gb.vertline.U80068.vertline.HPU-
80068 Helicobacter pylori strain 213, 622 e-175 vacuolating . . .
emb.vertline.AJ009434.vertline.HPAJ9434 Helicobacter pylori vacA
gene 618 e-174 (partial), . . . emb.vertline.AJ009441.vert-
line.HPAJ9441 Helicobacter pylori vacA gene 603 e-170 (partial), .
. . gb.vertline.AF035616.vertline.HPVCP2 Helicobacter pylori strain
R34A 599 e-168 vacuolating . . .
emb.vertline.AJ009447.vertline.HPAJ9447 Helicobacter pylori vacA
gene 587 e-165 (partial), . . .
emb.vertline.AJ009440.vertline.HPAJ- 9440 Helicobacter pylori vacA
gene 563 e-158 (partial), . . .
emb.vertline.AJ009436.vertline.HPAJ9436 Helicobacter pylori vacA
gene 555 e-155 (partial), . . . emb.vertline.AJ009443.vertline.-
HPAJ9443 Helicobacter pylori vacA gene 555 e-155 (partial), . . .
emb.vertline.AJ009446.vertline.HPAJ9446 Helicobacter pylori vacA
gene 555 e-155 (partial), . . . gb.vertline.U80067.vertlin-
e.HPU80067 Helicobacter pylori strain 184, 553 e-155 vacuolating .
. . gb.vertline.AF042735.vertline.AF042735 Helicobacter pylori JK22
553 e-155 vacuolating cytot . . .
emb.vertline.AJ009445.vertline.HPAJ9445 Helicobacter pylori vacA
gene 547 e-153 (partial), . . .
gb.vertline.AF035609.vertline.AF035- 609 Helicobacter pylori strain
R10A 547 e-153 vacuolatin . . .
gb.vertline.AF042734.vertline.AF042734 Helicobacter pylori JK1
vacuolating 537 e-150 cytoto . . . gb.vertline.AF035612.ve-
rtline.AF035612 Helicobacter pylori strain R26A 347 9e-93
vacuolatin . . . gb.vertline.AF035613.vertline.AF035613
Helicobacter pylori strain R40A 323 1e-85 vacuolatin . . .
emb.vertline.AJ006967.vertline.HPY6967 Helicobacter pylori vacA
gene, 317 8e-84 strain Mz19 . . .
gb.vertline.AF035615.vertline.HPVC- P1 Helicobacter pylori strain
R34A 315 3e-83 vacuolating . . .
gb.vertline.AF035614.vertline.AF035614 Helicobacter pylori strain
R50A 307 8e-81 vacuolatin . . . gb.vertline.U91578.vertline.HPU-
91578 Helicobacter pylori strain F37 vacA 109 4e-21 gene, pa . . .
gb.vertline.U91579.vertline.HPU91579 Helicobacter pylori strain F79
vacA 109 4e-21 gene, pa . . . gb.vertline.U91575.vertl-
ine.HPU91575 Helicobacter pylori strain F84 vacA 107 1e-20 gene, pa
. . . emb.vertline.Y14742.vertline.HPVACA26 Helicobacter pylori
partial vacA 101 9e-19 gene, stra . . .
gb.vertline.U91577.vertline.HPU91577 Helicobacter pylori strain F94
vacA 100 3e-18 gene, pa . . . gb.vertline.U91576.vertline.HPU91-
576 Helicobacter pylori strain F71 vacA 100 3e-18 gene, pa . . .
gb.vertline.AF035610.vertline.AF035610 Helicobacter pylori strain
R13A 94 2e-16 vacuolatin . . . gb.vertline.U91580.vertline-
.HPU91580 Helicobacter pylori strain F80 vacA 92 8e-16 gene, pa . .
. gb.vertline.AF0345611.vertline.AF035611 Helicobacter pylori
strain R59A 92 8e-16 vacuolatin . . .
emb.vertline.Y14744.vertline.HPVACA49 Helicobacter pylori partial,
VacA 88 1e-14 gene, stra . . .
[0269] Table 8 shows the results of a BLAST search using the H.
pylori CagA gene to identify homologous genes that can be used in a
family shuffling format to obtain improved antigens. Homologous
antigens have been cloned and sequenced from a number of related
yet distinct H. pylori strains and additional natural diversity can
be obtained by cloning antigen genes from other strains. These
genes and others or fragments thereof are be cloned by methods such
as PCR, shuffled and screened for improved antigens.
8TABLE 8 Sequences producing significant alignments Score E
Database/Accession No. Gene (bits) Value
gb.vertline.AF083352.vertline.AF083352 Helicobacter pylori
cytotoxin associated p . . . 7041 0.0
gb.vertline.AE000569.vertline.HPAE0005- 69 Helicobacter pylori
section 47 of 134 of . . . 5501 0.0
gb.vertline.L11714.vertline.HECMAJANT Helicobacter pylori major
antigen gene 4976 0.0 sequ . . . emb.vertline.X70039.vertline.HPCA-
I H. pylori cai gene for cytotoxicity associated 4294 0.0 . . .
gb.vertline.U60176.vertline.HPU60176 Helicobacter pylori cag
pathogenicity 4294 0.0 island . . .
dbj.vertline.AB003397.vertline.AB003397 Helicobacter pylori DNA for
CagA, 4274 0.0 complet . . . gb.vertline.U80066.vertline.HPU8006- 6
Helicobacter pylori strain 213, cytotoxin- 349 2e-93 as . . .
gb.vertline.U80065.vertline.HPU80065 Helicobacter pylori strain
184, cytotoxin- 343 1e-91 as . . . gb.vertline.AF043488.vertlin-
e.AF043488 Helicobacter pylori JK252 cytotoxicity 178 4e-42 ass . .
. gb.vertline.AF043487.vertline.AF043487 Helicobacter pylori JK25
cytotoxicity 170 1e-39 asso . . .
gb.vertline.AF043489.vertline.AF043489 Helicobacter pylori JK269
cytotoxicity 163 2e-37 ass . . . emb.vertline.X70038.vertl-
ine.HPCAIDUP H. pylori DNA duplication sequence within 159 4e-36 th
. . . gb.vertline.AF043490.vertline.AF043490 Helicobacter pylori
JK22 cytotoxicity 153 2e-34 asso . . .
Example 5
Development Of Broad-Spectrum Vaccines Against Malaria
[0270] This Example describes the use of DNA shuffling to generate
improved vaccines against malaria infection. An excellent target
for evolution by DNA shuffling is the Plasmodium falciparum
merozoite surface protein, MSP 1 (Hui et al. (1996) Infect. Immun.
64: 1502-1509). MSP 1 is expressed on the surface of merozoites as
an integral membrane protein. It is cleaved by parasite proteases
just before and concomitant with rupture and release from infected
cells. The cleavage appears to be obligatory for full function in
MSP 1 binding to RBC receptors. The cleaved fragments remain
attached to the membrane of the merozoite. Other membrane proteins
on merozoites also participate in the attachment and specific
invasion events. MSP1 is a proven candidate for inclusion in a
vaccine against the asexual blood stage of malaria.
