U.S. patent application number 13/866116 was filed with the patent office on 2017-03-23 for malaria antigen screening method.
The applicant listed for this patent is Joao Carlos Aguair, Douglas E. Brough, Joseph T. Bruder, Daniel John Carucci, Denise Louise Doolan, C. Richter King, Imre Kovesdi, Keith Limbach, Duncan L. McVey, Martha Sedegah, Walter R. Weiss. Invention is credited to Joao Carlos Aguair, Douglas E. Brough, Joseph T. Bruder, Daniel John Carucci, Denise Louise Doolan, C. Richter King, Imre Kovesdi, Keith Limbach, Duncan L. McVey, Martha Sedegah, Walter R. Weiss.
Application Number | 20170082607 13/866116 |
Document ID | / |
Family ID | 51729186 |
Filed Date | 2017-03-23 |
United States Patent
Application |
20170082607 |
Kind Code |
A9 |
Bruder; Joseph T. ; et
al. |
March 23, 2017 |
MALARIA ANTIGEN SCREENING METHOD
Abstract
The invention provides a method of identifying an antigen from a
pathogen or a disease antigen comprising the use of an adenoviral
vector array comprising two or more different adenoviral vectors,
wherein each adenoviral vector comprises a nucleic acid sequence
encoding a different antigen of a pathogen. The adenoviral vectors
are administered to antigen presenting cells (APCs) in vitro or to
an animal in vivo. The immunogenicity of the antigen is measured by
screening for an immune response from effector T lymphocytes in
vitro and by screening for the absence of pathogen-induced disease
onset in vivo.
Inventors: |
Bruder; Joseph T.;
(Ijamsville, MD) ; Kovesdi; Imre; (Rockville,
MD) ; McVey; Duncan L.; (Derwood, MD) ;
Brough; Douglas E.; (Gaithersburg, MD) ; King; C.
Richter; (Washington, DC) ; Doolan; Denise
Louise; (Camp Hill, AU) ; Aguair; Joao Carlos;
(Potomac, MD) ; Carucci; Daniel John; (Washington,
DC) ; Sedegah; Martha; (Gaithersburg, MD) ;
Weiss; Walter R.; (Bethesda, MD) ; Limbach;
Keith; (Gaithersburg, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bruder; Joseph T.
Kovesdi; Imre
McVey; Duncan L.
Brough; Douglas E.
King; C. Richter
Doolan; Denise Louise
Aguair; Joao Carlos
Carucci; Daniel John
Sedegah; Martha
Weiss; Walter R.
Limbach; Keith |
Ijamsville
Rockville
Derwood
Gaithersburg
Washington
Camp Hill
Potomac
Washington
Gaithersburg
Bethesda
Gaithersburg |
MD
MD
MD
MD
DC
MD
DC
MD
MD
MD |
US
US
US
US
US
AU
US
US
US
US
US |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20140314809 A1 |
October 23, 2014 |
|
|
Family ID: |
51729186 |
Appl. No.: |
13/866116 |
Filed: |
April 19, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11513439 |
Aug 25, 2006 |
8450055 |
|
|
13866116 |
|
|
|
|
60713001 |
Aug 31, 2005 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/505
20130101 |
International
Class: |
G01N 33/50 20060101
G01N033/50 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND
DEVELOPMENT
[0002] This invention was made in part with Government support
under Cooperative Research and Development Agreement (CRADA) Number
NMR-04-1869, and amendments thereto, executed between GenVec, Inc.
and the Naval Medical Research Center (NMRC). The Government may
have certain rights in this invention.
Claims
1. A method of identifying an antigen from a pathogen, which method
comprises (a) preparing an adenoviral vector array comprising two
or more different adenoviral vectors, wherein each adenoviral
vector comprises a nucleic acid sequence encoding a different
antigen of a pathogen, (b) contacting antigen presenting cells
(APCs) with the adenoviral vector array, wherein each different
adenoviral vector transduces an APC such that the nucleic acid
sequences of the different adenoviral vectors are expressed and the
different antigens are produced in the APCs, (c) incubating the
APCs with effector T lymphocytes obtained from a mammal immunized
with the pathogen, and (d) screening for an immune response from
the effector T lymphocytes, wherein an immune response from an
effector T lymphocyte contacting an APC indicates T lymphocyte
recognition of the antigen produced by the APC, whereupon the
antigen is identified.
2. The method of claim 1, wherein the pathogen is selected from a
Plasmodium species, human immune deficiency virus (HIV), severe
acute respiratory syndrome (SARS) virus, foot and mouth disease
(FMD) virus, and Mycobacterium tuberculosis.
3. The method of claim 3, wherein the pathogen is selected from the
group consisting of Plasmodium berghei, Plasmodium chabaudi,
Plasmodium vinckei, Plasmodium yoelii, Plasmodium falciparum,
Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale.
4. The method of any of claims 1-3, wherein the antigen presenting
cells are dendritic cells.
5. The method of any of claims 1-4, wherein the immune response is
selected from the group consisting of cytokine secretion from
effector T lymphocytes, cytotoxicity of effector T lymphocytes, and
immune activation of effector T lymphocytes.
6. The method of claim 5, wherein the cytokine is selected from the
group consisting of interferon gamma (IFN-.gamma.), TNF-.beta.,
TNF-.alpha., GM-CSF, CD40 ligand, Fas ligand, and interleukins.
7. The method of any of claims 1-6, wherein each of the multiple
adenoviral vectors exhibits reduced native binding to a
coxsackievirus and adenovirus receptor (CAR).
8. The method of claim 7, wherein each of the two or more different
adenoviral vectors comprises a fiber protein wherein a native
CAR-binding site is mutated.
9. The method of claim 8, wherein each of the two or more different
adenoviral vectors comprises a fiber protein comprising a normative
amino acid sequence.
