U.S. patent application number 10/328475 was filed with the patent office on 2003-06-19 for rotavirus enterotoxin adjuvant.
This patent application is currently assigned to Baylor College of Medicine. Invention is credited to Estes, Mary K..
Application Number | 20030113788 10/328475 |
Document ID | / |
Family ID | 22572414 |
Filed Date | 2003-06-19 |
United States Patent
Application |
20030113788 |
Kind Code |
A1 |
Estes, Mary K. |
June 19, 2003 |
Rotavirus enterotoxin adjuvant
Abstract
This invention relates to a method of potentiating an immune
response by administering a viral enterotoxin or derivative as an
adjuvant. More particularly it relates to administering a viral
enterotoxin or derivative as an adjuvant and an antigen to a
mucosal surface of a mammal.
Inventors: |
Estes, Mary K.; (Houston,
TX) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI, LLP
1301 MCKINNEY
SUITE 5100
HOUSTON
TX
77010-3095
US
|
Assignee: |
Baylor College of Medicine
|
Family ID: |
22572414 |
Appl. No.: |
10/328475 |
Filed: |
December 24, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10328475 |
Dec 24, 2002 |
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09687698 |
Oct 13, 2000 |
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6534067 |
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60159390 |
Oct 14, 1999 |
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Current U.S.
Class: |
435/6.16 ;
424/215.1; 435/345; 435/5 |
Current CPC
Class: |
A61K 2039/55588
20130101; A61K 2039/55511 20130101; Y02A 50/412 20180101; C12N
2720/12322 20130101; C07K 2319/00 20130101; C12N 2760/16134
20130101; A61K 39/12 20130101; A61K 39/15 20130101; C07K 14/005
20130101; A61K 2039/541 20130101; C12N 2720/12334 20130101; A61K
39/39 20130101; A61K 2039/55516 20130101; A61K 2039/5252 20130101;
Y02A 50/30 20180101; A61K 39/145 20130101; A61K 2039/5258
20130101 |
Class at
Publication: |
435/6 ; 435/5;
424/215.1; 435/345 |
International
Class: |
C12Q 001/70; A61K
039/15; C12Q 001/68; C12N 005/06; C12N 005/16 |
Goverment Interests
[0002] The work herein was supported by grants from the United
States Government. The United States Government may have certain
rights in the invention.
Claims
We claim:
1. A method of potentiating an immune response against an antigen
in an animal comprising the step of administering to said animal
said antigen and an adjuvant wherein said adjuvant is a rotavirus
enterotoxin or a derivative thereof.
2. The method of claim 1 wherein said antigen and said adjuvant are
administered to a mucosal surface of said animal.
3. The method of claim 2 wherein said mucosal surface is selected
from the group consisting of intranasal surface, oral surface,
rectal surface and genitourinary tract surface.
4. The method of claim 1 wherein said rotavirus enterotoxins
selected from the group consisting of NSP4 group A genotypes A, B,
C and D.
5. The method of claim 1 wherein said antigen and said adjuvant are
administered parenterally to said animal.
6. The method of claim 5 wherein said administration is
intraperitoneal, intravenous, and subcutaneous.
7. The method of claim 1 wherein said immune response is
systemic.
8. The method of claim 1 wherein said immune response is
mucosal.
9. The method of claim 1 wherein said antigen is selected from a
group consisting of rotavirus-like particles and influenza A
virus.
10. The method of claim 9 wherein said antigen is rotavirus-like
particles.
11. The method of claim 9 wherein said antigen is an inactivated
influenza A virus.
12. The method of claim 1 wherein said derivative of rotavirus is
OSU NSP4-P138S or SA11 NSP4 aa 112-175.
13. The method of claim 1 wherein said antigen and said adjuvant
are co-administered.
14. The method of claim 1 wherein the animal is a mammal.
15. The method of claim 1 wherein the animal is an avian
species.
16. The method of claim 1 wherein the animal is a human.
17. A method of potentiating an immune response against influenza A
virus in an animal comprising the steps of administering to a
mucosal surface of said animal, an inactivated influenza A virus,
and administering an adjuvant wherein said adjuvant is rotavirus
enterotoxin or a derivative thereof.
18. A method of potentiating an immune response to rotavirus-like
particles in an animal comprising the steps of administering to a
mucosal surface of said animal rotavirus-like particles and
administering an adjuvant wherein said adjuvant is rotavirus
enterotoxin or a derivative thereof.
Description
[0001] This application claims priority to U.S. Provisional
Application No. 60/159,390, which was filed Oct. 14, 1999.
FIELD OF THE INVENTION
[0003] This invention relates to the use of a viral enterotoxin or
derivative as an adjuvant to enhance immune responses. More
particularly it relates to the use of a viral enterotoxin or
derivative as an adjuvant at mucosal surfaces to potentiate immune
responses.
BACKGROUND OF THE INVENTION
[0004] Recombinant DNA technology has stimulated the pursuit of
new, safe and effective vaccines. Disadvantages of recombinant
vaccines include the need for large, repeated antigen doses, and a
general failure to generate major histocompatibility complex (MHC)
class I-restricted immune responses. To overcome these limitations,
recombinant vaccines require the use of systemic- or mucosal-active
immunostimulating agents, which are referred to as adjuvants
(Gradon, et al., 1999). Immunopotentiation by adjuvants can result
from quantitative enhancement of or qualitative alteration of
components of the immune response compared to the immune response
generated by an immunogen alone. Many compounds possess adjuvant
properties but the only adjuvants currently licensed for use in
humans by the Food and Drug Administration are aluminum salts
(aluminum hydroxide or aluminum phosphate), which are relatively
weak adjuvants approved only for systemic administration.
[0005] Development of mucosal vaccines has lagged behind systemic
vaccines because of our limited knowledge of mucosal immunity and
because no adjuvants have been licensed for use at mucosal
surfaces. Mucosal vaccines and adjuvants would be advantageous
because greater than 80% of pathogens enter the host at mucosal
sites. Localized infections of the mucosa are the most common cause
of mortality and morbidity in humans, and many pathogens that cause
systemic infections gain access to the body at mucosal sites,
including HIV, measles virus and polio virus (Gradon, et al.,
1999). Therefore, a vaccine strategy that can potentially prevent
the initial infection of the host is likely to be more successful
than one that resolves infection before the disease ensues.
[0006] The bacterial enterotoxins, cholera toxin (CT) and
Escherichia coli (E. coli) heat-labile enterotoxin (LT), are the
most potent mucosal adjuvants described to date, but their
enterotoxicity precludes their use in humans. Enterotoxins produced
by V. cholera, E. coli and Salmonella have similar modes of action.
Cholera toxin is a protein consisting of three polypeptides, A1,
A2, and B subunits. The B subunit contains the binding site by
which the cholera toxin binds to the ganglioside (G.sub.M1),
located on the cell membrane. The binding of the B subunit to the
G.sub.M1 receptor facilitates the translocation of the A subunit
through the membrane. The A1 subunit activates the cellular enzyme,
adenylcyclase, causing conversion of ATP to cyclic AMP (cAMP). The
increased levels of cAMP result in secretion of water and
electrolytes into the small intestine through interactions with
cAMP-sensitive NaCl transport mechanisms. It has been suggested
that the adjuvanticity of CT and LT is associated with their
ability to increase gut permeability; therefore, facilitating
access of luminal antigens to the gut mucosal immune system (Lycke,
et al., 1991). In addition to modulating the mucosal immune system
by stimulating synthesis of cAMP, it has also been suggested that
CT can modulate the mucosal immune response by stimulating cellular
syntheses of arachidonic acid metabolites (Peterson, et al,
1999).
[0007] Recent studies have examined the potential of CT and LT as
mucosal adjuvants against a variety of bacterial and viral
pathogens (Xu-Amano et al., 1994; Xu-Amno et al., 1993; Yamanoto et
al., 1996 and Wu, et al., 1997). However, prior art indicates that
as little as 5 .mu.g of purified CT, administered orally, was
sufficient to induce significant diarrhea in volunteers, while
ingestion of 25 .mu.g of CT elicited a full 20-liter cholera purge
(Levine, et al., 1983). Similar studies have shown that LT induces
fluid secretion at doses as low as 2.5%g when administered in
conjunction with a vaccine (Freytag and Clements, 1999).
[0008] A number of attempts have been made to alter the toxicity of
LT and CT, most of which focus on eliminating activity of subunit
A, which is associated with enterotoxicity. Recent studies have
shown that site-directed mutagenesis to change any amino acid in CT
or LT involved in the ADP-ribosylation results in a corresponding
loss of toxicity and adjuvanticity (Yamamoto, et al., 1997 and
Lycke, et al., 1992). Therefore, a logical conclusion is that
ADP-ribosylation and induction of cAMP are essential for the
enterotoxicity and adjuvanticity of LT and CT. As a result, a
linkage has been established between enterotoxicity and
adjuvanticity. Thus, there is a need for new, effective systemic
and mucosal adjuvants suitable for human use to enhance the
efficacy of vaccines to prevent life-threatening infections.
[0009] Although there are no sequence homologies between the
rotavirus non-structural protein (NSP4) and any known bacterial
enterotoxin, NSP4 by itself is immunogenic and antibodies against
NSP4 protect neonatal mice against diarrhea induced by a rotavirus
challenge (Ball, et al., 1996, and Zeng and Estes 1999). The
mechanism of adjuvanticity of NSP4 and CT or LT mutants may be
different. CT and LT activate cAMP which is required for
adjuvanticity and enterotoxicity (Freytag and Clements, 1999, and
Cheng, et al., 1999). In contrast, NSP4 enterotoxigenic activity
results from chloride secretion stimulated by a signal transduction
pathway that increases intracellular calcium through
receptor-mediated phospholipase C (PLC) activation and inositol
1,4,5-triphosphate (IP3) (Ball, et al., 1996, and Dong, et al.,
1997, and Morris et al., 1999). Activation of B and T lymphocytes
also involves PLC and IP3 that stimulate increases in intracellular
calcium (Freytag and Clements, 1999).
[0010] This invention demonstrates for the first time the use of
NSP4 as a new adjuvant. It is noteworthy that although the prior
art has used bacterial and aluminum compounds as adjuvants for
potentiating an immune response, the use of a viral enterotoxin or
derivative as an adjuvant has gone unrealized, suggesting that this
invention is indeed novel and nonobvious.
SUMMARY OF THE INVENTION
[0011] An embodiment of the present invention is a method of
potentiating an immune response against an antigen in an animal
comprising the step of administering the antigen and an adjuvant
wherein the adjuvant is a rotavirus enterotoxin or derivative
thereof. Exemplary rotavirus enterotoxins include, but are not
limited to the NSP4 group A genotypes A, B, C or D.
[0012] In specific embodiments, the adjuvant can be either a toxin
or a non-toxic derivative. More particularly, a derivative of
rotavirus can include, but is not limited to OSU NSP4-P138S or SA
11 NSP4 aa 112-175.
[0013] In a preferred embodiment of the present invention, the
antigen and the adjuvant are administered to an animal using
standard methods. They can be co-administered or administered
separately. Administration may be mucosal (e.g., intranasal,
ocular, gastrointestinal, oral, rectal and genitourinary tract) or
parenteral (e.g., intraperitoneal, intravenous, subcutaneous or
muscular.) Animals that may be treated using the method of the
invention include, but are not limited to humans, cows, horses,
pigs, dogs, cats, sheep goats, rabbits, rats, mice, birds, chickens
or fish.
[0014] Yet further, in specific embodiments, the immune response is
systemic or mucosal.
[0015] A further embodiment of the present invention is that the
antigen is rotavirus-like particles or influenza A. Additional
antigens that can be used in the present invention include, but are
not limited to cancer vaccines, viral vaccines, bacterial vaccines
or parasitic vaccines.
[0016] A specific embodiment of the present invention is a method
of potentiating an immune response against influenza A virus by
administering to a mucosal surface an inactivated influenza A virus
and an adjuvant. Specifically, the adjuvant is a rotavirus
enterotoxin or a derivative thereof.
[0017] Another specific embodiment of the present invention is a
method of potentiating an immune response to rotavirus-like
particles by administering to a mucosal surface rotavirus-like
particles and an adjuvant. In specific embodiments, the adjuvant is
a rotavirus enterotoxin or a derivative thereof.
[0018] Other and further objects, features and advantages would be
apparent and eventually more readily understood by reading the
following specification and by reference to the company drawings
forming a part thereof, or any examples of the present preferred
embodiments of the invention are given for the purpose of the
disclosure.
DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 shows the antibody responses evaluated by
enzyme-linked immunosorbent assays (ELISAs) of serum immunoglobulin
(IgG) immune response to keyhole limpet hemocyanin (KLH)
administered intranasally in the presence or absence of rotavirus
enterotoxin or LT.