[0271] The genes encoding MSP 1 can be isolated from various
isolates of Plasmodium falciparum merozoites by PCR technology.
Related naturally existing genes can be additionally used to
increase the diversity of the starting genes. A library of shuffled
MSP1 genes is generated by DNA shuffling, and this library is
screened for induction of efficient immune responses.
[0272] The screening can be done by injecting individual variants
into test animals, such as mice or monkeys. Either purified
recombinant proteins, or DNA vaccines or viral vectors encoding the
relevant genes are injected. Typically, a booster injection is
given 2-3 weeks after the first injection. Thereafter, the sera of
the test animals are collected and these sera are analyzed for the
presence of antibodies that reduce invasion of merozoites into
uninfected erythrocytes (RBC). RBC are infected by the merozoite,
immediately inside the RBC, the merozoite differentiates into a
ring and this matures to a schizont that contains several nascent
daughter merozoites, which then burst out of the infected cell,
destroying it, and go on to attach and invade another RBC. In vivo,
the merozoite is likely only extracellular for seconds. In vitro,
any blockade of this event can dramatically reduce the level of
reinfection. Antibodies against MSP 1 bind to the surface of
merozoites that are released from schizont infected RBC when they
rupture and thereby reduce the ability of these merozoites to
attach and engage cognate RBC receptors on the uninfected RBC
surface. Merozoite attachment is reduced, merozoite entry into new
RBC is reduced, and the numbers of newly invaded cells detected at
the early ring stage is therefore reduced if the culture is
examined several hours after the blockade of invasion test. In some
assay formats a surrogate of merozoite invasion inhibition is to
note the appearance of agglutinated merozoites, although this is an
indirect measure of antibodies that cause reduced invasion.
[0273] The shuffled antigens that induce the most potent antibody
responses reducing invasion of merozoites into uninfected
erythrocytes are selected for further testing and can be subjected
to new rounds of shuffling and selection. In subsequent studies,
the capacity of these antigens to induce antibodies in man is
investigated. Again, either purified recombinant antigens, or DNA
vaccines or viral vectors encoding the relevant genes are injected
and the protective immune responses are analyzed.
Example 6
Development of Broad-Spectrum Vaccines Against Viral Pathogens
[0274] This Example describes the use of DNA shuffling to obtain
vaccines that can induce an immune response against multiple
isolates of viral pathogens.
[0275] A. Venezuelan Equine Encephalitis Virus (VEE)
[0276] VEE belongs to the alphavirus genus, which are generally
transmitted by mosquitoes. However, VEE is an unusual alphavirus in
that it is also highly infectious by aerosol inhalation for both
humans and rodents. The disease manifestations in humans range from
subclinical or mild febrile disease to serious infection and
inflammation of the central nervous system. Virus clearance
coincides that of production of specific anti-VEE antibodies, which
are believed to be the primary mediators of protective immune
responses (Schmaljohn et al. (1982) Nature 297: 70). VEE is an
unusual virus also because its primary target outside the central
nervous system is the lymphoid tissue, and therefore, replication
defective variants may provide means to target vaccines or
pharmaceutically useful proteins to the immune system.
[0277] At least seven subtypes of VEE are known that can be
identified genetically and serologically. Based on epidemiological
data, the virus isolates fall into two main categories: I-AB and
I-C strains, which are associated with VEE epizootics/epidemics,
and the remaining serotypes, which are associated primarily with
enzootic vertebrate-mosquito cycles and circulate in specific
ecological zones (Johnston and Peters, In Fields Virology, Third
Edition, eds. B. N. Fields et al., Lippincott-Raven Publishers,
Philadelphia, 1996).
[0278] The envelope protein (E) appears to be the major antigen in
inducing neutralizing Abs. Accordingly, DNA shuffling is used to
obtain a library of recombinant E proteins by shuffling the
corresponding genes derived from various strains of VEE. These
libraries and individuals chimeras/mutants thereof are subsequently
screened for their capacity to induce widely cross-reacting and
protective Ab responses.
[0279] B. Flaviviruses
[0280] Japanese encephalitis virus (JE), Tick-borne encephalitis
virus (TBE) and Dengue virus are arthropod-borne viruses belonging
to the Flavivirus family, which comprises 69 related viruses. The
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 a tetravalent vaccine that induces
neutralizing Abs against all four serotypes is necessary. Moreover,
non-neutralizing antibodies induced by infection or vaccination by
one Dengue virus may cause enhancement of the disease during a
subsequent infection by another serotype. Therefore,
cross-protective, broad spectrum vaccines for TBE and JE would
provide significant improvements to the existing vaccines. In this
Example, the ability of DNA shuffling to efficiently-generate
chimeric and mutated genes is used to generate cross-protective
vaccines.
[0281] 1. Japanese Encephalitis Virus
[0282] Japanese encephalitis virus (JE) is a prototype of the JE
antigenic complex, which comprises St. Louis encephalitis virus,
Murray Valley encephalitis virus, Kunjin virus and West Nile virus
(Monath and Heinz, In Fields Virology, Third Edition, eds. B. N.
Fields et al., Lippincott-Raven Publishers, Philadelphia, pp
961-1034, 1996). Infections caused by JE are relatively rare, but
the case-fatality is 5-40% because no specific treatment is
available. JE is widely distributed in China, Japan, Philippines,
far-eastern Russia and India providing a significant threat to
those traveling in these areas. Currently available JE vaccine is
produced from brain tissues of mice infected with single virus
isolate. Side effects are observed in 10% to 30% of the
vaccinees.
[0283] To obtain chimeric and/or mutated antigens that provide a
protective immune response against all or most of the viruses
within the JE complex, DNA shuffling is performed on viral envelope
genes. The amino acid identity within the JE complex varies between
72% and 93%. In addition, significant antigenic variation has been
observed among JE strains by neutralization assays, agar gel
diffusion, antibody absorption and monoclonal antibody analysis
(Oda (1976) Kobe J. Med. Sci. 22: 123; Kobyashi et al. (1984)
Infect. Immun. 44: 117). Moreover, the amino acid divergence of the
envelope protein gene among 13 strains from different Asian
countries is as much as 4.2% (Ni and Barrett (1995) J. 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.
[0284] 2. Tick-Borne Encephalitis Virus
[0285] The tick-borne encephalitis virus complex comprises 14
antigenically related viruses, eight of which cause human disease,
including Powassan, Louping ill and Tick-borne encephalitis virus
(TBE) (Monath and Heinz, In Fields Virology, Third Edition, eds. B.
N. Fields et al., Lippincott-Raven Publishers, Philadelphia, pp.
961-1034, 1996). TBE has been recognized in all Central and Eastern
European countries, Scandinavia and Russia, whereas Powassan occurs
in Russia, Canada and the United States. The symptoms vary from
flu-like illness to severe meningitis, meningoencephalitis and
meningoencephalitis with a fatality rate of 1% to 2% (Gresikova and
Calisher, In Monath ed., The arboviruses: ecology and epidemiology,
vol. IV, Boca Raton, Fla., CRC Press, pp. 177-203, 1988).