10. The method of claim 9, wherein the normative amino acid
sequence comprises an RGD sequence.
11. The method of claim 9 or claim 10, wherein the normative amino
acid sequence is inserted into an exposed loop of the fiber
protein.
12. A method of identifying an antigen from a pathogen, which
method comprises (a) providing an adenoviral vector array
comprising two or more different adenoviral vectors, wherein each
adenoviral vector comprises a nucleic acid sequence encoding a
different antigen of a pathogen, (b) administering each of the
adenoviral vectors of the adenoviral vector array to a mammal, such
that the nucleic acid sequence is expressed and the antigen is
produced in the mammal, (c) infecting each mammal with the
pathogen, and (d) screening the infected mammal for onset of a
disease caused by the pathogen, wherein the absence in the infected
mammal of a disease caused by the pathogen indicates that the
adenoviral vector encodes an antigen of the pathogen, whereupon the
antigen is identified.
13. The method of claim 12, wherein the pathogen is selected from a
Plasmodium species, human immune deficiency virus (HIV), severe
acute respiratory syndrome (SARS) virus, foot and mouth disease
(FMD) virus, and Mycobacterium tuberculosis.
14. The method of claim 13, wherein the pathogen is selected from
the group consisting of Plasmodium berghei, Plasmodium chabaudi,
Plasmodium vinckei, Plasmodium yoelii, Plasmodium falciparum,
Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale.
15. The method of claim 12, wherein the adenoviral vector is
administered as part of an unpurified cell lysate comprising the
adenoviral vector.
16. A method of inducing an immune response against a pathogen in a
mammal, which method comprises (a) preparing an adenoviral vector
comprising a nucleic acid sequence encoding an antigen of a
pathogen identified by the method of any of claims 1-15, and (b)
administering the adenoviral vector to a mammal, wherein the
antigen is expressed in the mammal to induce an immune
response.
17. The method of any of claims 1-16, wherein the adenoviral
vectors are replication-deficient.
18. The method of claim 17, wherein each of the adenoviral vectors
requires complementation of the E1 region of the adenoviral genome
for replication.
19. The method of claim 17 or claim 18, wherein each of the
adenoviral vectors requires complementation of the E4 region of the
adenoviral genome for replication.
20. The method of any of claims 16-19, wherein each of the
adenoviral vectors lacks the entire E1 region and at least a
portion of the E4 region of the adenoviral genome.
21. The method of any of claims 16-20, wherein each of the
adenoviral vectors lacks all or part of the E3 region of the
adenoviral genome.
22. A method of identifying a disease antigen, which method
comprises (a) preparing an adenoviral vector array comprising two
or more different adenoviral vectors, wherein each adenoviral
vector comprises a nucleic acid sequence encoding a different
disease antigen, (b) contacting antigen presenting cells (APCs)
with the adenoviral vector array, wherein each different adenoviral
vector transduces an APC such that the nucleic acid sequences of
the different adenoviral vectors are expressed and the different
antigens are produced in the APCs, (c) incubating the APCs with
effector T lymphocytes obtained from a mammal affected by the
disease, and (d) screening for an immune response from the effector
T lymphocytes, wherein an immune response from an effector T
lymphocyte contacting an APC indicates T lymphocyte recognition of
the antigen produced by the APC, whereupon the disease antigen is
identified.
23. The method of claim 22, wherein the disease is cancer.
24. The method of claim 22, wherein the disease is an autoimmune
disease.
25. The method of any of claims 1-24, wherein the mammal is a
mouse.
26. The method of any of claims 1-24, wherein the mammal is a
human.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 11/513,439 (now allowed), filed on Aug. 25,
2006, which claims priority to U.S. Provisional application
60/713,001 filed on Aug. 31, 2005.
REFERENCE TO SEQUENCE LISTING
[0003] A sequence listing is provided in paper form and as in
computer readable form. I hereby state that the information
recorded in computer readable form is identical to the written
sequence listing.
BACKGROUND OF THE INVENTION
[0004] Malaria is one of the most devastating parasitic diseases
affecting humans. Indeed, 41% of the world's population lives in
areas where malaria is transmitted (e.g., parts of Africa, Asia,
the Middle East, Central and South America, Hispaniola, and
Oceania). The World Health Organization (WHO) and the Centers for
Disease Control (CDC) estimate that malaria infects 300-500 million
people and kills 700,000-3 million people annually, with the
majority of deaths occurring in children in sub-Saharan Africa.
Malaria also is a major health concern to U.S. military personnel
deployed to tropical regions of the world. For example, in August
2003, 28% of the 26.sup.th Marine Expeditionary Unit and Joint Task
Force briefly deployed to Monrovia, Liberia, were infected with the
malaria parasite Plasmodium falciparum. In addition, one 157-man
Marine Expeditionary Unit sustained a 44% malaria casualty rate
over a 12-day period while stationed at Robert International
Airport in Monrovia. In all conflicts during the past century
conducted in malaria endemic areas, malaria has been the leading
cause of casualties, exceeding enemy-inflicted casualties in its
impact on "person-days" lost from duty.
[0005] To combat malaria during U.S. military operations,
preventive drugs, insect repellants, and barriers have been used
with some success, but developing drug resistance by the malaria
parasite and insecticide resistance by mosquito vectors has limited
the efficacy of these agents. Moreover, the logistical burden and
side effects associated with the use of these agents often is
associated with high non-compliance rates. Vaccines are the most
cost effective and efficient therapeutic interventions for
infectious diseases. In this regard, vaccination has the advantage
of administration prior to military deployment and likely reduction
in non-compliance risks. However, decades of research and
development directed to a malaria vaccine have not proven
successful. Recent efforts have focused on developing vaccines
against several specific malaria genes and delivery vector systems
including adenovirus, poxvirus, and plasmids. The current status of
malaria vaccine development and clinical trials is reviewed in, for
example, Graves and Gelband, Cochrane Database Syst. Rev., 1:
CD000129 (2003), Moore et al., Lancet Infect. Dis., 2: 737-743
(2002), Carvalho et al., Scand. J. Immunol., 56: 327-343 (2002),
Moorthy and Hill, Br. Med. Bull., 62: 59-72 (2002), Greenwood and
Alonso, Chem. Immunol., 80: 366-395 (2002), and Richie and Saul,
Nature, 415: 694-701 (2002).