[0020] FIG. 2 shows the antibody responses evaluated by ELISAs of
intestinal immunoglobulin (IgA) immune response to KLH administered
intranasally in the presence or absence of rotavirus enterotoxin or
LT.
DETAILED DESCRIPTION OF THE INVENTION
[0021] It is readily apparent to one skilled in the art that
various embodiments and modifications may be made to the invention
disclosed in this application without departing from the scope and
the spirit of the invention.
[0022] As used herein the specification, "a" or "an" may mean one
or more. As used herein in the claim(s), when used in conjunction
with the word "comprising", the words "a" or "an" may mean one or
more than one. As used herein "another" may mean at least a second
or more.
[0023] The term "adjuvant" as used herein is defined as an agent
that, when administered with an antigen, enhances the immune
response to the antigen. Specific adjuvants used herein include,
but are not limited to rotavirus (groupA NSP4A, group A NSP4B,
group A NSP4C, group A NSP4D and non-group A NSP4, e.g., group B or
group C), rotavirus NSP4 derivatives, LT, LT derivatives, CT, CT
derivatives and aluminum compounds.
[0024] The term "antibody" as used herein is defined as a serum
immunoglobulin that has specific binding sites to combine with
antigens.
[0025] The term "antigen" as used herein is defined as a molecule
that provokes an immune response. This immune response may involve
either antibody production, the activation of specific
immunologically-competent cells, or both. An antigen can be derived
from organisms, subunits of proteins/antigens, killed or
inactivated whole cells or lysates. Exemplary organisms include but
are not limited to, Helicobacters, Campylobacters, Clostridia,
Corynebacterium diphtheriae, Bordetella pertussis, influenza virus,
parainfluenza viruses, respiratory syncytial virus, Borrelia
burgdorfei, Plasmodium, herpes simplex viruses, human
immunodeficiency virus, papilloma virus, Vibrio cholera, E. coli,
measles virus, rotavirus, shigella, Salmonella typhi, Neisseria
gonorrhea. Therefore, a skilled artisan realizes that any
marcromolecule, including virtually all proteins, can serve as
antigens.
[0026] The term "cDNA" is intended to refer to DNA prepared using
messenger RNA (mRNA) as template.
[0027] The term "derivative" as used herein is defined as an
altered form of a toxin or a protein. Exemplary derivatives as
contained herein include, but are not limited to OSU NSP4-P138S or
LT-R192G. One of skilled in the art recognizes that a derivative as
defined herein is an altered or mutated toxin or protein. Such
alterations or mutations are produced using standard techniques
well known in the art, e.g., site-directed mutagenesis or chemical
mutagenesis.
[0028] The term "DNA" as used herein is defined as deoxyribonucleic
acid.
[0029] The term "enterotoxin" as used herein is defined as a toxin
which affects the intestinal mucosal cells causing secretion of
fluid into the intestinal lumen which leads to the symptoms of
diarrhea.
[0030] The term "functionally equivalent codon" is used herein is
to refer to codons that encode the same amino acid, such as the six
codons for arginine or serine, and also refers to codons that
encode biologically equivalent amino acids.
[0031] The term "immunoglobulin" or "Ig", as used herein is defined
as a class of proteins which functions as antibodies. Two members
in this class of proteins are IgA and IgG. IgA functions as the
primary antibody that is present in body secretions, such as
saliva, tears, breast milk, gastrointestinal secretions and mucus
secretions of the respiratory and genitourinary tracts. IgG
functions as the most common circulating antibody.
[0032] The term "non-toxic derivative" or "toxoid" as used herein
is defined as a derivative of a toxin, which has limited toxicity,
but retains complete adjuvanticity. One of skilled in the art is
cognizant that "limited toxicity" as used herein refers to the
quality of retaining biological activity without being
poisonous.
[0033] The term "polynucleotide" as used herein is defined as a
chain of nucleotides. Furthermore, nucleic acids are polymers of
nucleotides. Thus, one of skill in the art is cognizant that
nucleic acids and polynucleotides as used herein are
interchangeable.
[0034] The term "polypeptide" as used herein is defined as a chain
of amino acid residues, usually having a defined squences. As used
herein the term polypeptide is mutually inclusive of the terms
"peptides" and "proteins".
[0035] The term "recombinant DNA" as used herein is defined as DNA
produced by joining pieces of DNA from different sources.
[0036] The term "recombinant polypeptide" as used herein is defined
as a hybrid protein produced by using recombinant DNA methods.
[0037] The term "RNA" as used herein is defined as ribonucleic
acid.
[0038] The term "toxin" as used herein is defined as a noxious or
poisonous substance that is produced by a pathogen and results in
damage to the infected host. Toxins that are considered cell bound
and released only upon lysis of the cell are referred to as
endotoxins. Toxins that are released extracellulary as the
organism/pathogen grows are referred to as exotoxins.
[0039] The term "vaccine" as used herein is defined as material
used to provoke an immune response (e.g., the production of
antibodies) on administration of the materials and thus conferring
immunity.
[0040] The term "vector" is used to refer to a carrier
polynucleotide molecule into which a polynucleotide sequence can be
inserted for introduction into a cell where it can be
replicated.
[0041] The term "virus" as used herein is defined as a particle
consisting of nucleic acid (RNA or DNA) enclosed in a protein coat,
with or without an outer lipid envelope, which is only capable of
replicating within a whole cell and spreading from cell to
cell.
[0042] One specific embodiment of the present invention is a method
of potentiating an immune response against an antigen in an animal
comprising the step of administering to the animal the antigen and
the adjuvant wherein the adjuvant is a rotavirus enterotoxin or a
derivative thereof. It is contemplated that the immune response is
systemic or mucosal.
[0043] In another specific embodiment of the present invention the
adjuvant and the antigen are administered to mucosal surfaces
(e.g., intranasal, ocular, gastrointestinal, oral, rectal and
genitourinary tract). A skilled artisan recognizes the importance
of developing mucosal immunization methods because the majority of
deaths from infectious diseases are caused by organisms that first
make contact with and either colonize or cross the mucosal surface
to infect the host. Therefore, a vaccine that does not prevent the
initial infection of the host will unlikely succeed in resolving
the infection before the disease ensues. Mucosal immunization
induces IgA antibodies, which are directed against specific
pathogens of mucosal surfaces. It is suggested that greater than
80% of all the antibodies produced in mucosal-associated lymphoid
tissues may block attachment of bacteria and viruses. This blockade
neutralizes bacterial toxins and inactivates invading viruses
inside the epithelial cells. Therefore, a skilled artisan can
readily recognize that mucosal immunization would actually prevent
the initial infection resulting in a decrease in the morbidity
caused by pathogens.
[0044] An additional embodiment includes administration of the
antigen and the adjuvant via parenteral routes (e.g.
intraperitoneal, intravenous, subcutaneous or muscular). To
demonstrate the range of applicability, different routes of
immunization are tested. The ability of the adjuvant to enhance the
immune response to multiple antigens and by more than one route of
immunization illustrate the potency and efficiency of the adjuvant.
One skilled in the art realizes that any given adjuvant may be more
effective with certain antigens via different routes of
immunization. Therefore, it is necessary to determine the range of
potency and efficiency of the adjuvant.
[0045] Yet further, in specific embodiments, the antigen and
adjuvant are co-administered or administered sequentially.
[0046] In other specific embodiments, the antigen is rotavirus-like
particles or influenza A antigen. Other exemplary antigens include,
but are not limited to cancer vaccines, viral vaccines or bacterial
vaccines. In certain embodiments, an antigenic composition's may be
chemically coupled to a carrier or recombinantly expressed with an
immunogenic carrier peptide or polypeptide (e.g., a antigen-carrier
fusion peptide or polypeptide) to enhance an immune reaction.
Exemplary and preferred immunogenic carrier amino acid sequences
include hepatitis B surface antigen, keyhole limpet hemocyannin
(KLH) and bovine serum albumin (BSA). Other albumins such as
ovalbumin, mouse serum albumin or rabbit serum albumin also can be
used as immunogenic carrier proteins. Means for conjugating a
polypeptide or peptide to a immunogenic carrier protein are well
known in the art and include, for example, glutaraldehyde,
m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide and
bis-biazotized benzidine.
[0047] Another embodiment of the present invention is that the
adjuvant can be an enterotoxin or a non-toxic derivative. Specific
enterotoxins used herein include, but are not limited to rotavirus
(groupA NSP4A, group A NSP4B, group A NSP4C, group A NSP4D and
non-group A NSP4), rotavirus NSP4 derivatives, LT, LT derivatives,
CT, or CT derivatives. One of skill in the art is cognizant of the
nomenclature of rotaviruses. For example, rotaviruses are
classified into groups A-F of which the viruses associated with the
most severe diseases are within group A. The NSP4 genes of group A
are further classified into groups A-D based on phylogenetic
analyses (Ciarlet, et al., 2000). Thus, in the present invention it
is also contemplated that non-group A NSP4 proteins may be used as
adjuvants. Non-group A NSP4 proteins, e.g., group B, have been
shown to mobilize intracellular calcium (Tian, et al., 1994).
Non-group A NSP4 proteins include, but are not limited to group B,
group C, group D, group E or group F.
[0048] The toxins used in the present invention may be purified
from standard bacterial or viral cultures or produced using
standard DNA recombinant methods and expression systems or chemical
modifications. Since the 1970's, a skilled artisan has used DNA
technology to synthesize and manipulate nucleic acids. These DNA
methods are standard techniques in the art.
[0049] Furthermore, derivatives of toxins included in the present
invention may contain insertions, substitutions and/or deletions of
the wild-type sequence using standard DNA recombinant methods,
e.g., site-directed mutagenesis. Exemplary derivatives include
without limitations OSU NSP4-P138S, which is a single amino acid
substitution at position 138 or SA 11 NSP4 aa 112-175, which is a
deletion of the first 111 amino acids. Standard chemical
modifications of amino acids, which are critical for toxicity, but
not adjuvanticity, may be used to generate non-toxic derivatives or
toxoids. One of skill in the art is cognizant of the specific
moiety or moieties that are associated with toxicity and
adjuvanticity in toxins. Thus, the moiety or moieties associated
with toxicity can be altered using standard mutagenesis techniques
resulting in a non-toxic derivative or toxoid that is an adjuvant.
An example of this dissociation of toxicity and adjuvanticity in a
toxin is provided in the reference by Dickinson and Clements,
Infection and Immunity (1995) 63:1617-1623, which is herein
incorporated by reference in its entirety. Thus, a skilled artisan
can appreciate the examples provided herein and extrapolate to
other toxins to dissociate the moieties associated with toxicity
and adjuvanticity to produce a non-toxic derivative or toxoid.
[0050] Other standard mutagenesis techniques that can be used in
the present invention include the use of reagents which modify
lysine, tyrosine or SH-containing amino acids. Furthermore,
standard photoaffinity labeling methods which use azido-linked
substrate derivatives, can be covalently linked to the toxin active
sites by ultraviolet irradiation.
[0051] Mutagenesis
[0052] Mutations can arise spontaneously as a result of events such
as errors in the fidelity of DNA replication or the movement of
transposable genetic elements (transposons) within the genome. They
also are induced following exposure to chemical or physical
mutagens. Such mutation-inducing agents include ionizing radiation,
ultraviolet light and a diverse array of chemical such as
alkylating agents and polycyclic aromatic hydrocarbons all of which
are capable of interacting either directly or indirectly (generally
following some metabolic biotransformations) with nucleic acids.
The DNA lesions induced by such environmental agents may lead to
modifications of base sequence when the affected DNA is replicated
or repaired and thus to a mutation. Mutation also can be
site-directed through the use of particular targeting methods.
[0053] A. Insertional Mutagenesis
[0054] Insertional mutagenesis is based on the inactivation of a
gene via insertion of a known DNA fragment. Because it involves the
insertion of some type of DNA fragment, the mutations generated are
generally loss-of-function, rather than gain-of-function mutations.
However, there are several examples of insertions generating
gain-of-function mutations (Oppenheimer et al., 1991). Insertional
mutagenesis has been very successful in bacteria and Drosophila
(Cooley et al., 1988) and recently has become a powerful tool in
corn (Schmidt et al., 1987); Arabidopsis; (Marks et al., 1991;
Koncz et al., 1990); and Antirrhinum (Sommer et al., 1990).
[0055] Transposable genetic elements are DNA sequences that can
move (transpose) from one place to another in the genome of a cell.
The first transposable elements to be recognized were the
Activator/Dissociation elements of Zea mays (McClintock, 1957).
Since then, they have been identified in a wide range of organisms,
both prokaryotic and eukaryotic.