[0286] Family DNA shuffling is used to generate chimeric envelope
proteins derived from the TBE complex to generate crossprotective
antigens. The 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 envelope protein of
Powassan is 78% identical at the amino acid level with that of TBE,
and cross-protection is unlikely, although epidemiological data is
limited.
[0287] Langat virus is used as a model system to analyze protective
immune responses in vivo (Iacono-Connors et al. (1996) Virus Res.
43: 125). Langat virus belongs to the TBE complex, and can be used
in challenge studies in BSL3 facilities. Serological studies based
on recombinant envelope proteins are performed to identify
immunogen variants that induce high levels of antibodies against
envelope proteins derived from most or all viruses of the TBE
complex.
[0288] 3. Dengue Viruses
[0289] Dengue viruses are transmitted though mosquito bites, posing
a significant threat to troops and civilian populations
particularly in tropical areas. There are four major serotypes of
Dengue virus, namely Dengue 1, 2, 3 and 4. A tetravalent vaccine
that induces neutralizing antibodies against all four strains of
Dengue is required to avoid antibody-mediated enhancement of the
disease when the individual encounters the virus of the other
strain.
[0290] The envelope protein of Dengue virus has been shown to
provide an immune response that protects from a future 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 genetic vaccines encoding envelope protein of Dengue-2 virus
developed neutralizing antibodies when analyzed by in vitro
neutralization assays, but the mice did not survive the challenge
with live Dengue-2 virus (Kochel et al. (1997) Vaccine 15:
547-552). However, protective immune responses were observed in
mice immunized with recombinant vaccinia virus expressing Dengue 4
virus structural proteins (Bray et al. (1989) J. Virol. 63: 2853).
These studies indicate that vaccinations with E proteins work, but
significant improvements in the immunogenicity of the protective
antigens are required.
[0291] In this Example, DNA shuffling is performed on the genes
encoding the envelope (E) protein from all four Dengue viruses and
their antigenic variants. Family DNA shuffling is used to generate
chimeric E protein variants that induce high titer neutralizing
antibodies against all serotypes of Dengue. The E-proteins of the
different dengue viruses share 62% to 77% of their amino acids.
Dengue I and Dengue 3 are most closely related (77% homologous),
followed by Dengue 2 (69%) and Dengue 4 (62%). These homologies are
well in the range that allows efficient family shuffling (Crameri
et al. (1998) Nature 391: 288-291).
[0292] The shuffled antigen sequences are incorporated into genetic
vaccine vectors, the plasmids purified, and subsequently injected
into mice. The sera are collected from the mice and analyzed for
the presence of high levels of cross-reactive antibodies. The best
antigens are selected for further studies using in vivo challenge
models to screen for chimeras/mutants that induce cross-protection
against all strains of Dengue.
[0293] C. Improved Expression and Immunogenicity of Hantaan Virus
Glycoproteins
[0294] One of the advantages of genetic vaccines is that vectors
expressing pathogen antigens can be generated even when the given
pathogen cannot be isolated in culture. An example of such
potential situation was an outbreak of severe respiratory disease
among rural residents of the Southwestern United States which was
caused by a previously unknown hantavirus, Sin Nombre virus (Hjelle
et al. (1994) J. Virol. 68: 592). Much RNA sequence information of
the virus was obtained well before the virus could be isolated and
characterized in vitro. In these situations, genetic vaccines can
provide means to generate efficient vaccines in a short period of
time by creating vectors encoding antigens encoded by the pathogen.
However, genetic vaccines can only work if these antigens can be
properly expressed in the host.
[0295] Hantaan virus belongs to the Bunyavirus family. A
characteristic feature of this family is that their glycoproteins
typically accumulate at the membranes of the Golgi apparatus when
expressed by cloned cDNAs, thereby reducing the efficacy of
corresponding genetic vaccines (Matsuoka et al. (1991) Curr. Top.
Microb. Immunol. 169: 161-179). Poor expression of Hantaan virus
glycoproteins on the cell surface is also one explanation for poor
immune responses following injections of Hantaan virus genetic
vaccines.
[0296] In this Example, family DNA shuffling is used to generate
recombinant Hantaan virus derived glycoproteins that are
efficiently expressed in human cells and that can induce protective
immune responses against the wild-type pathogen. Nucleic acids that
encode the Hantaan virus glycoprotein are shuffled with genes that
encode other homologous Bunyavirus glycoproteins. The resulting
library is screened to identify proteins that are readily expressed
in human cells. The screening is performed using a dual marker
expression vector that enables simultaneous analysis of
transfection efficiency and expression of fusion proteins that are
PIG-linked to the cell surface (Whitehorn et al. (1995)
Biotechnology (N Y) 13:1215-9).
[0297] Flow cytometry based cell sorting is used to select Hantaan
virus glycoprotein variants that are efficiently expressed in
mammalian cells. The corresponding sequences are then obtained by
PCR or plasmid recovery. These chimeras/mutants are further
analyzed for their capacity to protect wild mice against Hantaan
virus infections.
Example 7
DNA Shuffling of HSV-1 And HSV-2 Glycoproteins B and/or D as Means
to Induce Enhanced Protective Immune Responses
[0298] This Example describes the use of DNA shuffling to obtain
HSV glycoprotein B (gB) and glycoprotein D (gD) polypeptides that
exhibit improved ability to induce protective immune responses upon
administration to a mammal. Epidemiological studies have shown that
prior infections with HSV-1 give partial protection against
infections with HSV-2, indicating existence of cross-reactive
immune responses. Based on previous vaccination studies, the main
immunogenic glycoproteins in HSV appear to be gB and gD, which are
encoded by 2.7 kb and 1.2 kb genes, respectively. The gB and gD
genes of HSV-1 are about 85% identical to the corresponding gene of
HSV-2, and the gB genes of each share little sequence identity with
the gD genes. Baboon HSV-2 gB is appr. 75% identical to human HSV-1
or -2 gB, with rather long stretches of almost 90% identity. In
addition, 60-75% identity is found in portions of the genes of
equine and bovine herpesviruses.
[0299] Family shuffling is employed using as substrates nucleic
acids that encode gB and/or gD from HSV-1 and HSV-2. Preferably,
homologous genes are obtained from HSVs of various strains. An
alignment of gD nucleotide sequences from HSV-1 and two strains of
HSV-2 is shown in FIG. 7. Antigens encoded by the shuffled nucleic
acids are expressed and analyzed in vivo. For example, one can
screen for improved induction of neutralizing antibodies and/or CTL
responses against HSV-I/HSV-2. One can also detect protective
immunity by challenging mice or guinea pigs with the viruses.
Screening can be done using pools or individuals clones.
Example 8
Evolution of HIV Gp120 Proteins for Induction of Broad Spectrum
Neutralizing Ab Responses
[0300] This Example describes the use of DNA shuffling to generate
immunogens that crossreact among different strains of viruses,
unlike the wild-type immunogens. Shuffling two kinds of envelope
sequences can generate immunogens that induce neutralizing
antibodies against a third strain.