[0006] An unprecedented quantity of genomic data has emerged from
the sequencing and functional genomic analysis of many
disease-causing organisms, including malaria. Indeed, it has been
determined that the parasite Plasmodium falciparum encodes an
estimated 5,268 putative proteins (see Gardner et al., Nature, 419:
498-511 (2002)). This genetic information can be exploited for the
systematic discovery of novel antigens for vaccine development. In
the past, target antigens for genetic vaccines have been identified
based mainly on their abundance in the pathogen of interest and
their susceptibility to neutralization by antibodies generated in
infected individuals and animal models. This approach has failed to
yield effective vaccines against many of the most devastating
infectious diseases. With regard to malaria, less than 5% of the
Plasmodium falciparum genome is represented by antigens currently
in clinical development. A vaccine containing a recombinant P.
falciparum circumsporozoite protein (CSP) has been the most
successful vaccine tested to date, providing a protective efficacy
of 47-85% against experimental pathogen challenge (see, e.g.,
Stoute et al., N. Engl. J. Med., 336: 86-91 (1997), Stoute et al.,
J. Infect. Dis., 178: 1139-1144 (1998), and Kester et al., J.
Infect. Dis., 183: 640-647 (2001)) and pathogen challenge in the
field (see Bojang et al., Lancet, 358: 1927-1934 (2001)). The
protection afforded by this protein-based vaccine, however is short
lived (3-8 weeks). Other recent efforts at developing a malaria
vaccine have focused on several specific genes and their delivery
using various different vector systems including adenovirus,
poxvirus, and plasmid DNA. It is not apparent, however, whether
these recombinant vaccines are effective against malaria, or if
they encode the most potent protective antigens. It is clear that
protective antigens do exist for the malaria pathogen Plasmodium
falciparum, as evidenced by the ability of irradiated sporozoites
to induce cellular immune responses in human subjects and robust
sterile protection against parasite challenge (see, e.g.,
Nussenzweig and Nussenzweig, Adv. Immunol., 45: 283-334 (1989), and
Hoffman et al., J. Infect. Dis., 185: 1155-1164 (2002)).
[0007] Thus, there remains a need for improved methods for
identifying antigens that induce potent protective immunity against
pathogen challenge. The invention provides such a method. This and
other advantages of the invention will become apparent from the
detailed description provided herein.
BRIEF SUMMARY OF THE INVENTION
[0008] The invention provides a method of identifying an antigen
from a pathogen. The method comprises (a) preparing an adenoviral
vector array comprising two or more different adenoviral vectors,
wherein each adenoviral vector comprises a nucleic acid sequence
encoding a different antigen of a pathogen, (b) contacting antigen
presenting cells (APCs) with the adenoviral vector array, wherein
each different adenoviral vector transduces an APC such that the
nucleic acid sequences of the different adenoviral vectors are
expressed and the different antigens are produced in the APCs, (c)
incubating the APCs with effector T lymphocytes obtained from a
mammal immunized with the pathogen, and (d) screening for an immune
response from the effector T lymphocytes, wherein an immune
response from an effector T lymphocyte contacting an APC indicates
T lymphocyte recognition of the antigen produced by the APC,
whereupon the antigen is identified.
[0009] The invention also provides a method of identifying an
antigen from a pathogen, which method comprises (a) providing an
adenoviral vector array comprising two or more different adenoviral
vectors, wherein each adenoviral vector comprises a nucleic acid
sequence encoding a different antigen of a pathogen, (b)
administering each of the adenoviral vectors of the adenoviral
vector array to a mammal, such that the nucleic acid sequence is
expressed and the antigen is produced in the mammal, (c) infecting
each mammal with the pathogen, and (d) screening the infected
mammal for onset of a disease caused by the pathogen, wherein the
absence in the infected mammal of a disease caused by the pathogen
indicates that the adenoviral vector encodes an antigen of the
pathogen, whereupon the antigen is identified.
[0010] The invention further provides a method of identifying a
disease antigen, which method comprises (a) preparing an adenoviral
vector array comprising two or more different adenoviral vectors,
wherein each adenoviral vector comprises a nucleic acid sequence
encoding a different disease antigen, (b) contacting APCs with the
adenoviral vector array, wherein each different adenoviral vector
transduces an APC such that the nucleic acid sequences of the
different adenoviral vectors are expressed and the different
antigens are produced in the APCs, (c) incubating the APCs with
effector T lymphocytes obtained from a mammal affected by the
disease, and (d) screening for an immune response from the effector
T lymphocytes, wherein an immune response from an effector T
lymphocyte contacting an APC indicates T lymphocyte recognition of
the antigen produced by the APC, whereupon the disease antigen is
identified.
DETAILED DESCRIPTION OF THE INVENTION
[0011] The invention provides a method of identifying an antigen
from a pathogen comprising preparing an adenoviral vector array
comprising two or more different adenoviral vectors. Adenovirus
(Ad) is a 36 kb double-stranded DNA virus that efficiently
transfers DNA in vivo to a variety of different target cell types.
For use in the invention, the adenovirus is preferably made
replication-deficient by deleting, in whole or in part, select
genes required for viral replication. The expendable E3 region is
also frequently deleted to allow additional room for a larger DNA
insert. The vector can be produced in high titers and can
efficiently transfer DNA to replicating and non-replicating cells.