[0056] Transposable elements in the genome are characterized by
being flanked by direct repeats of a short sequence of DNA that has
been duplicated during transposition and is called a target site
duplication. Virtually all transposable elements whatever their
type, and mechanism of transposition, make such duplications at the
site of their insertion. In some cases the number of bases
duplicated is constant, in other cases it may vary with each
transposition event. Most transposable elements have inverted
repeat sequences at their termini. These terminal inverted repeats
may be anything from a few bases to a few hundred bases long and in
many cases they are known to be necessary for transposition.
[0057] Prokaryotic transposable elements have been most studied in
E. coli and Gram negative bacteria, but also are present in Gram
positive bacteria. They are generally termed insertion sequences if
they are less than about 2 kB long, or transposons if they are
longer. Bacteriophages such as mu and D108, which replicate by
transposition, make up a third type of transposable element.
Elements of each type encode at least one polypeptide a
transposase, required for their own transposition. Transposons
often further include genes coding for function unrelated to
transposition, for example, antibiotic resistance genes.
[0058] Transposons can be divided into two classes according to
their structure. First, compound or composite transposons have
copies of an insertion sequence element at each end, usually in an
inverted orientation. These transposons require transposases
encoded by one of their terminal IS elements. The second class of
transposon have terminal repeats of about 30 base pairs and do not
contain sequences from IS elements.
[0059] Transposition usually is either conservative or replicative,
although in some cases it can be both. In replicative
transposition, one copy of the transposing element remains at the
donor site, and another is inserted at the target site. In
conservative transposition, the transposing element is excised from
one site and inserted at another.
[0060] Eukaryotic elements also can be classified according to
their structure and mechanism of transposition. The primary
distinction is between elements that transpose via an RNA
intermediate, and elements that transpose directly from DNA to
DNA.
[0061] Elements that transpose via an RNA intermediate often are
referred to as retrotransposons, and their most characteristic
feature is that they encode polypeptides that are believed to have
reverse transcriptionase activity. There are two types of
retrotransposon. Some resemble the integrated proviral DNA of a
retrovirus in that they have long direct repeat sequences, long
terminal repeats (LTRs), at each end. The similarity between these
retrotransposons and proviruses extends to their coding capacity.
They contain sequences related to the gag and pol genes of a
retrovirus, suggesting that they transpose by a mechanism related
to a retroviral life cycle. Retrotransposons of the second type
have no terminal repeats. They also code for gag- and pol-like
polypeptides and transpose by reverse transcription of RNA
intermediates, but do so by a mechanism that differs from that or
retrovirus-like elements. Transposition by reverse transcription is
a replicative process and does not require excision of an element
from a donor site.
[0062] Transposable elements are an important source of spontaneous
mutations, and have influenced the ways in which genes and genomes
have evolved. They can inactivate genes by inserting within them,
and can cause gross chromosomal rearrangements either directly,
through the activity of their transposases, or indirectly, as a
result of recombination between copies of an element scattered
around the genome. Transposable elements that excise often do so
imprecisely and may produce alleles coding for altered gene
products if the number of bases added or deleted is a multiple of
three.
[0063] Transposable elements themselves may evolve in unusual ways.
If they were inherited like other DNA sequences, then copies of an
element in one species would be more like copies in closely related
species than copies in more distant species. This is not always the
case, suggesting that transposable elements are occasionally
transmitted horizontally from one species to another.
[0064] B. Chemical Mutagenesis
[0065] Chemical mutagenesis offers certain advantages, such as the
ability to find a full range of mutant alleles with degrees of
phenotypic severity, and is facile and inexpensive to perform. The
majority of chemical carcinogens produce mutations in DNA.
Benzo[a]pyrene, N-acetoxy-2-acetyl aminofluorene and aflotoxin B1
cause GC to TA transversions in bacteria and mammalian cells.
Benzo[a]pyrene also can produce base substitutions such as AT to
TA. N-nitroso compounds produce GC to AT transitions. Alkylation of
the 04 position of thymine induced by exposure to n-nitrosoureas
results in TA to CG transitions. Other chemical reagents can modify
lysine, tyrosine or SH-containing amino acids resulting in mutated
proteins.
[0066] C. Radiation Mutagenesis
[0067] The integrity of biological molecules is degraded by the
ionizing radiation. Adsorption of the incident energy leads to the
formation of ions and free radicals, and breakage of some covalent
bonds. Susceptibility to radiation damage appears quite variable
between molecules, and between different crystalline forms of the
same molecule. It depends on the total accumulated dose, and also
on the dose rate (as once free radicals are present, the molecular
damage they cause depends on their natural diffusion rate and thus
upon real time). Damage is reduced and controlled by making the
sample as cold as possible.
[0068] Ionizing radiation causes DNA damage and cell killing,
generally proportional to the dose rate. Ionizing radiation has
been postulated to induce multiple biological effects by direct
interaction with DNA, or through the formation of free radical
species leading to DNA damage (Hall, 1988).
[0069] In the present invention, the term ionizing radiation means
radiation comprising particles or photons that have sufficient
energy or can produce sufficient energy via nuclear interactions to
produce ionization (gain or loss of electrons). An exemplary and
preferred ionizing radiation is an x-radiation. The amount of
ionizing radiation needed in a given cell generally depends upon
the nature of that cell. Typically, an effective
expression-inducing dose is less than a dose of ionizing radiation
that causes cell damage or death directly. Means for determining an
effective amount of radiation are well known in the art.
[0070] In a certain embodiments, an-effective expression inducing
amount is from about 2 to about 30 Gray (Gy) administered at a rate
of from about 0.5 to about 2 Gy/minute. Even more preferably, an
effective expression inducing amount of ionizing radiation is from
about 5 to about 15 Gy. In other embodiments, doses of 2-9 Gy are
used in single doses. An effective dose of ionizing radiation may
be from 10 to 100 Gy, with 15 to 75 Gy being preferred, and 20 to
50 Gy being more preferred.
[0071] Any suitable means for delivering radiation to a tissue may
be employed in the present invention in addition to external means.
For example, radiation may be delivered by first providing a
radiolabeled antibody that immunoreacts with an antigen of the
tumor, followed by delivering an effective amount of the
radiolabeled antibody to the tumor. In addition, radioisotopes may
be used to deliver ionizing radiation to a tissue or cell.
[0072] D. In vitro Scanning Mutagenesis
[0073] Random mutagenesis also may be introduced using error prone
PCR (Cadwell and Joyce, 1992). The rate of mutagenesis may be
increased by performing PCR in multiple tubes with dilutions of
templates.
[0074] One particularly useful mutagenesis technique is alanine
scanning mutagenesis in which a number of residues are substituted
individually with the amino acid alanine so that the effects of
losing side-chain interactions can be determined, while minimizing
the risk of large-scale perturbations in protein conformation
(Cunningham et al., 1989).
[0075] In recent years, techniques for estimating the equilibrium
constant for ligand binding using minuscule amounts of protein have
been developed (Blackburn et al., 1991; U.S. Pat. Nos. 5,221,605
and 5,238,808). The ability to perform functional assays with small
amounts of material can be exploited to develop highly efficient,
in vitro methodologies for the saturation mutagenesis of
antibodies. The inventors bypassed cloning steps by combining PCR
mutagenesis with coupled in vitro transcription/translation for the
high throughput generation of protein mutants. Here, the PCR
products are used directly as the template for the in vitro
transcription/translation of the mutant single chain antibodies.
Because of the high efficiency with which all 19 amino acid
substitutions can be generated and analyzed in this way, it is now
possible to perform saturation mutagenesis on numerous residues of
interest, a process that can be described as in vitro scanning
saturation mutagenesis (Burks et al., 1997).
[0076] In vitro scanning saturation mutagenesis provides a rapid
method for obtaining a large amount of structure-function
information including: (i) identification of residues that modulate
ligand binding specificity, (ii) a better understanding of ligand
binding based on the identification of those amino acids that
retain activity and those that abolish activity at a given
location, (iii) an evaluation of the overall plasticity of an
active site or protein subdomain, (iv) identification of amino acid
substitutions that result in increased binding.
[0077] E. Random Mutagenesis by Fragmentation and Reassmbly
[0078] A method for generating libraries of displayed polypeptides
is described in U.S. Pat. No. 5,380,721. The method comprises
obtaining polynucleotide library members, pooling and fragmenting
the polynucleotides, and reforming fragments therefrom, performing
PCR amplification, thereby homologously recombining the fragments
to form a shuffled pool of recombined polynucleotides.
[0079] F. Site-Directed Mutagenesis
[0080] Structure-guided site-specific mutagenesis represents a
powerful tool for the dissection and engineering of protein-ligand
interactions (Wells, 1996, Braisted et al., 1996). The technique
provides for the preparations and testing of sequence variants by
introducing one or more nucleotide sequence changes into a selected
DNA.
[0081] Site-specific mutagenesis uses specific oligonucleotide
sequences which encode the DNA sequence of the desired mutation, as
well as a sufficient number of adjacent, unmodified nucleotides. In
this way, a primer sequence is provided with sufficient size and
complexity to form a stable duplex on both sides of the deletion
junction being traversed. A primer of about 17 to 25 nucleotides in
length is preferred, with about 5 to 10 residues on both sides of
the junction of the sequence being altered.
[0082] The technique typically employs a bacteriophage vector that
exists in both a single-stranded and double-stranded form. Vectors
useful in site-directed mutagenesis include vectors such as the M13
phage. These phage vectors are commercially available and their use
is generally well known to those skilled in the art.
Double-stranded plasmids are also routinely employed in
site-directed mutagenesis, which eliminates the step of
transferring the gene of interest from a phage to a plasmid.
[0083] In general, one first obtains a single-stranded vector, or
melts two strands of a double-stranded vector, which includes
within its sequence a DNA sequence encoding the desired protein or
genetic element. An oligonucleotide primer bearing the desired
mutated sequence, synthetically prepared, is then annealed with the
single-stranded DNA preparation, taking into account the degree of
mismatch when selecting hybridization conditions. The hybridized
product is subjected to DNA polymerizing enzymes such as E. coli
polymerase I (Klenow fragment) in order to complete the synthesis
of the mutation-bearing strand. Thus, a heteroduplex is formed,
wherein one strand encodes the original non-mutated sequence, and
the second strand bears the desired mutation. This heteroduplex
vector is then used to transform appropriate host cells, such as E.
coli cells, and clones are selected that include recombinant
vectors bearing the mutated sequence arrangement.
[0084] Comprehensive information on the functional significance and
information content of a given residue of protein can best be
obtained by saturation mutagenesis in which all 19 amino acid
substitutions are examined. The shortcoming of this approach is
that the logistics of multiresidue saturation mutagenesis are
daunting (Warren et al., 1996, Brown et al., 1996; Zeng et al.,
1996; Burton and Barbas, 1994; Yelton et al., 1995; Jackson et al.,
1995; Short et al., 1995; Wong et al., 1996; Hilton et al., 1996).
Hundreds, and possibly even thousands, of site specific mutants
must be studied. However, improved techniques make production and
rapid screening of mutants much more straightforward. See also,
U.S. Pat. Nos. 5,798,208 and 5,830,650, for a description of
"walk-through" mutagenesis.
[0085] Other methods of site-directed mutagenesis are disclosed in
U.S. Pat. Nos. 5,220,007; 5,284,760; 5,354,670; 5,366,878;
5,389,514; 5,635,377; and 5,789,166.
[0086] One skilled in the art will readily understand that in
making fragments or derivatives of toxins for use in the methods
and compositions of the invention, it is desirable to maintain
adjuvanticity and limit the toxicity or poisonous quality of the
toxin.
[0087] Nucleic Acids Encoding NSP4
[0088] In certain embodiments, one group of adjuvants of the
present invention are those that can be encoded by a nucleic acid
(e.g., DNA or RNA). It is contemplated that such adjuvants may be
encoded in an expression vector encoding the antigen, or in a
separate vector or other construct. These nucleic acids encoding
the adjuvants can be delivered directly, such as for example with
lipids or liposomes.
[0089] Specifically, nucleic acids according to the present
invention may encode an entire NSP4 gene, a domain of NSP4, or any
other fragment of NSP4 as set forth herein. The nucleic acid may be
derived from genomic DNA, i.e., cloned directly from the genome of
a particular organism. The following sequences are sequences
corresponding to NSP4 genes that are classified in the group A
rotaviruses and are within the scope of the invention and are
referenced with the corresponding GenBank Accession Numbers
http://www.ncbi.nlm.nih.gov/Genbank/GenbankSearch.html):
ALA(SEQ.ID.NO:1, AF144792); C-11 (SEQ.ID.NO:2, AF144793); R-2
(SEQ.ID.NO:3, AF144794); BAP-2 (SEQ.ID.NO:4, AF144795); BAPwt
(SEQ.ID.NO:5, AF144796);, A253 (SEQ.ID.NO:6, AF144797); A131
(SEQ.ID.NO:7, AF144798); A411 (SEQ.ID.NO:8, AF144799); A34
(SEQ.ID.NO:9, AF165219); H-1 (SEQ.ID.NO: 10, AF144801); FI-23
(SEQ.ID.NO:11, AF144802); FI-14 (SEQ.ID.NO:12, AF144803); BRV033
(SEQ.ID.NO:13, AF144804); B223 (SEQ.ID.NO:14, AF144805); CU-1
(SEQ.ID.NO:15, AF144806); OSU (SEQ.ID.NO:16, D88831); and SA11
(SEQ.ID.NO:17, AF0871678).