[0301] Antibody-mediated neutralization of HIV-1 is strictly
type-specific. Although neutralizing activity broadens in infected
individuals over time, induction of such antibodies by vaccination
has been shown to be extremely difficult. Antibody-mediated
protection from HIV-1 infection in vivo correlates with
antibody-mediated neutralization of virus in vitro.
[0302] FIG. 8 illustrates the generation of libraries of shuffled
gp120 genes. gp120 genes derived from HIV-1DH12 and HIV-11IIB(NL43)
are shuffled. The chimeric/mutant gp120 genes are then analyzed for
their capacity to induce antibodies that have broad spectrum
capacity to neutralize different strains of HIV. Individual
shuffled gp120 genes are incorporated into genetic vaccine vectors,
which are then introduced to mice by injection or topical
application onto the skin. These antigens can also be delivered as
purified recombinant proteins. The immune responses are measured by
analyzing the capacity of the mouse sera to neutralize HIV growth
in vitro. Neutralization assays are performed against HIV-1DH12,
HIV-11IIB and HIV-189.6. The chimeras/mutants that demonstrate
broad spectrum neutralization are chosen for further rounds of
shuffling and selection. Additional studies are performed in
monkeys to illustrate the capacity of the shuffled gp120 genes to
provide protection for subsequent infection with immunodeficiency
virus.
Example 9
Antigen Shuffling of the Hepadnavirus Envelope Protein
[0303] The Hepatitis B virus (HBV) is one of a member of a family
of viruses called hepadnaviruses. This Example describes the use of
genomes and individual genes from this family are used for DNA
shuffling, which results in antigens having improved
properties.
[0304] A. Shuffling of Hepadnavirus Envelope Protein Genes
[0305] The envelope protein of the HBV assembles to form particles
that carry the antigenic structures collectively known as the
Hepatitis B surface antigen (HBsAg; this term is also used to
designate the protein itself). Antibodies to the major antigenic
site, designated the "a" epitope (which is found in the envelope
domain called S), are capable of neutralizing the virus.
Immunization with the HBsAg-bearing protein thus serves as a
vaccine against viral infection. The HBV envelope also contains
other antigenic sites that can protect against viral infection and
are potentially vital components of an improved vaccine. The
epitopes are part of the envelope protein domains known as preS1
and preS2 (FIG. 9).
[0306] DNA shuffling of the envelope gene from several members of
the hepadnavirus family is used to obtain more immunogenic
proteins. Specifically, the genes from the following hepatitis
viruses are shuffled:
[0307] the human HBV viruses, subtypes ayw and adw2
[0308] a hepatitis virus isolated from chimpanzee
[0309] a hepatitis virus isolated from gibbon
[0310] a hepatitis virus isolated from woodchuck
[0311] If desired, genes from other genotypes of the human virus
are available for inclusion in the DNA shuffling reactions.
Likewise, other animal hepadnaviruses are available.
[0312] To promote the efficiency of the formation of chimeras
resulting from DNA shuffling, some artificial genes are made:
[0313] In one case, a synthetic gene is made that contains the HBV
envelope sequences, except for those codons which specify amino
acids found in the chimpanzee and gibbon genes. For those codons,
the chimpanzee or gibbon sequence is used.
[0314] In a second case, a synthetic gene is synthesized in which
the preS2 gene sequence from the human HBV adw2 strain is fused
with the woodchuck S region.
[0315] In a third case, all the oligonucleotides required to
chemically synthesize each of the hepadnavirus envelope genes are
mixed in approximately equal quantities and allowed to anneal to
form a library of sequences.
[0316] After DNA shuffling of the hepadnavirus envelope genes,
either or both of two strategies are used to obtain improved HBsAg
antigens.
[0317] Strategy A: Antigens are screened by immunizing mice using
two possible methods. The genes are injected in the form of DNA
vaccines, i.e., shuffled envelope genes carried by a plasmid that
comprises the genetic regulatory elements required for expression
of the envelope proteins. Alternatively, the protein is prepared
from the shuffled genes and used as the immunogen.
[0318] The sequences that give rise to greater immunogenicity for
either the preS1-, preS2- or S-borne HBV antigens are selected for
a second round of shuffling (FIG. 10). For the second round, the
best candidates are chosen based on their improved antigenicity and
their other properties such as higher expression level or more
efficient secretion. Screening and further rounds of shuffling are
continued until a maximum optimization for one of the antigenic
regions is obtained.
[0319] The individually optimized genes are then used as a
combination vaccine for the induction of optimal responses to
preS1-, preS2- and S-born epitopes.
[0320] Strategy B: After isolation of the individually optimized
genes as in Strategy A, the preS 1, preS2 and S candidates are
shuffled together, or in a pairwise fashion, in further rounds to
obtain genes which encode proteins that demonstrate improved
immunogenicity for at least two regions containing HBsAg epitopes
(FIG. 11).
[0321] B. Use of HBsAg to Carry Epitopes from Unrelated
Antigens
[0322] Several of the characteristics of the HBsAg make it a useful
protein to carry epitopes drawn from other, unrelated antigens. The
epitopes can be either B epitopes (which induce antibodies) or T
epitopes drawn from the class I type (which stimulate CD8.sup.+ T
lymphocytes and induced cytotoxic cells) or class II type (which
induce helper T lymphocytes and are important in providing
immunological memory responses.
[0323] 1. B cell Epitopes
[0324] Amino acid sequences of potential B epitopes are chosen from
any pathogen. Such sequences are often known to induce antibodies,
but the immunogenicity is weak or otherwise unsatisfactory for
preparation of a vaccine. These sequences can also be mimotopes,
which have been selected based on their ability to have a certain
antigenicity or immunogenicity.
[0325] The amino acid coding sequences are added to a hepadnavirus
envelope gene. The heterologous sequences can either replace
certain envelope sequences, or be added in addition to all the
envelope sequences. The heterologous epitope sequences can be
placed at any position in the envelope gene. A preferred position
is the region of the envelope gene that encodes the major "a"
epitope of the HBsAg (FIG. 12). This region is likely to be exposed
on the external side of the particles formed by the envelope
protein, and thus will expose the heterologous epitopes.
[0326] DNA shuffling is carried out on the envelope gene sequences,
keeping the sequence of the heterologous epitopes constant.
Screening is carried out to choose candidates that are secreted
into the culture medium after transfection of plasmids from the
shuffled library into cells in tissue culture.
[0327] Clones that encode a secreted protein are then tested for
immunogenicity in mice either as a DNA vaccine or as a protein
antigen, as described above. Clones that give an improved induction
of antibodies to the heterologous epitopes are chosen for further
rounds of DNA shuffling. The process is continued until the
immunogenicity of the heterologous epitope is sufficient for use as
a vaccine against the pathogen from which the heterologous epitopes
were derived.
[0328] 2. Class I Epitopes
[0329] MHC Class I epitopes are relatively short, linear peptide
sequences that are generally between 6 and 12 amino acids amino
acids in length, most often 9 amino acids in length. 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 cells.