The newly transferred genetic information remains epi-chromosomal,
thus eliminating the risks of random insertional mutagenesis and
permanent alteration of the genotype of the target cell. However,
if desired, the integrative properties of AAV can be conferred to
adenovirus by constructing an AAV-Ad chimeric vector. For example,
the AAV ITRs and nucleic acid encoding the Rep protein incorporated
into an adenoviral vector enables the adenoviral vector to
integrate into a mammalian cell genome. Therefore, AAV-Ad chimeric
vectors can be a desirable option for use in the invention.
[0012] Adenovirus from various origins, subtypes, or mixture of
subtypes can be used as the source of the viral genome for the
adenoviral vector. A non-human adenovirus (e.g., simian, avian,
canine, ovine, or bovine adenoviruses) can be used to generate the
adenoviral vector. Alternatively, a human adenovirus can be used as
the source of the viral genome for the adenoviral vector. For
instance, an adenovirus can be of subgroup A (e.g., serotypes 12,
18, and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34,
35, and 50), subgroup C (e.g., serotypes 1, 2, 5, and 6), subgroup
D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33,
36-39, and 42-48), subgroup E (e.g., serotype 4), subgroup F (e.g.,
serotypes 40 and 41), an unclassified serogroup (e.g., serotypes 49
and 51), or any other adenoviral serotype. Adenoviral serotypes 1
through 51 (i.e., Ad1 through Ad51) are available from the American
Type Culture Collection (ATCC, Manassas, Va.). Preferably, in the
context of the invention, the adenoviral vector is of human
subgroup C, especially serotype 2 or even more desirably serotype
5. However, non-group C adenoviruses can be used to prepare
adenoviral gene transfer vectors for delivery of gene products to
host cells. Preferred adenoviruses used in the construction of
non-group C adenoviral gene transfer vectors include Ad12 (group
A), Ad7 and Ad35 (group B), Ad30 and Ad36 (group D), Ad4 (group E),
and Ad41 (group F). Non-group C adenoviral vectors, methods of
producing non-group C adenoviral vectors, and methods of using
non-group C adenoviral vectors are disclosed in, for example, U.S.
Pat. Nos. 5,801,030, 5,837,511, and 5,849,561, and International
Patent Application Publications WO 97/12986 and WO 98/53087.
[0013] The adenoviral vector can comprise a mixture of subtypes and
thereby be a "chimeric" adenoviral vector. A chimeric adenoviral
vector can comprise an adenoviral genome that is derived from two
or more (e.g., 2, 3, 4, etc.) different adenovirus serotypes. In
the context of the invention, a chimeric adenoviral vector can
comprise approximately different or equal amounts of the genome of
each of the two or more different adenovirus serotypes. When the
chimeric adenoviral vector genome is comprised of the genomes of
two different adenovirus serotypes, the chimeric adenoviral vector
genome preferably comprises no more than about 70% (e.g., no more
than about 65%, about 50%, or about 40%) of the genome of one of
the adenovirus serotypes, with the remainder of the chimeric
adenovirus genome being derived from the genome of the other
adenovirus serotype. In one embodiment, the chimeric adenoviral
vector can contain an adenoviral genome comprising a portion of a
serotype 2 genome and a portion of a serotype 5 genome. For
example, nucleotides 1-456 of such an adenoviral vector can be
derived from a serotype 2 genome, while the remainder of the
adenoviral genome can be derived from a serotype 5 genome.
[0014] The adenoviral vector of the invention can be
replication-competent. For example, the adenoviral vector can have
a mutation (e.g., a deletion, an insertion, or a substitution) in
the adenoviral genome that does not inhibit viral replication in
host cells. The adenoviral vector also can be conditionally
replication-competent. Preferably, however, the adenoviral vector
is replication-deficient in host cells.
[0015] By "replication-deficient" is meant that the adenoviral
vector requires complementation of one or more regions of the
adenoviral genome that are required for replication, as a result
of, for example, a deficiency in at least one replication-essential
gene function (i.e., such that the adenoviral vector does not
replicate in typical host cells, especially those in a human
patient that could be infected by the adenoviral vector in the
course of the inventive method). A deficiency in a gene, gene
function, gene, or genomic region, as used herein, is defined as a
mutation or deletion of sufficient genetic material of the viral
genome to obliterate or impair the function of the gene (e.g., such
that the function of the gene product is reduced by at least about
2-fold, 5-fold, 10-fold, 20-fold, 30-fold, or 50-fold) whose
nucleic acid sequence was mutated or deleted in whole or in part.
Deletion of an entire gene region often is not required for
disruption of a replication-essential gene function. However, for
the purpose of providing sufficient space in the adenoviral genome
for one or more transgenes, removal of a majority of a gene region
may be desirable. While deletion of genetic material is preferred,
mutation of genetic material by addition or substitution also is
appropriate for disrupting gene function. Replication-essential
gene functions are those gene functions that are required for
replication (e.g., propagation) and are encoded by, for example,
the adenoviral early regions (e.g., the E1, E2, and E4 regions),
late regions (e.g., the L1-L5 regions), genes involved in viral
packaging (e.g., the IVa2 gene), and virus-associated RNAs (e.g.,
VA-RNA 1 and/or VA-RNA-2).