[0090] Also within the scope of the invention is the NSP4 sequences
of the different virus strains and groups which are discussed in
Ciarlet, et al., 2000, which is hereby incorporated by reference.
Thus any strain can be used to incorporate the NSP4 sequence into
vectors to make recombinant molecules.
[0091] In further embodiments, the following NSP4 sequences that
are classified in the non-group A rotavirus NSP4 s are within the
scope of the present invention and are referenced with the
corresponding GenBank Accession Numbers
http://www.ncbi.nlm.nih.gov/Genbank/GenbankSearch.html)- : group B
IDIR (SEQ.ID.NO: 18, U03557); group C (SEQ.ID.NO: 19.times.83967);
group C (SEQ.ID.NO: 20, D88353) and group C (SEQ.ID.NO: 21,
L12391)
[0092] In preferred embodiments, however, the nucleic acid would
comprise complementary DNA (cDNA). The genome of rotavirus is a
double-stranded RNA. Thus, one of skill in the art is cognizant
that cDNA is produced of the RNA genome to allow for cloning into
vectors. Typically, RT-PCR is used to generate copies of cDNA from
RNA.
[0093] A. Vectors for Cloning, Gene Transfer and Expression
[0094] Within certain embodiments, expression vectors are employed
to express a NSP4 peptide product or derivative thereof, which can
then be purified and, for example, be used to as an adjuvant.
Expression requires that appropriate signals be provided in the
vectors, and which include various regulatory elements, such as
enhancers/promoters from both viral and mammalian sources that
drive expression of the genes of interest in host cells.
[0095] 1. Regulatory Elements
[0096] In certain embodiments, the polynucleotide sequence encoding
a gene product is under transcriptional control of a promoter. A
promoter refers to a DNA sequence recognized by the synthetic
machinery of the cell, or introduced synthetic machinery, required
to initiate the specific transcription of a gene. One of skill in
the art further recognizes that under transcriptional control means
that the promoter is in the correct location and orientation in
relation to the polynucleotide sequence to control RNA polymerase
initiation and expression of the gene.
[0097] In certain embodiments, the bacterial phage promoters are
used to obtain high-level expression of the coding sequence of
interest. Yet further other viral or mammalian promoters may be
used. All of these promoters systems are well-known in the art.
[0098] 2. Selectable Markers
[0099] In certain embodiments of the invention, the cells contain
polynucleotide sequence constructs of the present invention. A cell
may be identified in vitro or in vivo by including a marker in the
expression construct. Such markers would confer an identifiable
change to the cell permitting easy identification of cells
containing the expression construct. Usually the inclusion of a
drug selection marker aids in cloning and in the selection of
transformants, for example, genes that confer resistance to
neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol
are useful selectable markers. Alternatively, enzymes such as
herpes simplex virus thymidine kinase (tk) or chloramphenicol
acetyltransferase (CAT) may be employed. Immunologic markers also
can be employed. The selectable marker employed is not believed to
be important, so long as it is capable of being expressed
simultaneously with the polynucleotide sequence encoding a gene
product. Further examples of selectable markers are well known to
one of skill in the art.
[0100] 3. Vectors
[0101] In certain embodiments, vectors may be employed to produce
the adjuvants of the present invention. A polynucleotide sequence
can be exogenous, which means that it is foreign to the cell into
which the vector is being introduced or that the sequence is
homologous to a sequence in the cell but in a position within the
host cell polynucleotide sequence in which the sequence is
ordinarily not found. Vectors include plasmids, cosmids, viruses
(bacteriophage, animal viruses, and plant viruses), and artificial
chromosomes (e.g., YACs). One of skill in the art would be well
equipped to construct a vector through standard recombinant
techniques, which are described in Maniatis et al., 1988 and
Ausubel et al., 1994, both incorporated herein by reference.
[0102] An expression vector is a vector containing a polynucleotide
sequence coding for at least part of a gene product capable of
being transcribed. In some cases, RNA molecules are then translated
into a protein, polypeptide, or peptide. In other cases, these
sequences are not translated, for example, in the production of
antisense molecules or ribozymes. Expression vectors can contain a
variety of "control sequences," which refer to polynucleotide
sequences necessary for the transcription and possibly translation
of an operably linked coding sequence in a particular host
organism. In addition to control sequences that govern
transcription and translation, vectors and expression vectors may
contain polynucleotide sequences that serve other functions as well
and are described infra.
[0103] 4. Expression Systems
[0104] Numerous expression systems exist that comprise at least a
part or all of the compositions discussed above. Prokaryote- and/or
eukaryote-based systems can be employed for use with the present
invention to produce polynucleotide sequences, or their cognate
polypeptides, proteins and peptides. Many such systems are
commercially and widely available.
[0105] The insect cell/baculovirus system can produce a high level
of protein expression of a heterologous polynucleotide segment,
such as described in U.S. Pat. No. 5,871,986, 4,879,236, both
herein incorporated by reference, and which can be bought, for
example, under the name MAXBAC.RTM. 2.0 from INVITROGEN.RTM. and
BACPACK.TM. BACULOVIRUS EXPRESSION SYSTEM FROM CLONTECH.RTM..
[0106] Other examples of expression systems include
STRATAGENE.RTM.'s COMPLETE CONTROL.TM. Inducible Mammalian
Expression System, which involves a synthetic ecdysone-inducible
receptor, or its pET Expression System, an E. coli expression
system. Another example of an inducible expression system is
available from INVITROGEN.RTM., which carries the T-REX.TM.
(tetracycline-regulated expression) System, an inducible mammalian
expression system that uses the full-length CMV promoter.
INVITROGEN.RTM. also provides a yeast expression system called the
Pichia methanolica Expression System, which is designed for
high-level production of recombinant proteins in the methylotrophic
yeast Pichia methanolica. One of skill in the art would know how to
express a vector, such as an expression construct, to produce a
polynucleotide sequence or its cognate polypeptide, protein, or
peptide.
[0107] 5. Delivery of Expression Vectors
[0108] There are a number of ways in which expression vectors may
be introduced into cells. In certain embodiments of the invention,
the expression construct comprises a virus or engineered construct
derived from a viral genome. The ability of certain viruses to
enter cells via receptor-mediated endocytosis, to integrate into
host cell genome and express viral genes stably and efficiently
have made them attractive candidates for the transfer of foreign
genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein,
1988; Baichwal and Sugden, 1986; Temin, 1986). The first viruses
used as gene vectors were DNA viruses including the papovaviruses
(simian virus 40, bovine papilloma virus, and polyoma) (Ridgeway,
1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988;
Baichwal and Sugden, 1986). These have a relatively low capacity
for foreign DNA sequences and have a restricted host spectrum.
Furthermore, their oncogenic potential and cytopathic effects in
permissive cells raise safety concerns. They can accommodate only
up to 8 kB of foreign genetic material but can be readily
introduced in a variety of cell lines and laboratory animals
(Nicolas and Rubenstein, 1988; Temin, 1986).
[0109] Several non-viral methods for the transfer of expression
constructs into cultured mammalian cells are contemplated by the
present invention. These include calcium phosphate precipitation
(Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al.,
1990) DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et
al., 1986; Potter et al., 1984), direct microinjection (Harland and
Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982;
Fraley et al., 1979) and lipofectamine-DNA complexes, cell
sonication (Fechheimer et al., 1987), gene bombardment using high
velocity microprojectiles (Yang et al., 1990), and
receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988).
Some of these techniques may be successfully adapted for in vivo or
ex vivo use.
[0110] 6. Host Cells
[0111] As used herein, the terms "cell," "cell line," and "cell
culture" may be used interchangeably. All of these terms also
include their progeny, which is any and all subsequent generations.
It is understood that all progeny may not be identical due to
deliberate or inadvertent mutations. In the context of expressing a
heterologous polynucleotide sequence, "host cell" refers to a
prokaryotic or eukaryotic cell, and it includes any transformable
organisms that is capable of replicating a vector and/or expressing
a heterologous gene encoded by a vector. A host cell can, and has
been, used as a recipient for vectors. A host cell may be
"transfected" or "transformed," which refers to a process by which
exogenous polynucleotide is transferred or introduced into the host
cell. A transformed cell includes the primary subject cell and its
progeny.
[0112] Some vectors may employ control sequences that allow it to
be replicated and/or expressed in both prokaryotic and eukaryotic
cells. One of skill in the art would further understand the
conditions under which to incubate all of the above described host
cells to maintain them and to permit replication of a vector. Also
understood and known are techniques and conditions that would allow
large-scale production of vectors, as well as production of the
polynucleotides encoded by vectors and their cognate polypeptides,
proteins, or peptides.
[0113] Polypeptides, Peptides or Proteins
[0114] The present invention also relates to the production and/or
purification of polypeptides, peptides or proteins that are used as
an adjuvant. The peptides of the invention can be synthesized in
solution or on a solid support in accordance with conventional
techniques. Various automatic synthesizers are commercially
available and can be used in accordance with known protocols. See,
for example, Stewart and Young, (1984); Tam et al., (1983);
Merrifield, (1986); and Barany and Merrifield (1979), each
incorporated herein by reference. Short peptide sequences, or
libraries of overlapping peptides, usually from about 6 up to about
35 to 50 amino acids, which correspond to the selected regions
described herein, can be readily synthesized and then screened in
screening assays designed to identify reactive peptides.
Alternatively, recombinant DNA technology may be employed wherein a
polynucleotide sequence which encodes a peptide of the invention is
inserted into an expression vector, transformed or transfected into
an appropriate host cell and cultivated under conditions suitable
for expression.
[0115] A. Purification of Proteins
[0116] In further embodiments, it may be desirable to purify the
polypeptides, peptide, proteins or variants thereof. Protein
purification techniques are well known to those of skill in the
art. These techniques involve, at one level, the crude
fractionation of the cellular milieu to polypeptide and
non-polypeptide fractions. Having separated the polypeptide from
other proteins, the polypeptide of interest may be further purified
using chromatographic and electrophoretic techniques to achieve
partial or complete purification (or purification to homogeneity).
Analytical methods particularly suited to the preparation of a pure
peptide are ion-exchange chromatography, exclusion chromatography;
polyacrylamide gel electrophoresis; isoelectric focusing. A
particularly efficient method of purifying peptides is fast protein
liquid chromatography or even HPLC.
[0117] Certain aspects of the present invention concern the
purification, and in particular embodiments, the substantial
purification, of an encoded protein or peptide. The term purified
protein or peptide as used herein, is intended to refer to a
composition, isolatable from other components, wherein the protein
or peptide is purified to any degree relative to its
naturally-obtainable state. A purified protein or peptide therefore
also refers to a protein or peptide, free from the environment in
which it may naturally occur.
[0118] Generally, purified will refer to a protein or peptide
composition that has been subjected to fractionation to remove
various other components, and which composition substantially
retains its expressed biological activity. Where the term
substantially purified is used, this designation will refer to a
composition in which the protein or peptide forms the major
component of the composition, such as constituting about 50%, about
60%, about 70%, about 80%, about 90%, about 95% or more of the
proteins in the composition.
[0119] Various methods for quantifying the degree of purification
of the protein or peptide will be known to those of skill in the
art in light of the present disclosure. These include, for example,
determining the specific activity of an active fraction, or
assessing the amount of polypeptides within a fraction by SDS/PAGE
analysis. A preferred method for assessing the purity of a fraction
is to calculate the specific activity of the fraction, to compare
it to the specific activity of the initial extract, and to thus
calculate the degree of purity, herein assessed by a "-fold
purification number." The actual units used to represent the amount
of activity will, of course, be dependent upon the particular assay
technique chosen to follow the purification and whether or not the
expressed protein or peptide exhibits a detectable activity.
[0120] Various techniques suitable for use in protein purification
will be well known to those of skill in the art. These include, for
example, precipitation with ammonium sulphate, PEG, antibodies and
the like or by heat denaturation, followed by centrifugation;
chromatography steps such as ion exchange, gel filtration, reverse
phase, hydroxylapatite and affinity chromatography; isoelectric
focusing; gel electrophoresis; and combinations of such and other
techniques. As is generally known in the art, it is believed that
the order of conducting the various purification steps may be
changed, or that certain steps may be omitted, and still result in
a suitable method for the preparation of a substantially purified
protein or peptide.