[0330] Polynucleotide sequences that encode one or more class I
epitopes are inserted into the sequence of a hepadnavirus envelope
gene either by replacing certain envelope sequences, or by
inserting the epitope sequences into the envelope gene. This is
typically done by modifying the gene before DNA shuffling or by
including in the shuffling reaction certain oligonucleotide
fragments that encode the heterologous epitopes as well as
sufficient flanking hepadnavirus sequences to be incorporated into
the shuffled products.
[0331] Preferably, the heterologous class I epitopes are placed
into different positions in the several hepadnavirus genes used for
the DNA shuffling reaction. This will optimize the chances for
finding chimeric gene carrying the epitopes in an optimal position
for efficient presentation.
[0332] 3. Class II Epitopes
[0333] MHC Class II epitopes are generally required to be part of a
protein which is taken up by antigen presenting cells, rather than
synthesized within the cell. Preferably, such epitopes are
incorporated into a carrier protein such as the HBV envelope that
can be produced in a soluble form or which can be secreted if the
gene is delivered in the form of a DNA vaccine.
[0334] Polynucleotides that encode heterologous class II epitopes
are inserted into regions of the hepadnavirus envelope genes that
are not involved in the transmembrane structure of the protein. DNA
shuffling is performed to obtain a secreted protein that also
carries the class II epitopes. When injected as a protein, or when
the gene is delivered as a DNA vaccine, the protein can be taken up
by antigen presenting cells for processing of the class II
epitopes.
Example 10
Evolution of Broad Spectrum Vaccines Against Hepatitis C Virus.
[0335] Antigenic heterogeneity of different strains of Hepatitis C
Virus (HCV) is a major problem in development of efficient vaccines
against HCV. Antibodies or CTLs specific for one strain of HCV
typically do not protects against other strains. Multivalent
vaccine antigens that simultaneously protect against several
strains of HCV would be of major importance when developing
efficient vaccines against HCV.
[0336] The HCV envelope genes, which encode envelope proteins E1
and E2, have been shown to induce both antibody and
lymphoproliferative responses against these antigens (Lee et al.
(1998) J. Virol. 72: 8430-6), and these responses can be optimized
by DNA shuffling. The hypervariable region I (HVR1) of the envelope
protein E2 of HCV is the most variable antigenic fragment in the
whole viral genome and is primarily responsible for the large
inter- and intra-individual heterogeneity of the infecting virus
(Puntoriero et al. (1998) EMBO J. 17: 3521-33). Therefore, the gene
encoding E2 is a particularly useful target for evolution by DNA
shuffling.
[0337] DNA shuffling of HCV antigens, such as nucleocapsid or
envelope proteins E1, E2, provides a means to generate multivalent
HCV vaccines that simultaneously protect against several strains of
HCV. These antigens are shuffled using the family DNA shuffling
approach. The starting genes will be obtained from various natural
isolates of HCV. In addition, related genes from other viruses can
be used to increase the number of different recombinants that are
generated. A library of related, chimeric variants of HCV antigens
are then generated and this library will be screened for induction
of widely crossreactive immune responses. The screening can be done
directly in vivo by injecting individual variants into test
animals, such as mice or monkeys. Either purified recombinant
proteins or DNA vaccines encoding the relevant genes are injected.
Typically, a booster injection is given 2-3 weeks after the first
injection. Thereafter, the sera of the test animals are collected
and these sera are tested for the presence of antibodies that react
against multiple HCV virus isolates.
[0338] Before the in vivo testing is initiated, the antigens can be
pre-enriched in vitro for antigens that are recognized by
polyclonal antisera derived from previously infected patients or
test animals. Alternatively, monoclonal antibodies that are
specific for various strains of HCV are used. The screening is
performed using phage display or ELISA assays. For example, the
antigen variants are expressed on bacteriophage M13 and the phage
are then incubated on plates coated with antisera derived from
patients or test animals infected with various HCV isolates. The
phage that bind to the antibodies are then eluted and further
analyzed in test animals for induction of crossreactive
antibodies.
Example 11
Evolution of Chimeric Allergens that Induce Broad Immune Responses
and have Reduced Risk of Inducing Anaphylactic Reactions
[0339] Specific immunotherapy of allergy is performed by injecting
increasing amounts of the given allergens into the patients. The
therapy typically alters the types of allergen-specific immune
responses from a dominating T helper 2 (T.sub.H2) type response to
a dominating T helper I (T.sub.H1) type response. However, because
allergic patients have increased levels of IgE antibodies specific
for the allergens, the immunotherapy of allergy involves a risk of
IgE receptor mediated anaphylactic reactions.
[0340] T helper (T.sub.H) cells are capable of producing a large
number of different cytokines, and based on their cytokine
synthesis pattern T.sub.H cells are divided into two subsets (Paul
and Seder (1994) Cell 76: 241-251). T.sub.H1 cells produce high
levels of IL-2 and IFN-gamma and no or minimal levels of IL-4, IL-5
and IL-13. In contrast, T.sub.H2 cells produce high levels of IL-4,
IL-5 and IL-13, whereas IL-2 and IFN-gamma production is minimal or
absent. T.sub.H1 cells activate macrophages, dendritic cells and
augment the cytolytic activity of CD8+ cytotoxic T lymphocytes and
NK cells (Id.), whereas T.sub.H2 cells provide efficient help for B
cells and they also mediate allergic responses due to the capacity
of T.sub.H2 cells to induce IgE isotype switching and
differentiation of B cells into IgE secreting cell (De Vries and
Punnonen (1996) In Cytokine regulation of humoral immunity: basic
and clinical aspects. Eds. Snapper, C. M., John Wiley & Sons,
Ltd., West Sussex, UK, pp. 195-215
[0341] This Example describes methods to generate chimeric
allergens that can broadly modulate allergic immune responses. This
can be achieved by DNA shuffling of related allergen genes to
generate chimeric genes. In addition, chimeric/mutated allergens
are less likely to be recognized by preexisting IgE antibodies of
the patients. Importantly, allergen variants that are not
recognized by IgE antibodies can be selected using patient sera and
negative selection (FIG. 13).
[0342] As one example, chimeric allergen variants of Der p2, Der Q,
Tyr p2 Lep d2 and Gly d2 allergens are generated. These house dust
mite allergens are very common in exacerbating allergic and
asthmatic symptoms, and improved means to downregulate such
allergic immune responses are desired. House dust mites can be used
as sources of the genes. The corresponding genes are shuffled using
family DNA shuffling and a shuffled library is generated. Phage
display is used to exclude allergens that are recognized by
antibodies from allergic individuals. It is particularly important
is to exclude variants that are recognized by IgE antibodies. Phage
expressing the allergen variants are incubated with pools of sera
derived from allergic individuals. The phage that are recognized by
IgE antibodies are removed, and the remaining allergens are further
tested in vitro and in vivo for their capacity to activate
allergen-specific human T cells (FIG. 14). Because immunotherapy of
allergy is believed to function through induction of a dominating
TH I response as compared to allergic T.sub.H2 response, efficient
T cell activation and induction of a TH I type response by allergen
variants is used as a measure of the efficacy of the allergens to
modulate allergic T cell responses.