[0016] The replication-deficient adenoviral vector desirably
requires complementation of at least one replication-essential gene
function of one or more regions of the adenoviral genome for viral
replication. Preferably, the adenoviral vector requires
complementation of at least one gene function of the E1A region,
the E1B region, or the E4 region of the adenoviral genome required
for viral replication (denoted an E1-deficient or E4-deficient
adenoviral vector). In addition to a deficiency in the E1 region,
the recombinant adenovirus also can have a mutation in the major
late promoter (MLP), as discussed in International Patent
Application Publication WO 00/00628. Most preferably, the
adenoviral vector is deficient in at least one
replication-essential gene function (desirably all
replication-essential gene functions) of the E1 region and at least
one gene function of the nonessential E3 region (e.g., an Xba I
deletion of the E3 region) (denoted an E1/E3-deficient adenoviral
vector). With respect to the E1 region, the adenoviral vector can
be deficient in part or all of the E1A region and/or part or all of
the E1B region, e.g., in at least one replication-essential gene
function of each of the E1A and E1B regions, thus requiring
complementation of the E1A region and the E1B region of the
adenoviral genome for replication. The adenoviral vector also can
require complementation of the E4 region of the adenoviral genome
for replication, such as through a deficiency in one or more
replication-essential gene functions of the E4 region.
[0017] When the adenoviral vector is E1-deficient, the adenoviral
vector genome can comprise a deletion beginning at any nucleotide
between nucleotides 335 to 375 (e.g., nucleotide 356) and ending at
any nucleotide between nucleotides 3,310 to 3,350 (e.g., nucleotide
3,329) or even ending at any nucleotide between 3,490 and 3,530
(e.g., nucleotide 3,510) (based on the adenovirus serotype 5
genome). When E2A-deficient, the adenoviral vector genome can
comprise a deletion beginning at any nucleotide between nucleotides
22,425 to 22,465 (e.g., nucleotide 22,443) and ending at any
nucleotide between nucleotides 24,010 to 24,050 (e.g., nucleotide
24,032) (based on the adenovirus serotype 5 genome). When
E3-deficient, the adenoviral vector genome can comprise a deletion
beginning at any nucleotide between nucleotides 28,575 to 29,615
(e.g., nucleotide 28,593) and ending at any nucleotide between
nucleotides 30,450 to 30,490 (e.g., nucleotide 30,470) (based on
the adenovirus serotype 5 genome). When E4-deficient, the
adenoviral vector genome can comprise a deletion beginning at, for
example, any nucleotide between nucleotides 32,805 to 32,845 (e.g.,
nucleotide 32,826) and ending at, for example, any nucleotide
between nucleotides 35,540 to 35,580 (e.g., nucleotide 35,561)
(based on the adenovirus serotype 5 genome). The endpoints defining
the deleted nucleotide portions can be difficult to precisely
determine and typically will not significantly affect the nature of
the adenoviral vector, i.e., each of the aforementioned nucleotide
numbers can be +/-1, 2, 3, 4, 5, or even 10 or 20 nucleotides.
[0018] When the adenoviral vector is deficient in at least one
replication-essential gene function in one region of the adenoviral
genome (e.g., an E1- or E1/E3-deficient adenoviral vector), the
adenoviral vector is referred to as "singly replication-deficient."
A particularly preferred singly replication-deficient adenoviral
vector is, for example, a replication-deficient adenoviral vector
requiring, at most, complementation of the E1 region of the
adenoviral genome, so as to propagate the adenoviral vector (e.g.,
to form adenoviral vector particles).
[0019] The adenoviral vector can be "multiply
replication-deficient," meaning that the adenoviral vector is
deficient in one or more replication-essential gene functions in
each of two or more regions of the adenoviral genome, and requires
complementation of those functions for replication. For example,
the aforementioned E1-deficient or E1/E3-deficient adenoviral
vector can be further deficient in at least one
replication-essential gene function of the E4 region (denoted an
E1/E4- or E1/E3/E4-deficient adenoviral vector), and/or the E2
region (denoted an E1/E2- or E1/E2/E3-deficient adenoviral vector),
preferably the E2A region (denoted an E1/E2A- or
E1/E2A/E3-deficient adenoviral vector). When the adenoviral vector
is multiply replication-deficient, the deficiencies can be a
combination of the nucleotide deletions discussed above with
respect to each individual region.
[0020] If the adenoviral vector of the invention is deficient in a
replication-essential gene function of the E2A region, the vector
preferably does not comprise a complete deletion of the E2A region,
which deletion preferably is less than about 230 base pairs in
length. Generally, the E2A region of the adenovirus codes for a DBP
(DNA binding protein), a polypeptide required for DNA replication.
DBP is composed of 473 to 529 amino acids depending on the viral
serotype. It is believed that DBP is an asymmetric protein that
exists as a prolate ellipsoid consisting of a globular Ct with an
extended Nt domain. Studies indicate that the Ct domain is
responsible for DBP's ability to bind to nucleic acids, bind to
zinc, and function in DNA synthesis at the level of DNA chain
elongation. However, the Nt domain is believed to function in late
gene expression at both transcriptional and post-transcriptional
levels, is responsible for efficient nuclear localization of the
protein, and also may be involved in enhancement of its own
expression. Deletions in the Nt domain between amino acids 2 to 38
have indicated that this region is important for DBP function
(Brough et al., Virology, 196: 269-281 (1993)). While deletions in
the E2A region coding for the Ct region of the DBP have no effect
on viral replication, deletions in the E2A region which code for
amino acids 2 to 38 of the Nt domain of the DBP impair viral
replication. It is preferable that any multiply
replication-deficient adenoviral vector contains this portion of
the E2A region of the adenoviral genome. In particular, for
example, the desired portion of the E2A region to be retained is
that portion of the E2A region of the adenoviral genome which is
defined by the 5' end of the E2A region, specifically positions
Ad5(23816) to Ad5(24032) of the E2A region of the adenoviral
serotype 5 genome. This portion of the adenoviral genome desirably
is included in the adenoviral vector because it is not complemented
in current E2A complementing cell lines so as to provide the
desired level of viral propagation.