[0121] There is no general requirement that the protein or peptide
always be provided in the most purified state. Indeed, it is
contemplated that less substantially purified products will have
utility in certain embodiments. Partial purification may be
accomplished by using fewer purification steps in combination, or
by utilizing different forms of the same general purification
scheme. For example, it is appreciated that a cation-exchange
column chromatography performed utilizing an HPLC apparatus will
generally result in a greater "-fold" purification than the same
technique utilizing a low pressure chromatography system. Methods
exhibiting a lower degree of relative purification may have
advantages in total recovery of protein product, or in maintaining
the activity of an expressed protein.
[0122] It is known that the migration of a polypeptide can vary,
sometimes significantly, with different conditions of SDS/PAGE
(Capaldi et al., 1977). It will therefore be appreciated that under
differing electrophoresis conditions, the apparent molecular
weights of purified or partially purified expression products may
vary.
[0123] High Performance Liquid Chromatography (HPLC) is
characterized by a very rapid separation with extraordinary
resolution of peaks. This is achieved by the use of very fine
particles and high pressure to maintain an adequate flow rate.
Separation can be accomplished in a matter of minutes, or at most
an hour. Moreover, only a very small volume of the sample is needed
because the particles are so small and close-packed that the void
volume is a very small fraction of the bed volume. Also, the
concentration of the sample need not be very great because the
bands are so narrow that there is very little dilution of the
sample.
[0124] Gel chromatography, or molecular sieve chromatography, is a
special type of partition chromatography that is based on molecular
size. The theory behind gel chromatography is that the column,
which is prepared with tiny particles of an inert substance that
contain small pores, separates larger molecules from smaller
molecules as they pass through or around the pores, depending on
their size. As long as the material of which the particles are made
does not adsorb the molecules, the sole factor determining rate of
flow is the size. Hence, molecules are eluted from the column in
decreasing size, so long as the shape is relatively constant. Gel
chromatography is unsurpassed for separating molecules of different
size because separation is independent of all other factors such as
pH, ionic strength, temperature, etc. There also is virtually no
adsorption, less zone spreading and the elution volume is related
in a simple matter to molecular weight.
[0125] Affinity Chromatography is a chromatographic procedure that
relies on the specific affinity between a substance to be isolated
and a molecule that it can specifically bind to. This is a
receptor-ligand type interaction. The column material is
synthesized by covalently coupling one of the binding partners to
an insoluble matrix. The column material is then able to
specifically adsorb the substance from the solution. Elution occurs
by changing the conditions to those in which binding will not occur
(alter pH, ionic strength, temperature, etc.).
[0126] A particular type of affinity chromatography useful in the
purification of carbohydrate containing compounds is lectin
affinity chromatography. Lectins are a class of substances that
bind to a variety of polysaccharides and glycoproteins. Lectins are
usually coupled to agarose by cyanogen bromide. Conconavalin A
coupled to Sepharose was the first material of this sort to be used
and has been widely used in the isolation of polysaccharides and
glycoproteins other lectins that have been include lentil lectin,
wheat germ agglutinin which has been useful in the purification of
N-acetyl glucosaminyl residues and Helix pomatia lectin. Lectins
themselves are purified using affinity chromatography with
carbohydrate ligands. Lactose has been used to purify lectins from
castor bean and peanuts; maltose has been useful in extracting
lectins from lentils and jack bean; N-acetyl-D galactosamine is
used for purifying lectins from soybean; N-acetyl glucosaminyl
binds to lectins from wheat germ; D-galactosamine has been used in
obtaining lectins from clams and L-fucose will bind to lectins from
lotus.
[0127] The matrix should be a substance that itself does not adsorb
molecules to any significant extent and that has a broad range of
chemical, physical and thermal stability. The ligand should be
coupled in such a way as to not affect its binding properties. The
ligand should also provide relatively tight binding. And it should
be possible to elute the substance without destroying the sample or
the ligand. One of the most common forms of affinity chromatography
is immunoaffinity chromatography. The generation of antibodies that
would be suitable for use in accord with the present invention is
discussed below.
[0128] Fusion Proteins
[0129] Also contemplated in the present invention is a fusion
protein or chimera. This molecule generally has all or a
substantial portion of the native molecule, linked at the N- or
C-terminus, to all or a portion of a second polypeptide. The second
polypeptide may include a second enterotoxin or another protein
that comprises adjuvant activity. Yet further, the second
polypeptide may comprise the antigen of interest. For example, a
fusion protein can comprise NSP4 protein and influenza A antigen.
Another useful fusion includes the addition of an immunologically
active domain, such as an antibody epitope, to facilitate
purification of the fusion protein. Inclusion of a cleavage site at
or near the fusion junction will facilitate removal of the
extraneous polypeptide after purification.
[0130] Dosage and Formulation
[0131] The adjuvants of this invention can be formulated and
administered to potentiate the immune response in the body of an
animal. They can be administered by any conventional means
available for use in conjunction with potential vaccines. They can
be administered alone, but are generally administered with a
pharmaceutical carrier selected on the basis of the chosen route of
administration and standard pharmaceutical practice.
[0132] The adjuvant can be administered in liquid dosage forms such
as elixirs, syrups, emulsions and suspensions. The adjuvant can
also be formulated for administration parenterally by injection,
rapid infusion, nasopharyngeal absorption or dermoabsorption. The
adjuvant may be administered intramuscularly, intravenously or as a
suppository.
[0133] Liquid dosage forms for oral administration can contain
coloring and flavoring to increase patient acceptance.
[0134] In general, water, a suitable oil, saline, aqueous dextrose
(glucose), and related sugar solutions and glycols such as
propylene glycol or polyethylene glycols are suitable carriers for
parenteral solutions. Suitable pharmaceutical carriers are
described in Remington's Pharmaceutical Sciences, a standard
reference text in this field.
[0135] Accordingly, the pharmaceutical composition of the present
invention may be delivered via various routes to various sites in
the animal to achieve a particular effect. One skilled in the art
will recognize that although more than one route can be used for
administration, a particular route can provide a more immediate and
more effective reaction than another route. Local or systemic
delivery can be accomplished by administration comprising
application or installation of the formulation into body cavities
inhalation or insufflation of an aerosol, or by parenteral
introduction, comprising intramuscular, intravenous,
intraperitoneal, subcutaneous, intradermal, as well as topical
administration.
[0136] The adjuvant of the present invention can be provided in
unit dosage form wherein each dosage unit, e.g., a teaspoonful,
solution, or suppository, contains a predetermined amount of the
adjuvant, alone or in appropriate combination with a vaccine. The
term "unit dosage form" as used herein refers to physically
discrete units suitable as unitary dosages for animals, each unit
containing a predetermined quantity of the adjuvant, alone or in
combination with a vaccine, calculated in an amount sufficient to
produce the desired effect, in association with a pharmaceutically
acceptable diluent, carrier, or vehicle, where appropriate. The
specifications for the unit dosage forms of the present invention
depend on the particular effect to be achieved and the particular
pharmacodynamics associated with the pharmaceutical composition in
the particular host.
[0137] These methods described herein are by no means all
inclusive, and further methods to suit the specific application
will be apparent to the ordinary skilled artisan. Moreover, the
effective amount of the compositions can be further approximated
through analogy to compounds known to exert the desired affect.
[0138] The following examples are offered by way of example, and
are not intended to limit the scope of the invention in any
manner.
EXAMPLE 1
Animal Vaccination and Pathogen Challenge
[0139] Standard protocols have been established to immunize
animals. Animals can be immunized orally, intranasally or
parentally with test antigens (Ciarlet, et al., 1998, O'Neal, et
al., 1998, Mbawuike, et al., 1990, Mbawuike, et al., 1993, Ciarlet,
et al., 2000, and Ciarlet, et al., 1999b). The regime for
immunization can vary depending on the route of administration, the
type of vaccine administered or the amount of the vaccine
administered. To confer immunity (e.g., specific antibody
production), the animals are challenged with a pathogen. For
example, an influenza vaccine was administered at sub-optimal doses
as an intraperitoneal injection to mice. Four to six weeks
following the vaccination, mice were infected or challenged with
influenza virus and mortality was recorded (Mbawuike, et al.,
1990). In another example, rabbits were vaccinated twice
intramuscularly at 0 and 21 or 28 days with a given volume of
rotavirus cell lysate. After 56-69 days postvaccination, the
rabbits were challenged with rotavirus and serumand fecal samples
were collected to measure antirotavirus antibodies (Ciarlet, et
al., 1998).
[0140] A skilled artisan will recognize the established methods of
animal immunizations. Furthermore, one skilled in the art realizes
that the test antigens can be derived from organisms, subunits of
proteins/antigens, killed or inactivated whole cells or lysates.
Examples of potential vaccines to be used with this invention
include, but are not limited to cancer vaccines, bacterial
vaccines, viral vaccines or parasitic vaccines.
EXAMPLE 2
Determination of the Capability of the Rotavirus Enterotoxin NSP4
to Potentiate Mucosal Immune Response to Keyhole Limpet Hemocyanin
(KLH)
[0141] NSP4 viral enterotoxin was demonstrated to modulate mucosal
immune response to KLH. Balb/c mice were immunized intranasally at
days 0 and 21 with 6, 10, or 20 .mu.g of NSP4 proteins of simian
(SA11, SEQ.ID.NO:24 or NSP4 aa 112-175, SEQ.ID.NO:22) or porcine
(OSU, SEQ.ID.NO:25) origin, 10 .mu.g of mutant LT (LT-R192G), or
buffer. The antibody responses were evaluated using ELISAs to
determine the total fecal IgA (ug/ml), KLH-specific fecal IgA
(ng/ml), and KLH-specific serum IgG antibody titers.
[0142] The data show that 6 .mu.g of simian SA11 NSP4 enterotoxin
can act as an effective mucosal adjuvant when administered
intranasally to mice with KLH as the test antigen. FIGS. 1 and 2
illustrate that NSP4 enhanced the specific serum IgG and fecal IgA
immune response to KLH significantly (p<0.03, Mann Whitney U)
compared to the immune response induced by KLH alone. KLH-specific
serum IgG and fecal IgA were not detected in mice immunized with
NSP4 alone. Although the NSP4 adjuvant activity with KLH was weaker
than that of a nontoxic mutant of LT (LT-R192G), there is no
evidence that NSP4 binds G.sub.M1, making NSP4 a safer and more
practical adjuvant candidate than CT and LT mutants.
EXAMPLE 3
Determination of the Capability of the Rotavirus Enterotoxin NSP4
to Potentiate Mucosal and Systemic Immune Responses to
Non-Replicating Antigens
[0143] NSP4 possess immunopotentiating activity with a broad
variety of antigens. Non-replicating model antigen, ovalbumin
(OVA), a particulate non-replicating candidate vaccine,
rotavirus-like particles (RV-VLPs) (Ciarlet, et al., 1998, and
O'Neal, et al., 1998), and an inactivated influenza A virus
(Mbawuike, et al., 1990) are used in the presence or absence of
NSP4 as the adjuvant. Rotaviruses are the major agents that cause
severe gastroenteritis in children (Ciarlet, et al., 1998), and
influenza A viruses cause a serious respiratory illness in both
children and adults (Mbawuike, et al., 1990). RV-VLPs and influenza
A antigens were chosen as test antigens because well established
mouse models are available, both systemic and mucosal immunization
routes are efficacious in both models, protection at two distinct
mucosal surfaces (respiratory and intestinal tracts) can be tested
and the test antigens are under vaccine development or are in use
in humans (Ciarlet, et al., 1998, O'Neal, et al., 1998, Mbawuike,
et al., 1990, Mbawuike, et al., 1993, and Ciarlet, et al.,
2000).
[0144] One example to study the adjuvanticity of NSP4, but not
limited to this example, is to use adult Balb/c mice (6 per group)
immunized orally, intranasally or parenterally at days 0 and 21
with test antigens at sub-optimal antigen doses using the
established protocols stated in example 1 (Ciarlet, et al., 1998,
O'Neal, et al., 1998, Mbawuike, et al., 1990, Mbawuike, et al.,
1993, Ciarlet, et al., 2000, and Ciarlet, et al., 1999b). The test
antigens are administered in conjunction with increasing
concentrations (5, 10, or 20 .mu.g) of SA11 NSP4, PBS, or 10 .mu.g
of mutant LT (LT-R192G) or 20 .mu.g of QS-21, as control adjuvants
for mucosal or systemic routes of immunization, respectively.
Already established specific IgG and IgA ELISAs, and neutralization
(rotavirus) or hemagglutination-inhibition (influenza A) assays
(Ciarlet, et al., 1998, O'Neal, et al., 1998, Mbawuike, et al.,
1990, Mbawuike, et al., 1993, and Ciarlet, et al., 2000) are used
to evaluate antibody responses to each combination of test antigens
and adjuvants in serum and fecal samples.