[0343] The optimal allergen variants are then further tested in
vivo by studying skin responses after injections to the skin. A
strong inflammatory response around the injection site is an
indication of efficient T cell activation, and the allergen
variants that induce the most efficient delayed type T cell
response (typically observed 24 hours after the injection) are the
best candidates for further studies in vivo to identify allergens
that effectively downregulate allergic immune responses.
Accordingly, these allergen variants re analyzed for their capacity
to inhibit allergic responses in allergic, atopic and asthmatic
individuals. The screening of allergen variants is further
illustrated in FIG. 13 and FIG. 14.
Example 12
Evolution of Cancer Antigens that Induce Efficient Anti-Tumor
Immune Responses
[0344] Several cancer cells express antigens that are present at
significantly higher levels on the malignant cells than on other
cells in the body. Such antigens provide excellent targets for
preventive cancer vaccines and immunotherapy of cancer. The
immunogenicity of such antigens can be improved by DNA shuffling.
In addition, DNA shuffling provides means to improve expression
levels of cancer antigens.
[0345] This Example describes methods to generate cancer antigens
that can efficiently induce anti-tumor immune responses by DNA
shuffling of related cancer antigen genes. Libraries of shuffled
melanoma-associated glycoprotein (gp 100/pmel 17) genes (Huang et
al. (1998) J. Invest. Dermatol. 111: 662-7) are generated. The
genes can be isolated from melanoma cells obtained from various
patients, who may have mutations of the gene, increasing the
diversity in the starting genes. In addition, a gp 100 gene can be
isolated from other mammalian species to further increase the
diversity of starting genes. A typical method for the isolation of
the genes is RT-PCR. The corresponding genes are shuffled using
single gene DNA shuffling or family DNA shuffling and a shuffled
library is generated.
[0346] The shuffled gp100 variants, either pools or individual
clones, are subsequently injected into test animals, and the immune
responses are studied (FIG. 15). The shuffled antigens are either
expressed in E. coli and recombinant, purified proteins are
injected, or the antigen genes are used as components of DNA
vaccines. The immune response can be analyzed for example by
measuring anti-gp 100 antibodies, as previous studies indicate that
the antigen can induce specific antibody responses (Huang et al.,
supra.). Alternatively, the test animals that can be challenged by
malignant cells expressing gp100. Animals that have been
efficiently immunized will generate cytotoxic T cells specific for
gp 100 and will survive the challenge, whereas in non-immunized or
poorly immunized animals the malignant cells will efficiently grow
eventually resulting in lethal expansion of the cells. Furthermore,
antigens that induce cytotoxic T cells that have the capacity to
kill cancer cells can be identified by measuring the capacity of T
cells derived from immunized animals to kill cancer cells in vitro.
Typically the cancer cells are first labeled with radioactive
isotopes and the release of radioactivity is an indication of tumor
cell killing after incubation in the presence of T cells from
immunized animals. Such cytotoxicity assays are known in the
art.
[0347] The antigens that induce highest levels of specific
antibodies and/or can protect against the highest number of
malignant cells can be chosen for additional rounds of shuffling
and screening. Mice are useful test animals because large numbers
of antigens can be studied. However, monkeys are a preferred test
animal, because the MHC molecules of monkeys are very similar to
those of humans.
[0348] To screen for antigens that have optimal capacity to
activate antigen-specific T cells, peripheral blood mononuclear
cells from previously infected or immunized humans individuals can
be used. This is a particularly useful method, because the MHC
molecules that will present the antigenic peptides are human MHC
molecules. Shuffled cancer antigens that induce cytotoxic T cells
that have the capacity to kill cancer cells can be identified by
measuring the capacity of T cells derived from immunized animals to
kill cancer cells in vitro. Typically the cancer cells are first
labeled with radioactive isotopes and the release of radioactivity
is an indication of tumor cell killing after incubation in the
presence of T cells from immunized animals. Such cytotoxicity
assays are known in the art.
Example 13
Evolution of Autoantigens that Induce Efficient Immune
Responses
[0349] Autoimmune diseases are characterized by an immune response
directed against self antigens expressed by the host. Autoimmune
responses are generally mediated by T.sub.H1 cells that produce
high levels of IL-2 and IFN-gamma. Vaccines that can direct
autoantigen specific T cells towards T.sub.H2 phenotype producing
increased levels of IL-4 and IL-5 would be beneficial. For such
vaccines to work, the vaccine antigens have to be able to
efficiently activate specific T cells. DNA shuffling can be used to
generate antigens that have such properties. To optimally induce
T.sub.H2 cell differentiation it may be beneficial to coadminister
cytokines that have been shown to enhance T.sub.H2 cell activation
and differentiation, such as IL-4 (Racke et al. (1994) J. Exp. Med.
180: 1961-66).
[0350] This Example describes methods for generating autoantigens
that can efficiently induce immune responses. DNA shuffling is
performed on related autoantigen genes. For example, libraries of
shuffled myelin basic proteins, or fragments thereof (Zamvil and
Steinman (1990) Ann. Rev. Immunol. 8: 579-621); Brocke et al.
(1996) Nature 379: 343-46) are generated. MBP is considered to be
an important autoantigen in patients with multiple sclerosis (MS).
The genes encoding MBP from at least bovine, mouse, rat, guinea pig
and human have been isolated providing an excellent starting point
for family shuffling. A typical method for the isolation of the
genes is RT-PCR. The shuffled MBP variants, either pools or
individual clones, are subsequently injected into test animals, and
the immune responses are studied. The shuffled antigens are either
expressed in E. coli and recombinant, purified proteins are
injected, or the antigen genes are used as components of DNA
vaccines or viral vectors. The immune response can be analyzed for
example by measuring anti-MBP antibodies by ELISA. Alternatively,
the lymphocytes derived from immunized test animals are activated
with MBP, and the T cell proliferation or cytokine synthesis is
studies. A sensitive assays for cytokine synthesis is ELISPOT
(McCutcheon et al. (1997) J. Immunol. Methods 210: 149-66). Mice
are useful test animals because large numbers of antigens can be
studied. However, monkeys are a preferred test animal, because the
MHC molecules of monkeys are very similar to those of humans.
[0351] To screen for antigens that have optimal capacity to
activate MBP specific T cells peripheral blood mononuclear cells
from patients with MS can also be used. This is a particularly
useful method, because the MHC molecules that will present the
antigenic peptides are human MHC molecules. Shuffled antigens that
activate MBP specific T cells can be identified by measuring the
capacity of T cells derived from MS patients to proliferate or
produce cytokines upon culture in the presence of the antigen
variants. Such assays are known in the art. One such assay is
ELISPOT (McCutcheon et al., stipra.). An indication of the efficacy
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
a patient with MS. Strong inflammation is correlated with strong
activation of antigen-specific T cells. Improved activation of MBP
specific T cells, particularly in the presence of IL-4, is likely
to result in enhanced T.sub.H2 cell responses, which are beneficial
in the treatment of MS patients.
Example 14
Method of Optimizing the Immunogenicity of Hepatitis B Surface
Antigen
[0352] This Example describes methods by which the envelope protein
sequence of the hepatitis B virus can be evolved to provide a more
immunogenic surface antigen. Such a protein is important for
vaccination of low responders and for immunotherapy of chronic
hepatitis B.