[0021] While the above-described deletions are described with
respect to an adenovirus serotype 5 genome, one of ordinary skill
in the art can determine the nucleotide coordinates of the same
regions of other adenovirus serotypes, such as an adenovirus
serotype 2 genome, without undue experimentation, based on the
similarity between the genomes of various adenovirus serotypes,
particularly adenovirus serotypes 2 and 5.
[0022] In one embodiment of the invention, the adenoviral vector
can comprise an adenoviral genome deficient in one or more
replication-essential gene functions of each of the E1 and E4
regions (i.e., the adenoviral vector is an E1/E4-deficient
adenoviral vector), preferably with the entire coding region of the
E4 region having been deleted from the adenoviral genome. In other
words, all the open reading frames (ORFs) of the E4 region have
been removed. Most preferably, the adenoviral vector is rendered
replication-deficient by deletion of all of the E1 region and by
deletion of a portion of the E4 region. The E4 region of the
adenoviral vector can retain the native E4 promoter,
polyadenylation sequence, and/or the right-side inverted terminal
repeat (ITR).
[0023] It should be appreciated that the deletion of different
regions of the adenoviral vector can alter the immune response of
the mammal. In particular, deletion of different regions can reduce
the inflammatory response generated by the adenoviral vector. An
adenoviral vector deleted of the entire E4 region can elicit a
lower host immune response. Furthermore, the adenoviral vector's
coat protein can be modified so as to decrease the adenoviral
vector's ability or inability to be recognized by a neutralizing
antibody directed against the wild-type coat protein, as described
in International Patent Application WO 98/40509. Such modifications
are useful for long-term treatment of persistent disorders.
[0024] The adenoviral vector, when multiply replication-deficient,
especially in replication-essential gene functions of the E1 and E4
regions, can include a spacer sequence to provide viral growth in a
complementing cell line similar to that achieved by singly
replication-deficient adenoviral vectors, particularly an
E1-deficient adenoviral vector. In a preferred E4-deficient
adenoviral vector of the invention wherein the L5 fiber region is
retained, the spacer is desirably located between the L5 fiber
region and the right-side ITR. More preferably in such an
adenoviral vector, the E4 polyadenylation sequence alone or, most
preferably, in combination with another sequence exists between the
L5 fiber region and the right-side ITR, so as to sufficiently
separate the retained L5 fiber region from the right-side ITR, such
that viral production of such a vector approaches that of a singly
replication-deficient adenoviral vector, particularly a singly
replication-deficient E1 deficient adenoviral vector.
[0025] The spacer sequence can contain any nucleotide sequence or
sequences which are of a desired length, such as sequences at least
about 15 base pairs (e.g., between about 15 base pairs and about
12,000 base pairs), preferably about 100 base pairs to about 10,000
base pairs, more preferably about 500 base pairs to about 8,000
base pairs, even more preferably about 1,500 base pairs to about
6,000 base pairs, and most preferably about 2,000 to about 3,000
base pairs in length. The spacer sequence can be coding or
non-coding and native or non-native with respect to the adenoviral
genome, but does not restore the replication-essential function to
the deficient region. The spacer can also contain a
promoter-variable expression cassette. More preferably, the spacer
comprises an additional polyadenylation sequence and/or a passenger
gene. Preferably, in the case of a spacer inserted into a region
deficient for E4, both the E4 polyadenylation sequence and the E4
promoter of the adenoviral genome or any other (cellular or viral)
promoter remain in the vector. The spacer is located between the E4
polyadenylation site and the E4 promoter, or, if the E4 promoter is
not present in the vector, the spacer is proximal to the right-side
ITR. The spacer can comprise any suitable polyadenylation sequence.
Examples of suitable polyadenylation sequences include synthetic
optimized sequences, BGH (Bovine Growth Hormone), polyoma virus, TK
(Thymidine Kinase), EBV (Epstein Barr Virus) and the
papillomaviruses, including human papillomaviruses and BPV (Bovine
Papilloma Virus). Preferably, particularly in the E4 deficient
region, the spacer includes an SV40 polyadenylation sequence. The
SV40 polyadenylation sequence allows for higher virus production
levels of multiply replication deficient adenoviral vectors. In the
absence of a spacer, production of fiber protein and/or viral
growth of the multiply replication-deficient adenoviral vector is
reduced by comparison to that of a singly replication-deficient
adenoviral vector. However, inclusion of the spacer in at least one
of the deficient adenoviral regions, preferably the E4 region, can
counteract this decrease in fiber protein production and viral
growth. Ideally, the spacer is composed of the glucuronidase gene.
The use of a spacer in an adenoviral vector is further described
in, for example, U.S. Pat. No. 5,851,806 and International Patent
Application Publication WO 97/21826.
[0026] It has been observed that an at least E4-deficient
adenoviral vector expresses a transgene at high levels for a
limited amount of time in vivo and that persistence of expression
of a transgene in an at least E4-deficient adenoviral vector can be
modulated through the action of a trans-acting factor, such as HSV
ICP0, Ad pTP, CMV-IE2, CMV-IE86, HIV tat, HTLV-tax, HBV-X, AAV Rep
78, the cellular factor from the U205 osteosarcoma cell line that
functions like HSV ICP0, or the cellular factor in PC12 cells that
is induced by nerve growth factor, among others, as described in
for example, U.S. Pat. Nos. 6,225,113, 6,649,373, and 6,660,521,
and International Patent Application Publication WO 00/34496. In
view of the above, a replication-deficient adenoviral vector (e.g.,
the at least E4-deficient adenoviral vector) or a second expression
vector can comprise a nucleic acid sequence encoding a trans-acting
factor that modulates the persistence of expression of the nucleic
acid sequence. Persistent expression of antigenic DNA can be
desired when generating immune tolerance.