EXAMPLE 4
Different Types of NSP4 and Potency
[0145] Based on amino acid sequence divergence (60-85%), there are
4 known genotypes of group A NSP4: A, B, C and D (Ciarlet, et al.,
1999b). These NSP4 s have been cloned and expressed in the
baculovirus expression system (BES) using standard procedures well
known in the art (Zhang, et al., 1998).
[0146] Briefly, NSP4 that was cloned into a vector was subcloned
into the baculovirus transfer vector. Recombinant baculoviruses
expressing NSP4 were generated and recombinant virus stocks were
plaque purified. NSP4 was purified from Sf9 cells infected with the
recombinant baculovirus. Infected cells were harvested and lysed
with lysis buffer.
[0147] NSP4 was first semipurified by fast-performance liquid
chromatography (FPLC) using a quaternary methylamine anion-exchange
column. The NSP4-rich fractions were pooled for futher purification
by using an agarose imunoaffinity column onto which rabbit
immunoglobulin G (IgG) against SA11 NSP4 had been immobilized. NSP4
was eluted from the column and the eluate was then dialyzed and
lyophilized.
[0148] The adjuvanticity of all NSP4 types is determined using the
experimental design described in Example 3. The choice of test
antigen, dose of NSP4, route of immunization, and control adjuvant
tested is the regimen identified in Example 3 with the most
enhanced immune response.
EXAMPLE 5
Challenge Studies Using the Most Potent NSP4 Adjuvant
[0149] The experimental design is the same as described in Example
3, except mice are challenged at a determined time point after
vaccination. The optimal dose, type of NSP4, route of immunization,
and control adjuvant are chosen based on results obtained in
Example 4. Antibody responses are analyzed by ELISAs, and
hemagglutination-inhibition or neutralization assays. Protective
efficacy is evaluated by protection from rotavirus infection
(reduction in virus shedding) or influenza A disease (reduction in
mortality) (Ciarlet, et al., 1998, O'Neal, et al., 1998, Mbawuike,
et al., 1990, Mbawuike, et al., 1993, and Ciarlet, et al.,
2000).
EXAMPLE 6
Development of Nontoxic Derivatives of NSP4 which Retain
Adjuvanticity and Identification of the Domain in NSP4 Responsible
for Adjuvant Activity
[0150] The region between amino acid residues 131 to 140 of the
wild-type OSU NSP4 (type B) (SEQ.ID.NO: 25) is important in
enterotoxicity because a single amino acid change at position 138
(P.fwdarw.S) renders the protein (OSU NSP4-P138S, SEQ.ID.NO:23)
nondiarrheagenic in neonatal mice (Zhang, et al., 1998). Recently,
a secreted non-glycosylated protease cleavage product of SA11 NSP4
aa 112-175 (SEQ.ID.NO:22) that retains enterotoxin function in mice
has been cloned and produced in the baculovirus expression system.
The data in FIGS. 1 and 2 show that 10 .mu.g of SA11 NSP4 aa
112-175 retains similar (p=0.89, Mann Whitney U) adjuvant activity
to that of the full-length SA11 NSP4 (SEQ.ID.NO: 24) when
administered intranasally to Balb/c mice with KLH as the test
antigen. SA11 NSP4 aa 112-175 enhanced the specific serum IgG and
fecal IgA immune response to KLH significantly (p<0.02, Mann
Whitney U) compared to the immune response induced by KLH alone.
These data indicate that the adjuvant and enterotoxic domain in
NSP4 are contained in the C-terminus of the protein. Additionally,
expressed NSP4 aa 112-175 is more soluble than the glycosylated and
hydrophobic full-length SA11 NSP4.
EXAMPLE 7
Adjuvant Activity of Rotavirus Fusion Proteins or Cross-Linked
Proteins
[0151] Fusion proteins containing rotavirus enterotoxin or a
derivative are fused to an antigen and prepared using standard
methods well known in the art. In addition to fusion proteins,
rotavirus enterotoxin or a derivative may be covalently coupled or
cross-linked to antigens (Cryz et al., 1994, Liang et al., 1988,
Czerkinsky, et al., 1989) using established protocols well known in
the art.
[0152] The adjuvanticity of fusion proteins or cross-linked
adjuvant or adjuvant peptides to an antigen is determined using the
experimental design in Example 3.
REFERENCES
[0153] All patents and publications mentioned in the specification
are indicative of the level of those skilled in the art to which
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[0182] One skilled in the art readily appreciates that the present
invention is well adapted to carry out the objectives and obtain
the ends and advantages mentioned as well as those inherent
therein. Methods, procedures and techniques described herein are
presently representative of the preferred embodiments and are
intended to be exemplary and are not intended as limitations of the
scope. Changes therein and other uses will occur to those skilled
in the art which are encompassed within the spirit of the invention
or defined by the scope of the pending claims.
Sequence CWU 1
1
25 1 751 DNA Rotavirus strain ALA 1 ggcttttaaa agttctgttc
cgagagagcg cgtgcggaaa gatggataaa cttaccgacc 60 tcaattacac
attgagcgta atcactttaa tgaatagtac attgcatgca atattggaag 120
atccagggat ggcgtatttc ccatacatag catctgtgtt gactgttctg ttcactttac
180 ataaagcatc aattccaaca atgaaaattg cgttaaaaac atctagatgt
tcctacaaag 240 ttattaaata ttgcattgta accatattta atacattgtt
gaaattagct ggatataaag 300 aacaaataac tactaaagat gaaattgaaa
aacagatgga tagagtaatc agagaaatga 360 gacgtcagtt ggaaatgatt
gataaattga caactcgtga aattgaacag gtagaactac 420 taagacgtat
atatgacaga ttaacggtac gaaagactga tgagatagat atgtcgaagg 480
agatcaatca gaaaaatata cgaacgctag atgaatggga gaatggaaaa aatccatatg
540 aaccaagcga agtgaccgca tcattgtgag aggttggact gccgtcgact
gtctctggaa 600 gcggcggagt ccttcacagt aagtcccatc ggacctgatg
actggctgag aagccacagt 660 cagtcatatc gcgtgtggct caagccttaa
tcccgtttaa ccaatccggt gagcgccgga 720 cgttaatgga aggaatggtc
ttagtgtgac c 751 2 751 DNA Lapine rotavirus strain C-11 2
ggcttttaaa agttctgttc cgagagagcg cgtgcggaaa gatggataaa cttaccgacc
60 tcaattacac attgagcgtg atcactttaa tgaatagtac attgcataca
atattggaag 120 atccagggat ggcgtatttc ccatacatag catctgtgtt
gactgttctg ttcactttgc 180 ataaagcatc aattccaaca atgaaaattg
cgttaaaaac atctagctgt tcctacaaag 240 ttattaaata ttgtcttgtt
actatattta atacattgcc taaattagct ggatataaag 300 aacaaataac
tactaaacgt gaaattgaaa aacagatgga tagagttatc agagaaatga 360
gacgtcagtt agaaatgatt gataaattga caactcgtga aattgaacag gtagaactac
420 taagacgtat atatgacaaa ttaacggtac gaaagactga tgagataggt
atgttgaagg 480 agatcaatca gaaaaatata cggacgctag atgaatggga
gaatggaaag aatccatacg 540 aaccaagcaa agtgaccgca tcattgtgag
aggttggact gccgtcgact gtcctggaag 600 cggcggagtc cttcacagta
agtcccatcg gacctgatga ctggctgaga agccacagtc 660 atatcatatc
gcgtgtggct caagccttaa tcccgtttaa ccaatccggt gagcgccgga 720
cgttaatgga aggaatggtc ttagtgtgac c 751 3 751 DNA Lapine rotavirus
strain R-2 3 ggcttttaaa agttctgttc cgagagagcg cgtgcggaaa gatggaaaag
cttaccgacc 60 tcaactatac attgaatgtg atcactttat tgaacagtac
attgcataca atattggagg 120 atccagggat ggcgtacttt ccttacattg
catctgtcct aacagtttta ttcacattac 180 acaaagcgtc gattccaacg
atgaaaattg ccttaagaac atcaaaatgt tcctataaag 240 tgataaagta
ttgtattgta acaattttca atacgctact aaagttagcc ggctataaag 300
aacagattac tactaaagaa tggattgaaa aacagttgga caaagtaata aaagaaatga
360 gacgtcagct agaaatgata gataaattga caactcgaga aattgaacag
gtagagctac 420 taaaacgtat atacgacaaa ctaatgatac gaaagactga
tgaaatagat atgacgaagg 480 agatcaatca aaaaaatgta aaaacgctag
atgaatggga gaatgggaag aatccatatg 540 aatcaaaaga agtgactgca
gcaatgtaag aggttgggct gccgtcgact gtcttcggaa 600 gcggcggagt
tcttcacagt aagttccatc ggacctgatg agtggctgag aagccacagt 660
cagtcatatc gcgtgtggct caagccttaa tcccgtttaa ccaatccggt gagcgccgga
720 cgttaatgga aggaagggtc ttagtgtgac c 751 4 751 DNA Lapine
rotavirus strain BAP-2 4 ggcttttaaa agttctgttc cgagagagcg
cgtgcggaaa gatggataaa cttaccgacc 60 tcaattacac attgagcgta
atcactttaa tgaatagtac attgcatgca atattggaag 120 atccagggat
ggcgtatttc ccatacatag catctgtgtt gactgtactg ttcactttac 180
ataaagcatc aattccaaca atgaaaattg cgttaaaaac atctagatgt tcctacaaag
240 ttattaaata ttgcattgta accatattta atacattgtt gaaattagct
ggatataaag 300 aacaaataac tactaaagat gaaattgaaa gacagatgga
cagagtagtc cgagaaatga 360 gacgtcagtt ggaaatgatt gataaattga
caacacgtga aattgaacag gtagaactac 420 taagacgtat atacgacaga
ctaacggtgc gaaagactga tgagatagat atgtcgaagg 480 agatcaatca
gaaaaatata cggacgttag atgaatggga gaatggaaaa aatccatatg 540
aaccaagcga ggtgaccgca tcattgtgag aggttggact gccgtcgact gtccctggaa
600 gcggcggagt cctttacagt aagtcccatc ggacctgatg actggctgag
aagccacagt 660 cagtcatatc gcgtgtggct caagccttaa tcccgcttaa
ccaatccggt gagcgccgga 720 cgttaatgga aggaatggtc ttagtgtgac c 751 5
751 DNA Lapine rotavirus strain BAP (wildtype) 5 ggcttttaaa
agttctgttc cgagagagcg cgtgcggaaa gatggataaa cttaccgacc 60
tcaattacac attgagcgta atcactttaa tgaatagtac attgcatgca atattggaag
120 atccagggat ggcgtatttc ccatacatag catctgtgtt gactgtactg