[0353] Background
[0354] Current HBV vaccines (Merck, SKB) are based on the
immunogenicity of the viral envelope protein and contain the Major
(or Small) form of the envelope protein produced as particles in
yeast. These particles induce antibodies to the major surface
antigen (HBsAg) which can protect against infection when antibody
levels are at least 10 milli-International Units per milliliter
(mU/ml). These recombinant protein preparations are not capable of
inducing humoral immunity in chronic carriers (some 300 million
cases worldwide) the induction of which would be important to
control virus spread. Moreover, certain individuals respond poorly
to the vaccine (up to 30-50% of vaccinees in some groups) and do
not develop protective levels of antibody. The inclusion of the
natural epitope sequences contained in the Middle or Large forms of
the viral envelope protein has been-used as a method to increase
the immunogenicity of vaccine preparations. An alternative method
is to introduce new (i.e., not present in the natural virus
sequence) helper T-cell epitopes into the HBsAg sequence using DNA
shuffling technology.
[0355] Method
[0356] DNA sequences of HBsAg from different subtypes of HBV (e.g.,
ayw and adr) and the related woodchuck hepatitis virus are prepared
for shuffling. Comparison of the genes encoding these proteins
suggests that recombination would occur at least ten times within
850 base pairs when shuffling the ayw and woodchuck hepatitis virus
(WHV) DNA sequences. Nucleotide and amino acid sequences of
portions of different subtypes of HBV are shown in FIG. 17.
[0357] The sequence of the main HBsAg B-cell antigenic site (the
"a" epitope) can be retained in the protein sequence by including
the coding sequences of the external "a" loop in the final protein
preparation. Peptide analogue(s) for the "a" epitope of HBsAg have
been described (Neurath et al. (1984) J. Virol. Methods 9:341-346),
and the immunogenicity of the "a" epitope has been demonstrated
(Bhatnagar et al. (1982) Proc. Nat'l. Acad. Sci. USA 79:
4400-4404). HBsAg and WHsAg share the major "a" determinant, and
chimps can be protected by both antigens (Cote et al. (1986) J.
Virol. 60: 895-901). Likewise, important CTL epitopes can be
included in the protein in a defined way.
[0358] One can also easily introduce B or T (helper or CTL)
epitopes from other antigens into the shuffled HBsAg sequence. This
may focus the immune response to certain epitopes, independent of
other potentially dominant epitopes from the same protein.
Furthermore, the availability of the "a" loop on the HBsAg may
provide a region of the envelope protein into which other
artificial antigens or mimotopes could be included.
[0359] In all cases where a novel HBV envelope sequence is prepared
to include a specific epitope (from HBV, another pathogen or a
tumor cell), shuffling of the surrounding sequences in the HBV
envelope will serve to optimize expression of the protein and help
to ensure that the immune response is directed to the desired
epitope.
[0360] Several methods of analyzing and utilizing shuffled HBsAg
sequences are described below.
[0361] A. Modulating Expression Levels of HBsAg
[0362] Shuffled HBsAg sequences are introduced into cells in
culture and the ability to direct expression of secreted HBsAg
(measured with clinical kits for HBsAg expression) is evaluated.
This can be used to identify shuffled HBsAg sequences which exhibit
optimized HBsAg expression levels. Such coding sequences are
particularly interesting for DNA vaccination.
[0363] B. Circumventing Low Responsiveness to the HBsAg
[0364] Shuffled HBsAg sequences are evaluated for their ability to
induce an immune response to the clinically relevant HBsAg
epitopes. This can be done using mice of the H-2s and H-2f
haplotypes, which respond poorly or not at all to HBsAg protein
immunization. In these experiments, one can verify that antibodies
are generated to the main "a" epitope in the S protein, and a
second protective epitope in the PreS2 region (a linear
sequence).
[0365] The PreS2 and S coding sequences for the envelope protein
(HBsAg) from the HBV ayw subtype (plasmid pCAG-M-Kan; Whalen) and
the WHV (plasmid pWVHV8 from ATCC) are amplified from the two
plasmids by PCR and shuffled. Examples of suitable primers for PCR
amplification are shown in FIG. 18. The shuffled library of
sequences is cloned into an HBsAg-expression vector and individual
colonies are chosen for preparation of plasmid DNA. The DNA is
administered to the test animals and vectors which induce the
desired immune response are identified and recovered.
[0366] C. Presentation of Natural HBsAg CTL Epitopes by Evolved
HBsAg Proteins
[0367] This example describes methods of using the evolved HBsAg
protein to present natural HBsAg CTL epitopes. Shuffling is used to
increase overall immunogenicity of the HBsAg protein, as discussed
above. However, some of the evolved HBsAg sequences are replaced
with class I or class II epitope sequences from the natural HBsAg
protein in order to stimulate immunoreactivity specifically to
these natural viral epitopes. Alternatively, the natural viral
epitopes can be added to the evolved protein without loss of
immunogenicity of the evolved HBsAg.
[0368] D. Expression of Tumor-Derived CTL Epitopes by Evolved HBsAg
Proteins
[0369] This example describes methods of using the evolved HBsAg
protein is used to express tumor-derived CTL epitopes. 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 in order to
stimulate immunoreactivity specifically to these natural viral
epitopes. Alternatively, the tumor cells epitopes can be added to
the evolved protein without loss of immunogenicity of the evolved
HBsAg.
[0370] E. Expression of Mimotope Sequences by the HBsAg
[0371] This example describes the use of an evolved HBsAg protein
for expression of mimotope sequences. Again, the evolved HBsAg
protein is used to increase overall immunogenicity of the protein.
However, some of the evolved HBsAg sequences are replaced with
mimotope sequences to stimulate immunoreactivity specifically to
the natural sequence which cross reacts with the mimotope.
Alternatively, the mimotope sequences can be added to the evolved
protein without loss of immunogenicity of the evolved HBsAg.
Example 15
Fusion Proteins of the HBsAg Polypeptide and HIV gp120 Protein
[0372] This Example describes the preparation of fusion proteins
("chimeras") formed from the HBsAg polypeptide and the
extracellular fragment gp120 of the HIV envelope protein, and their
use as vaccines.
[0373] Background
[0374] When used as a vaccine, recombinant monomeric gp120 has
failed to induce antibodies that have strong neutralizing activity
with primary isolates of the HIV virus. It has been suggested that
oligomeric forms of the HIV envelope protein which expose certain
regions of the tertiary structure would be better able to elicit
virus-neutralizing antibodies (Parrin et al. (1997) Immunol. Lett.
57: 105-112; VanCott et al. (1997) J. Virol. 71: 4319-4330;
[0375] In this Example, DNA shuffling is applied to this problem,
in order to obtain gp120 polypeptides which adopt conformations
slightly different from those of previous preparations of
recombinant gp120. To allow the individual gp120 molecules to
interact as oligomers, a fusion is prepared between gp120 sequences
(on the N-terminus of the fusion) and HBsAg sequences (on the
C-terminal of the fusion).