[0027] Desirably, the adenoviral vector requires, at most,
complementation of replication-essential gene functions of the E1,
E2A, and/or E4 regions of the adenoviral genome for replication
(i.e., propagation). However, the adenoviral genome can be modified
to disrupt one or more replication-essential gene functions as
desired by the practitioner, so long as the adenoviral vector
remains deficient and can be propagated using, for example,
complementing cells and/or exogenous DNA (e.g., helper adenovirus)
encoding the disrupted replication-essential gene functions. In
this respect, the adenoviral vector can be deficient in
replication-essential gene functions of only the early regions of
the adenoviral genome, only the late regions of the adenoviral
genome, both the early and late regions of the adenoviral genome,
or all adenoviral genes (i.e., a high capacity adenovector (HC-Ad),
see Morsy et al., Proc. Natl. Acad. Sci. USA, 95: 965-976 (1998),
Chen et al., Proc. Natl. Acad. Sci. USA, 94: 1645-1650 (1997), and
Kochanek et al., Hum. Gene Ther., 10: 2451-2459 (1999)). Suitable
replication-deficient adenoviral vectors, including singly and
multiply replication-deficient adenoviral vectors, are disclosed in
U.S. Pat. Nos. 5,837,511, 5,851,806, 5,994,106, 6,127,175, and
6,482,616; U.S. Patent Application Publications 2001/0043922 A1,
2002/0004040 A1, 2002/0031831 A1, 2002/0110545 A1, and 2004/0161848
A1; and International Patent Application Publications WO 94/28152,
WO 95/02697, WO 95/16772, WO 95/34671, WO 96/22378, WO 97/12986, WO
97/21826, and WO 03/022311.
[0028] By removing all or part of, for example, the E1, E3, and E4
regions of the adenoviral genome, the resulting adenoviral vector
is able to accept inserts of exogenous nucleic acid sequences while
retaining the ability to be packaged into adenoviral capsids. The
nucleic acid sequence can be positioned in the E1 region, the E3
region, or the E4 region of the adenoviral genome. Indeed, the
nucleic acid sequence can be inserted anywhere in the adenoviral
genome so long as the position does not prevent expression of the
nucleic acid sequence or interfere with packaging of the adenoviral
vector.
[0029] If the adenoviral vector is not replication-deficient,
ideally the adenoviral vector is manipulated to limit replication
of the vector to within a target tissue. The adenoviral vector can
be a conditionally-replicating adenoviral vector, which is
engineered to replicate under conditions pre-determined by the
practitioner. For example, replication-essential gene functions,
e.g., gene functions encoded by the adenoviral early regions, can
be operably linked to an inducible, repressible, or tissue-specific
transcription control sequence, e.g., promoter. In this embodiment,
replication requires the presence or absence of specific factors
that interact with the transcription control sequence. In
autoimmune disease treatment, it can be advantageous to control
adenoviral vector replication in, for instance, lymph nodes, to
obtain continual antigen production and control immune cell
production. Conditionally-replicating adenoviral vectors are
described further in U.S. Pat. No. 5,998,205.
[0030] In addition to modification (e.g., deletion, mutation, or
replacement) of adenoviral sequences encoding replication-essential
gene functions, the adenoviral genome can contain benign or
non-lethal modifications, i.e., modifications which do not render
the adenovirus replication-deficient, or, desirably, do not
adversely affect viral functioning and/or production of viral
proteins, even if such modifications are in regions of the
adenoviral genome that otherwise contain replication-essential gene
functions. Such modifications commonly result from DNA manipulation
or serve to facilitate expression vector construction. For example,
it can be advantageous to remove or introduce restriction enzyme
sites in the adenoviral genome. Such benign mutations often have no
detectable adverse effect on viral functioning. For example, the
adenoviral vector can comprise a deletion of nucleotides 10,594 and
10,595 (based on the adenoviral serotype 5 genome), which are
associated with VA-RNA-1 transcription, but the deletion of which
does not prohibit production of VA-RNA-1.
[0031] Similarly, the coat protein of a viral vector, preferably an
adenoviral vector, can be manipulated to alter the binding
specificity or recognition of a virus for a viral receptor on a
potential host cell. For adenovirus, such manipulations can include
deletion of regions of the fiber, penton, or hexon, insertions of
various native or non-native ligands into portions of the coat
protein, and the like. Manipulation of the coat protein can broaden
the range of cells infected by the adenoviral vector or enable
targeting of the adenoviral vector to a specific cell type.
[0032] For example, in one embodiment, the adenoviral vector
comprises a chimeric coat protein (e.g., a fiber, hexon pIX, pIIIa,
or penton protein), which differs from the wild-type (i.e., native)
coat protein by the introduction of a normative amino acid
sequence, preferably at or near the carboxyl terminus. Preferably,
the normative amino acid sequence is inserted into or in place of
an internal coat protein sequence. One of ordinary skill in the art
will understand that the normative amino acid sequence can be
inserted within the internal coat protein sequence or at the end of
the internal coat protein sequence. The non-native amino acid
sequence of the chimeric adenoviral coat protein allows an
adenoviral vector comprising the chimeric adenoviral coat protein
to bind and, desirably, infect host cells not naturally infected by
the corresponding adenovirus without the non-native amino acid
sequence (i.e., host cells not infected by the corresponding
wild-type adenovirus), to bind to host cells naturally infected by
the corresponding adenovirus with greater affinity than the
corresponding adenovirus without the non-native amino acid
sequence, or to bind to particular target cells with greater
affinity than non-target cells. By "preferentially binds" is meant
that the non-native amino acid sequence binds a receptor, such as,
for instance, .alpha..sub.v.beta..sub.3 integrin, with at least
about 3-fold greater affinity (e.g., at least about 5-fold,
10-fold, 15-fold, 20-fold, 25-fold, 35-fold, 45-fold, or 50-fold
greater affinity) than the non-native ligand binds a different
receptor, such as, for instance, .alpha..sub.v.beta..sub.1
integrin.