ttcactttac 180 ataaagcatc aattccaaca atgaaaattg cgttaaaaac
atctagatgt tcctacaaag 240 ttattaaata ttgcattgta accatattta
atacattgtt gaaattagct ggatataaag 300 aacaaataac tactaaagat
gaaattgaaa agcagatgga cagagtaatc cgagaaatga 360 gacgtcagtt
ggaaatgatt gataaattga caactcgtga aattgaacag gtagaactac 420
taagaagaat atacgacaga ctaacggtac gtaagactga tgagatagat atgtcgaagg
480 aaatcaatca gaaaaatata cggacgttag atgaatggga gaatggaaaa
aatccatatg 540 aaccaagcga ggtgaccgca tcattgtgag aggttggact
gccgtcgact gtccctggaa 600 gcggcggagt ccttcacagt aagtcccatc
ggacctgatg actggctgag aagccacagt 660 cagtcatatc gcgtgtggct
caagccttaa tcccgtttaa ccaatccggt gagcgccgga 720 cgttaatgga
aggaatggtc ttagtgtgac c 751 6 750 DNA Porcine rotavirus strain A253
6 ggcttttaaa agttctattt cgagagagcg cgtgcggaaa gatggataag cttgcagacc
60 ttaattatac tttgagcgtt atcactttaa tgaatgatac actacactct
ataattcaag 120 atccagggat ggcgtacttc ccatatattg catctgtact
gactgtatta tttactctac 180 ataaggcatc aattcccaca atgaaaattg
cgttaaaaac gtcaaagtgt tcgtacaaag 240 taattaagta ttgcatggtt
acaatcatta atactcttct gaagttggct ggttacaagg 300 aacaggttac
tactaaggac gaaattgaac aacagatgga tagaattgta aaagagatga 360
gacgtcaact ggaaatgatt gataaattga ctactcgtga aattgaacag gtagaattac
420 ttaaacgtat acacgataaa ttggtagtta gacctgtaga cgttatagac
atgtcgaaag 480 aatttaacca gaaaaatatt agaacgctag acgaatggga
aagtgggaaa aatccatacg 540 aaccctcgga agttactgcg tctatgtgag
aggttgagtt gccgtcgtct gtcttcggaa 600 gcggcggaac tcttcaccgc
aagccccatt ggacacgatg gtttactgac aaaccccagt 660 caatcatttc
gcgtgtagca catccctaat cccgaataac caatccagcg aatgttggac 720
gttaatggaa ggaatggtct taatgtgacc 750 7 750 DNA Porcine rotavirus
strain A131 7 ggcttttaaa agttctgttt cgagagagcg cgtgcggaaa
gatggataag cttgcagacc 60 ttaattacac tttgagcgtt attactttaa
tgaatgacac actacattct attattcaag 120 atccagggat ggcgatcttc
ccatatatag catctgtact gactgtatta tttactctac 180 ataaggcatc
aatacccaca atgaaaattg cgttaaaaac gtcaaagtgt tcgtataaag 240
taataaagta ctgcattgtt acaattatca atactcttct gaaattggct ggttacaagg
300 aacaggttac tacaaaggat gaaattgaac aacagatgga cagaatcatt
aaagagatga 360 gacgtcaact ggaaatgata gataagttga ctactcgtga
aattgaacag gtagaattac 420 ttaagcgtat tcatgataag ttggttgtaa
ggccagtaga cgttattgac atgtcgaaag 480 aatttaatca gaagaatata
cgaacgcttg acgaatggga aagtggaaaa aatccatacg 540 aaccgtcgga
agtaactgca tctatgtgag aggttgagtt accctcgtct gtatttggga 600
gcggcgggac tcttcatcgc aaaccacatt ggacacgatg gtttactgac aaaccccagt
660 caatcatatc gcgtgtagca cagccataat cccgtataac aaatcctgcg
aatgttggac 720 gttaatggaa ggaatggtct taatgtgacc 750 8 750 DNA
Porcine rotavirus strain A411 8 ggcttttaaa agttctgttc cgagagagcg
cgtgcggaaa gatggataag cttgacgatc 60 ttaattatac tttgagcgtc
atcactttaa tgaatgacac actacattct ataattcaag 120 atccaggaat
ggcgtacttc ccatacatag catctgtact gactgtttta tttactctac 180
ataaggcatc aattcccaca atgaaaattg cgttaagaac gtcaaagtgt tcgtataaag
240 taataaaata ctgcattgtt acaattttta atactcttct gaaattggct
ggttacaaag 300 aacaggttac tactaaagac gaaattgaac aacagatgga
cagaattatc aaagagatga 360 gacgtcaact ggaaatgatt gacaaattga
ctactcgtga aattgaacag gtagaattac 420 ttaaacgtat tcacgataaa
ctggttgcaa ggtcagttga cgttatagac atgtcgaaag 480 aatttaatca
gaaaaatata agaacgctag atgaatggga aagtggaaaa aatccctacg 540
aaccgtcgga agtaactgca tctatgtgag aggttgagtt gccgtcatca gtctttggga
600 gcggcggaac tcttcatcgc aagccccatt ggacccgatg gttgactgag
aagccacagt 660 caatcatttc tcgtgtagca cagccctaat cccgattaac
caatccagcg aatgttggac 720 gttaatggaa ggaatggtct taatgtgacc 750 9
675 DNA Porcine rotavirus strain A34 9 gatggataag cttgccgacc
tcaactacac attgagtgta atcactttaa tgaatgatac 60 gttacactct
attattcaag atccaggaat ggcgtatttt ccatatatcg catctgttct 120
aactgtttta tttactctac ataaagcatc aattccaacg atgaaaatag cattaagaac
180 gtcaaaatgt tcatacaaag taattaaata ttgtatggtt acgatcatta
atactcttct 240 aaagttggct ggttataaag aacaggttac taccaaggat
gaaatcgaac aacagatgga 300 cagaattgtt aaagagatga gacgtcaact
ggagatgatt gacaaattga caactcgtga 360 aattgaacag gtcgaattac
ttaagcgtat acatgataaa ttagttacta gaccagttga 420 tgctatagac
atgtcgaaag aatttaatca gaagaatatc agaacgctag atgaatggga 480
aagcggaaaa aatccatatg aaccatcaga agtgactgca tctatgtgag aggttgagtt
540 gccgtcgtct gtcttcggaa gcggcggaac tcttcaccgc aagccccatt
ggacctgatg 600 gttgactgag aagccacagt caatcatatc gcgtgtggct
cagccttaat cccgtttaac 660 caatccagcg aatgt 675 10 751 DNA Equine
rotavirus strain H-2 10 ggcttttaaa agttctgttc cgagagagcg cgtgcggaaa
gatggataag cttaccgacc 60 tcaactatac attgaatgtg atcactttat
tgaacagtac attgcataca atattggagg 120 atccagggat ggcgtacttt
ccttacattg catctgtcct aacagtttta ttcacattac 180 acaaagcgtc
gattccaacg atgaaaattg ccttaagaac atcaaaatgt tcgtataaag 240
tgataaagta ttgtattgta acaattttca atacgctact aaagttagca ggctataaag
300 aacagattac tactaaagat gaaatagaaa aacaaatgga tagagtagtt
aaagaaatga 360 gacgtcattt agagatgatt gataaattga ctacacgtga
aattgaacaa gtagaattac 420 ttaaacgtat ttatgataaa ctgatgatac
gggcaacaga cgaaatagat atgacgaaag 480 aaatcaatca aaagaacgtg
aaaacgctag aagaatggga aaatggaaag aatccttatg 540 aatcaaaaga
agtgactgca gcaatgtaag aggttgagct gccgtcgact atcttcggaa 600
gcggcggagt tctttacagt aagctccatc agacctgatg gctggctgag aagccacagt
660 cagccatatc gcgtgtggct caagccttaa tcccgtttaa ccaatccggt
cagtaccgga 720 cgttaatgga aggagtggtc ttagtgtgaa g 751 11 751 DNA
Equine rotavirus strain FI-23 11 ggcttttaaa agttctgttc cgagagagcg
cgtgcggaaa gatggataag cttaccgacc 60 ttaattatac attgaatgta
attactctat tgaacagtac attgcataca attttagagg 120 atccagggat
ggcgtatttc ccttacattg catctgtact aacagtatta ttcacattac 180
acaaagcgtc gattccaacg atgaagattg ccttaagaac atcaaaatgt tcgtacaagg
240 tgattaagta ttgtatagtt acaattttca atacgctact aaagttagca
ggctataagg 300 aacagattac tactaaggac gaaatagaaa aacaaatgga
tagagttgtt aaagaaatga 360 ggcgtcacct agagatgata gataagttga
ctacacgtga aatagagcaa gttgaattac 420 ttaaacgtat atacgataag
ctgatggcac gagcaacaga tgaaattgat atgactaaag 480 aaataaatca
gaagaacgtg aaaacgttag aagaatggga aaatggaaag aatccttacg 540
aatcaaaacg aatgactgca gcaatgtaag aggttgaact gccgtcgact atctttggaa
600 gcgggggggt actatatagt aagctccatc agacctaata gctggctgag
aagccacagt 660 cagcaattta aaaagtggct caagccttaa ttcccttcaa
ccaatccggt cagtaccgga 720 cgttaatgga aggagtggtc ttagtgtgaa g 751 12
751 DNA Equine rotavirus strain FI-14 12 ggcttttaaa agttctgttc
cgagagagcg cgtgcggaaa gatggataaa ctaaccgacc 60 tcaactatac
attgaacgta atcactttaa ttaacagcac attgcataca attttagagg 120
atcccggaat ggcgtatttc ccttacattg catctgtatt aacagtatta ttcacattac
180 acaaggcatc gataccaacg atgaagatag ccttgaaaac atcaaagtgt
tcgtataaag 240 tagtaaaata ctgtatagtt acaattttta atacgctact
aaaattagca ggctacaaag 300 aacaaataac tactaaagat gaaattgaga
agcaaatgga cagagtaatt aaagaaatga 360 gacgtcattt agagatgata
gacaagttga caactcgtga gatagagcaa gttgaactac 420 ttaagcgtat
atacgataag ctaatgattc gggctacgga cgaaattgat atgtcgaaag 480
aaattaacca aaagaacgta agaacgttag aagaatggga aaacggaaag aatccttatg
540 aatcaaaaga agttactgca gcaatgtaag aggttgagct gccgtcgact
atcttcggaa 600 gcggcggagt attttacagt aagctccacc aaacctgatg
gctggcagaa aaaccccatt 660 cagcaatttc gcgtgtggct cataacttaa
ttccgttcaa tcactccggt cagtaccgga 720 cgttaatgga aggagtggtc
ttagtgtgaa g 751 13 751 DNA Bovine rotavirus strain BRV033 13
ggctttaaaa agttctgttc cgagagagtg tgtgcgggaa gatggagaag cttaccgacc
60 tcaactacac atcgagtgtt atcactctaa tgaacaacac attgcatacg
attcttgagg 120 accccggaat ggcgtacttc ccatacattg catctgtcct
aacagttttg tttacgttgc 180 acaaggcatc tatacctaca atgaagatag
cactgaaaac gtccaagtgt tcatacaaag 240 tagtaaaata ctgtatagta
acgatattca atacgttgtt gaaattggca ggttacaaag 300 aacagataac
tactaaagat gagatagaaa agcaaatgga cagggttgtt aaagagatga 360
gacgtcagtt tgaaatgatt gataagttga ctacacgtga aatagagcag gtagagttgc
420 taaagcgcat acacgacaag ttgatggttc gagcaacaga tgagattgat
atgacgaagg 480 aaataaacca aaagaacgta agaacgctag aagaatggga
aaatggaaaa aatccttatg 540 aacccaagga ggtgactgca gcgatgtaag
aggttgagct gccctcgact gtcttcggaa 600 gcggcggagt tcttcacagt
aagccacatc ggacatgatg acttactgaa aagccccagt 660 cagtcatttc
ccgagtggct taagccttaa tccccttcaa ccattcaggt cagcaccgga 720
cgttaatgga gggaacggtc ttaatgtgac a 751 14 751 DNA Bovine rotavirus
strain B223 14 ggctttaaaa agttctgttc cgagagagtg tgtgcgggaa
gatggaaaag ctaaccgacc 60 tcaactatac attgagtgtt atcactctaa
tgaactccac attgcatacg attcttgagg 120 accccgggat ggcgtacttc
ccatatattg catcagtttt aacagtatta ttcacgttgc 180 acaaggcatc
tatacccaca atgaagattg ctctaaagac gtccaagtgt tcatacaaag 240
tagtaaaata ttgcattgtt acgattttca atacgttgtt gaaattggct gggtacaaag
300 aacagataac tactaaagat gagatagaga aacagatgga aagggtagta
aaggaaatga 360 gacgtcactt caaaatgata gacaaattga caactcgtga
aattgagcag gtaggattgc 420 taaagcgcat tcacgacaag ttggatatac
gggctgttga tgaaatagac atgacgaaag 480 aaattaacca gaaaaacgtt
agaacgctag aagaatggga gtggggaaaa aatccctatg 540 aacccaaaga
agttactgct gcaatgtaag aggttgagct accttcgaca gtattcggaa 600
gcgggggggt actacacagt aagcctcaac ggttatgttg actaactgag aaacctcaat
660 cagtcatttc cagagttttt taagccttaa tccccttcaa ccattcaggt