[0376] The N-terminal peptide sequence of the S region of the HBsAg
polypeptide is a transmembrane structure which is locked into the
membrane of the endoplasmic reticulum. The actual N-terminus of the
S region as well as the preS2 sequences are located in the lumenal
part of the ER. They are found on the outside of the final HBsAg
particles. By placing the gp120 sequences on the N-terminus of the
HBsAg preS2 or S sequences, the gp120 sequences are also located on
the outside of the particles. The gp120 molecules can thus be
brought together in three-dimensional space to interact as in the
virus.
[0377] Since the exact conformation of the final chimera which will
have the most appropriate immunogenicity cannot be predicted, DNA
shuffling is employed. The sequences of the HBsAg polypeptide,
which functions as a scaffold, and of gp120 are both shuffled.
Screening of the shuffled products can be performed by ELISA assay
using antibodies (polyclonal or monoclonal) which have previously
been determined to have virus neutralizing activity.
[0378] Method
[0379] The sequences encoding the gp120 fragment of the HIV
envelope protein are preferably prepared as a synthetic gene to
include codons which are optimal for gene expression in mammals.
The gp120 sequence will typically include a signal sequence on its
N-terminal end.
[0380] The gp120 sequences are inserted into the preS2 region of an
HBsAg-expressing plasmid. In the preS2 region of the plasmid pMKan
and its derivatives, an EcoRI site and an XhoI site are available
for cloning. The gp120 sequences can be inserted between these two
sites, which brings the gp120 closer to the start of the S coding
sequences, or into the EcoRI site alone, which leaves a spacer
sequence of about 50 amino acids between the gp120 sequence and the
start of the S region of the HBsAg. These two different cloning
strategies will give rise to chimeric molecules in which the gp120
sequences are located at different distances from the transmembrane
region of the HBsAg sequence. This may be advantageous in allowing
the gp120 sequences to adopt conformations which are more suitable
immunogens than monomeric gp120.
[0381] DNA shuffling of the entire chimeric sequence is carried
out. Family shuffling is preferred; this involves the preparation
of several gp120-HBsAg fusion proteins in which different gp120 and
HBsAg (or WHV) sequences are used. An alignment of HBsAg nucleotide
sequences is shown in FIG. 19. After shuffling of the different
sequences, the products are cloned into an expression vector such
as pMKan. Pools of clones from the library of shuffled products are
transfected into cultured cells and the secretion of chimeric
proteins is assayed with broadly reactive antibodies to gp120.
Positive clones can be further evaluated with particular antibodies
that have demonstrated HIV neutralizing activity, for example the
anti-CD4 binding domain recombinant human monoclonal antibody,
IgG1b12 (Kessler et al. (1997) AIDS Res. Hum. Retroviruses. 1: 13:
575-582; Roben et al. (1994) J. Virol. 68: 4821-4828). Candidate
clones can then be used to immunize mice and the antiserum obtained
is evaluated for HIV virus-neutralizing activity in in vitro
assays.
[0382] Because the gp120 molecule (approx. 1100 amino acids) is
larger in size than the monomeric HBsAg preS2+S protein (282 amino
acids), it is likely that not every HBsAg monomer in an aggregated
particle will contain a gp120 sequence. Internal initiation of
protein synthesis can take place on the HBsAg coding sequences at
the initiator methionine that marks the beginning of the S region.
Thus, the chimeric molecule (which contains the gp120 sequences)
will be mixed in the cell with the S region and the multimeric
particles should assemble with an appropriate number of chimeric
polypeptides and native HBsAg S monomers. Alternatively, an
S-expressing plasmid can be mixed with the plasmid expressing the
chimera, or a single plasmid which expresses the chimera and the S
form can be constructed. A diagram of the resulting particles is
shown in FIG. 20.
Example 16
DNA Shuffling of HSV-1 and HSV-2 Glycoproteins B and/or D as Means
to Induce Enhanced Protective Immune Responses
[0383] This Example describes the use of DNA shuffling to obtain
HSV glycoprotein B (gB) and glycoprotein D (gD) polypeptides that
exhibit improved ability to induce protective immune responses upon
administration to a mammal. Epidemiological studies have shown that
prior infections with HSV-1 give partial protection against
infections with HSV-2, indicating existence of cross-reactive
immune responses. Based on previous vaccination studies, the main
immunogenic glycoproteins in HSV appear to be gB and gD, which are
encoded by 2.7 kb and 1.2 kb genes, respectively. The gB and gD
genes of HSV-1 are about 85% identical to the corresponding gene of
HSV-2, and the gB genes of each share little sequence identity with
the gD genes. Baboon HSV-2 gB is appr. 75% identical to human HSV-1
or -2 gB, with rather long stretches of almost 90% identity. In
addition, 60-75% identity is found in portions of the genes of
equine and bovine herpes viruses.
[0384] Family shuffling is employed using as substrates nucleic
acids that encode gB and/or gD from HSV-1 and HSV-2. Preferably,
homologous genes are obtained from HSVs of various strains. An
alignment of gD nucleotide sequences from HSV-1 and two strains of
HSV-2 is shown in FIG. 7. Antigens encoded by the shuffled nucleic
acids are expressed and analyzed in vivo. For example, one can
screen for improved induction of neutralizing antibodies and/or CTL
responses against HSV-1/HSV-2. One can also detect protective
immunity by challenging mice or guinea pigs with the viruses.
Screening can be done using pools or individuals clones.
Example 17
Evolution Of HIV Gp120 Proteins for Induction of Broad Spectrum
Neutralizing Ab Responses
[0385] This Example describes the use of DNA shuffling to generate
immunogens that crossreact among different strains of viruses,
unlike the wild-type immunogens. Shuffling two kinds of envelope
sequences can generate immunogens that induce neutralizing
antibodies against a third strain.
[0386] Antibody-mediated neutralization of HIV-1 is strictly
type-specific. Although neutralizing activity broadens in infected
individuals over time, induction of such antibodies by vaccination
has been shown to be extremely difficult. Antibody-mediated
protection from HIV-1 infection in vivo correlates with
antibody-mediated neutralization of virus in vitro.
[0387] FIG. 8 illustrates the generation of libraries of shuffled
gp120 genes. gp120 genes derived from HIV-1DH12 and HIV-11IIB(NL43)
are shuffled. The chimeric/mutant gp120 genes are then analyzed for
their capacity to induce antibodies that have broad spectrum
capacity to neutralize different strains of HIV. Individual
shuffled gp120 genes are incorporated into genetic vaccine vectors,
which are then introduced to mice by injection or topical
application onto the skin. These antigens can also be delivered as
purified recombinant proteins. The immune responses are measured by
analyzing the capacity of the mouse sera to neutralize HIV growth
in vitro. Neutralization assays are performed against HIV-1DH12,
HIV-11IIB and HIV-189.6. The chimeras/mutants that demonstrate
broad spectrum neutralization are chosen for further rounds of
shuffling and selection. Additional studies are performed in
monkeys to illustrate the capacity of the shuffled gp120 genes to
provide protection for subsequent infection with immunodeficiency
virus.
[0388] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference for all purposes.
* * * * *
References