cagcaccgga 720 cgttaatgga aggaacggtc ttaatgtgac a 751 15 750 DNA
Canine rotavirus strain CU-1 15 ggcttttaaa agttctgttc cgagaaagcg
catgcggaaa gatggagaag cttgcagacc 60 tcaactatac cctgagtgta
atcacgctaa tgaatgatac tttgcacact attatggagg 120 atcccggaat
ggcatacttc ccatatattg catctgttct aactgtacta tttacattac 180
ataaggcatc aatcccaacc atgaaaatcg cacttaaaac atcaagatgt tcatacaagg
240 ttatcaagta ctgcatagta tcagtattta acactctatt gaagttggct
ggatacaaag 300 agcagataac tactaaagat gaaatagaaa aacaaatgga
cagagttgtt aaagaaatga 360 ggcgtcagct ggaaatgatt gataaactaa
ccacaaggga gatagaacag gttgaacttc 420 ttaaacgaat acacgatatg
ttaattgcaa agcccgtaga caagatagat atgtcgcaag 480 agttcaacca
aaagcatttc aaaacactaa acgagtgggc agagggtgaa aatccatacg 540
aaccgagaga agtaactgca tctttatgag aggttgaact gccgtcttcg gtatgcggga
600 gcggaggagt aataaacaga aaatctcatc gaacttgatg aatggtagag
aaacctcatt 660 cagtaatttc gcgggtgact tagtcttatt cacgttttac
cattccagcc agtgctggac 720 gttaatggaa ggaatggtct taatgtgacc 750 16
750 DNA Porcine rotavirus A 16 ggcttttaaa agttctgttc cgagagagcg
cgtgcggaaa gatggataag cttgccgacc 60 tcaattacac attgagcgta
atcactttaa tgaatgacac actacactct attattcaag 120 atccaggaat
ggcgtatttt ccatatattg catctgttct gactgtttta tttactctac 180
ataaagcatc aattccaaca atgaaaatag cgttaaaaac gtcaaagtgt tcgtacaaag
240 taattaaata ttgcatggtt acaatcatta atactcttct gaagttggct
ggttataaag 300 aacaggttac tactaaggat gaaattgaac aacagatgga
cagaattatt aaagagatga 360 gacgtcaact ggaaatgatt gacaaattga
cgactcgtga aattgaacag gttgaattac 420 ttaaacgtat acatgacaaa
ttagctgcta gatcagttga cgctatagat atgtcgaaag 480 aatttaatca
gaaaaatatt cgaacgctag atgaatggga aagtggaaaa aatccatatg 540
aaccgtcgga agtaactgcg tctatgtgag aggttgagtt gccgtcgtct gtcttcggaa
600 gcggcggaac tcttcaccgc aagccccatt ggacccgatg gttgactgag
aagccacagt 660 caatcatatc gcgtgtggct cagccttaat cccgtttaac
caatccagcg aatgttggac 720 gttaatggaa ggaatggtct taatgtgacc 750 17
528 DNA Simian 11 rotavirus (strain SA11) 17 atggaaaagc ttaccgacct
caattataca ttgagtgtaa tcactctaat gaacaataca 60 ttgcacacaa
tacttgagga tccaggaatg gcgtattttc cttatatagc atctgtctta 120
acagttttgt ttgcgctaca taaagcatcc attccaacaa tgaaaattgc attgaaaacg
180 tcaaaatgtt catataaagt ggtgaaatat tgtattgtaa caatttttaa
tacgttgtta 240 aaattggcag gttataaaga gcagataact actaaagatg
agatagaaaa gcaaatggac 300 agagtagtca aagaaatgag acgccagcta
gaaatgattg acaaattgac tacacgtgaa 360 attgaacaag tagagttgct
taaacgcatt tacgataaat tgacggtgca aacgacaggc 420 gaaatagata
tgacaaaaga gatcaatcaa aaaaacgtga gaacgctaga agaatgggaa 480
agtggaaaaa atccttatga accaagagaa gtgactgcag caatgtaa 528 18 750 DNA
IDIR agent 18 ggcaaaataa aacccaaaga tgactgagaa taacgagatg
caacaactat tcgtacaagc 60 agcgtatgaa gaaatcctaa agttagctga
cagcgttgat catgaacaaa tacgcgagtc 120 catttcgaac tcatcgccac
aaaaattgtt gactggtgcg ctactaacag tgacggctct 180 atttacaaca
ttgatggtca gaaagaaagg aactcaattt ctaattcaaa aatttcagtc 240
aaatgtggtt catctgtcag aaatgttagt ttggaaagca agtcaaacag ttaaacaact
300 atgtgatgaa gtacttaatc aacatgaggt gctgcagaaa ttgcagtgtc
tggatcaact 360 atgtgaagat gtaaggaaac tgagatataa cgtagaacac
attaaaggtt tggatatttc 420 aaatgaattg ataagtttaa ccgaacgtaa
aatggctgat atagacggaa gaatacgaga 480 tgttgagcgt tcatgcgata
ggaagatcag agactatgat tggaaactag cagcactaac 540 ggcaaaccca
gttcaccaaa ttgcagcgca cgtggacatg ataagtcaac acgaagaaaa 600
tgaggctgaa gcacaagata tccaacaaca cgtaaacaaa caagcaagag taaaaatgtc
660 atctagaagg ctttaacgat ccgtgggata gctaggaggc gtaaactctg
tggttgtccc 720 tccccatcag atcaaacgag ataaaaaccc 750 19 613 DNA
Human rotavirus C 19 ggctttaaat ttttcagatc actttgctct acgaagtaat
ggatttcatc aatcaaactt 60 tgttctcaaa gtatactgaa agtaatgtag
atacaattcc ttatcttttg ggtcttattc 120 ttgcattaac taatggatca
agaatactta gattcattaa ctcattcata atcatatgta 180 agcacatagt
gactacgtct aaatcagcta ttgacaaaat gagaaaaatt aataattcgg 240
aacataacac aaagaatgcg catgaagaat atgaagaggt aatgaagcag ataagagaaa
300 tgcgtattca tatgactgca ttgtttaata gtttacatga tgataatgtt
aaatggagaa 360 tgagtgaatc tattcgcaga gaaaagaaac atgaaatgaa
gatgagtgat aatagaaatg 420 aattcaaaca ttcacataat gatacaaata
tatgtgaaaa atctggatta gagacggaag 480 tttgtctatg aaatccctgc
gcttcctgct ggtgaacgga cgccatcccg ttcatttcta 540 gcgagtagag
aaaaacattg tacccgaaac gctgagttga ggatcaatgt agatatgaaa 600
aattcatgtg
gct 613 20 613 DNA Human rotavirus C strain Ehime 9301 20
ggctttaaat ttttcagatc actttgctct acgaagtaat ggatttcatc aatcaaactt
60 tgttctcaaa gtatactgaa agttatgtag atacaattcc ttatcttttg
ggtcttattc 120 ttgcattaac taatggatca agagtactta gatttattaa
ctcattcatc accatatgta 180 agcatatagt gattacgtct aaatcagcca
ttgacaaaat gagaaaaatt aataattcgg 240 aacataacac aacgaatgcg
catgaagaat atgaagaggt aatgaagcag ataagagaaa 300 tgcgtattca
tatgactgca ttgtttaata gtttacatga tgataatgtt aaatggagaa 360
tgagcgaatc tattcgtcga gaaaagaaac atgaaatgaa gatgagtaat aatagaaatg
420 aattcaaaca ttcacataat gatacaaata tatgtgaaaa atctggatta
gagacggaag 480 tttgtctatg aaattcctgc gcttcctgct ggtgaacgga
cgccatcccg ttcatttcta 540 gcgagtagag aaaaacattg tacccgaaac
gctgagttga ggatcaatgt agatatgaaa 600 aattcatgag gct 613 21 719 DNA
Bovine rotavirus strain Shintoku 21 ggctttaaaa attgcgacaa
tgtccgattt cggaattaat cttgatgcca tttgcgacaa 60 tgtcagaaga
aattcatcaa attcaagtat caaatctcaa gtatcaaatc ggagttcacg 120
aaagatggat tttgttgatg aagatgagtt gagcacgtac ttcaattcaa aaacatccgt
180 gacacaatca gattcttgtt caaatgatct aaatgtgaaa cattcaatta
tagcagaagc 240 tgtggtatgt gacgaatctg cgcatgtgtc tgcggatgcg
gttcaagaaa aggatgtggt 300 tgttccaaaa atggatgaaa gtgttatgaa
gtggatgatg gacagtcatg atggaatatg 360 tgtgaatgga ggtctgaact
tttcaaaatt aaaaaataaa agtaatgaac atgaaactaa 420 agtaacgtca
gaaacaaatg tatcagctca cgtttcagca ggaatcaatt cacaattggg 480
gatgttcaac ccaattcagc acaaaataaa gaaagaggct ataccagaaa tgtttgaaga
540 tgaagataca gatgaatgta cttgtagaaa ttgcccatat aaagaaaaat
atcttaaact 600 ccgaaaaaaa ctgaaaaatg tgttagtcga tatcataact
gaaatgtagt cgagtacttg 660 cccgtactgc atcaggtgac tggaatcggc
attgagggga tccccaaccc gatctgtgg 719 22 64 PRT Simian strain SA11 22
Met Ile Asp Lys Leu Thr Thr Arg Glu Ile Glu Gln Val Glu Leu Leu 1 5
10 15 Lys Arg Ile Tyr Asp Lys Leu Thr Val Gln Thr Thr Gly Glu Ile
Asp 20 25 30 Met Thr Lys Glu Ile Asn Gln Lys Asn Val Arg Thr Leu
Glu Glu Trp 35 40 45 Glu Ser Gly Lys Asn Pro Tyr Glu Pro Arg Glu
Val Thr Ala Ala Met 50 55 60 23 175 PRT Porcine 23 Met Asp Lys Leu
Ala Asp Leu Asn Tyr Thr Leu Ser Val Ile Thr Leu 1 5 10 15 Met Asn
Asp Thr Leu His Ser Ile Ile Gln Asp Pro Gly Met Ala Tyr 20 25 30
Phe Pro Tyr Ile Ala Ser Val Leu Thr Val Leu Phe Thr Leu His Lys 35
40 45 Ala Ser Ile Pro Thr Met Lys Ile Ala Leu Lys Thr Ser Lys Cys
Ser 50 55 60 Tyr Lys Val Ile Lys Tyr Cys Met Val Thr Ile Ile Asn
Thr Leu Leu 65 70 75 80 Lys Leu Ala Gly Tyr Lys Glu Gln Val Thr Thr
Lys Asp Glu Ile Glu 85 90 95 Gln Gln Met Asp Arg Ile Ile Lys Glu
Met Arg Arg Gln Leu Glu Met 100 105 110 Ile Asp Lys Leu Thr Thr Arg
Glu Ile Glu Gln Val Glu Leu Leu Lys 115 120 125 Arg Ile His Asp Lys
Leu Ala Ala Arg Pro Val Asp Ala Ile Asp Met 130 135 140 Ser Lys Glu
Phe Asn Gln Lys Asn Ile Arg Thr Leu Asp Glu Trp Glu 145 150 155 160
Ser Gly Lys Asn Pro Tyr Glu Pro Ser Glu Val Thr Ala Ser Met 165 170
175 24 175 PRT Simian 11 rotavirus (strain SA11) 24 Met Glu Lys Leu
Thr Asp Leu Asn Tyr Thr Leu Ser Val Ile Thr Leu 1 5 10 15 Met Asn
Asn Thr Leu His Thr Ile Leu Glu Asp Pro Gly Met Ala Tyr 20 25 30
Phe Pro Tyr Ile Ala Ser Val Leu Thr Val Leu Phe Ala Leu His Lys 35
40 45 Ala Ser Ile Pro Thr Met Lys Ile Ala Leu Lys Thr Ser Lys Cys
Ser 50 55 60 Tyr Lys Val Val Lys Tyr Cys Ile Val Thr Ile Phe Asn
Thr Leu Leu 65 70 75 80 Lys Leu Ala Gly Tyr Lys Glu Gln Ile Thr Thr
Lys Asp Glu Ile Glu 85 90 95 Lys Gln Met Asp Arg Val Val Lys Glu
Met Arg Arg Gln Leu Glu Met 100 105 110 Ile Asp Lys Leu Thr Thr Arg
Glu Ile Glu Gln Val Glu Leu Leu Lys 115 120 125 Arg Ile Tyr Asp Lys
Leu Thr Val Gln Thr Thr Gly Glu Ile Asp Met 130 135 140 Thr Lys Glu
Ile Asn Gln Lys Asn Val Arg Thr Leu Glu Glu Trp Glu 145 150 155 160
Ser Gly Lys Asn Pro Tyr Glu Pro Arg Glu Val Thr Ala Ala Met 165 170
175 25 175 PRT Porcine rotavirus A 25 Met Asp Lys Leu Ala Asp Leu
Asn Tyr Thr Leu Ser Val Ile Thr Leu 1 5 10 15 Met Asn Asp Thr Leu
His Ser Ile Ile Gln Asp Pro Gly Met Ala Tyr 20 25 30 Phe Pro Tyr
Ile Ala Ser Val Leu Thr Val Leu Phe Thr Leu His Lys 35 40 45 Ala
Ser Ile Pro Thr Met Lys Ile Ala Leu Lys Thr Ser Lys Cys Ser 50 55
60 Tyr Lys Val Ile Lys Tyr Cys Met Val Thr Ile Ile Asn Thr Leu Leu
65 70 75 80 Lys Leu Ala Gly Tyr Lys Glu Gln Val Thr Thr Lys Asp Glu
Ile Glu 85 90 95 Gln Gln Met Asp Arg Ile Ile Lys Glu Met Arg Arg
Gln Leu Glu Met 100 105 110 Ile Asp Lys Leu Thr Thr Arg Glu Ile Glu
Gln Val Glu Leu Leu Lys 115 120 125 Arg Ile His Asp Lys Leu Ala Ala
Arg Ser Val Asp Ala Ile Asp Met 130 135 140 Ser Lys Glu Phe Asn Gln
Lys Asn Ile Arg Thr Leu Asp Glu Trp Glu 145 150 155 160 Ser Gly Lys
Asn Pro Tyr Glu Pro Ser Glu Val Thr Ala Ser Met 165 170 175
* * * * *
References