[0033] Desirably, the adenoviral vector comprises a chimeric coat
protein comprising a non-native amino acid sequence that confers to
the chimeric coat protein the ability to bind to an immune cell
more efficiently than a wild-type adenoviral coat protein. In
particular, the adenoviral vector can comprise a chimeric
adenoviral fiber protein comprising a non-native amino acid
sequence which facilitates uptake of the adenoviral vector by
immune cells, preferably antigen presenting cells, such as
dendritic cells, monocytes, and macrophages. In a preferred
embodiment, the adenoviral vector comprises a chimeric fiber
protein comprising an amino acid sequence (e.g., a non-native amino
acid sequence) comprising an RGD motif including, but not limited
to, CRGDC (SEQ ID NO: 1), CXCRGDCXC (SEQ ID NO: 2), wherein X
represents any amino acid, and CDCRGDCFC (SEQ ID NO: 3), which
increases transduction efficiency of an adenoviral vector into
dendritic cells. The RGD-motif, or any non-native amino acid
sequence ligand, preferably is inserted into the adenoviral fiber
knob region, ideally in an exposed loop of the adenoviral knob,
such as the HI loop. A non-native amino acid sequence also can be
appended to the C-terminus of the adenoviral fiber protein,
optionally via a spacer sequence.
[0034] Where dendritic cells are the desired target cell, the
non-native amino acid sequence can recognize a protein typically
found on dendritic cell surfaces such as adhesion proteins,
chemokine receptors, complement receptors, co-stimulation proteins,
cytokine receptors, high level antigen presenting molecules, homing
proteins, marker proteins, receptors for antigen uptake, signaling
proteins, virus receptors, etc. Examples of such potential
ligand-binding sites in dendritic cells include
.alpha..sub.v.beta..sub.3 integrins, .alpha..sub.v.beta..sub.5
integrins, 2A1, 7-TM receptors, CD1, CD11a, CD11b, CD11c, CD21,
CD24, CD32, CD4, CD40, CD44 variants, CD46, CD49d, CD50, CD54,
CD58, CD64, ASGPR, CD80, CD83, CD86, E-cadherin, integrins, M342,
MHC-I, MHC-II, MIDC-8, MMR, OX62, p200-MR6, p55, S100, TNF-R, etc.
Preferably, where dendritic cells are targeted, the ligand
recognizes the CD40 cell surface protein, such as, for example, a
CD-40 (bi)specific antibody fragment or a domain derived from the
CD40L polypeptide.
[0035] Where macrophages are the desired target, the non-native
amino acid sequence can recognize a protein typically found on
macrophage cell surfaces, such as phosphatidylserine receptors,
vitronectin receptors, integrins, adhesion receptors, receptors
involved in signal transduction and/or inflammation, markers,
receptors for induction of cytokines, or receptors up-regulated
upon challenge by pathogens, members of the group B scavenger
receptor cysteine-rich (SRCR) superfamily, sialic acid binding
receptors, members of the Fc receptor family, B7-1 and B7-2 surface
molecules, lymphocyte receptors, leukocyte receptors, antigen
presenting molecules, and the like. Examples of suitable macrophage
surface target proteins include, but are not limited to, heparin
sulfate proteoglycans, .alpha..sub.v.beta..sub.3 integrins,
.alpha..sub.v.beta..sub.5 integrins, B7-1, B7-2, CD11c, CD13, CD16,
CD163, CD1a, CD22, CD23, CD29, Cd32, CD33, CD36, CD44, CD45, CD49e,
CD52, CD53, CD54, CD71, CD87, CD9, CD98, Ig receptors, Fc receptor
proteins (e.g., subtypes of Fc.alpha., Fc.gamma., Fc.epsilon.,
etc.), folate receptor b, HLA Class I, Sialoadhesin, siglec-5, and
the toll-like receptor-2 (TLR2).
[0036] Where B-cells are the desired target, the ligand can
recognize a protein typically found on B-cell surfaces, such as
integrins and other adhesion molecules, complement receptors,
interleukin receptors, phagocyte receptors, immunoglobulin
receptors, activation markers, transferrin receptors, members of
the scavenger receptor cysteine-rich (SRCR) superfamily, growth
factor receptors, selectins, MHC molecules, TNF-receptors, and
TNF-R associated factors. Examples of typical B-cell surface
proteins include .beta.-glycan, B cell antigen receptor (BAC),
B7-2, B-cell receptor (BCR), C3d receptor, CD1, CD18, CD19, CD20,
CD21, CD22, CD23, CD35, CD40, CD5, CD6, CD69, CD69, CD71,
CD79a/CD79b dimer, CD95, endoglin, Fas antigen, human Ig receptors,
Fc receptor proteins (e.g., subtypes of Fca, Fcg, Fc.epsilon.,
etc.), IgM, gp200-MR6, Growth Hormone Receptor (GH-R), ICAM-1,
ILT2, CD85, MHC class I and H molecules, transforming growth factor
receptor (TGF-R), .alpha..sub.4.beta..sub.7 integrin, and
.alpha..sub.v.beta..sub.3 integrin.
[0037] In another embodiment of the invention, the adenoviral
vector comprises a chimeric virus coat protein not selective for a
specific type of eukaryotic cell. The chimeric coat protein differs
from the wild-type coat protein by an insertion of a normative
amino acid sequence into or in place of an internal coat protein
sequence. In this embodiment, the chimeric adenovirus coat protein
efficiently binds to a broader range of eukaryotic cells than a
wild-type adenovirus coat, such as described in International
Patent Application WO 97/20051.
[0038] Specificity of binding of an adenovirus to a given cell also
can be adjusted by use of an adenovirus comprising a short-shafted
adenoviral fiber gene, as discussed in U.S. Pat. No. 5,962,311. Use
of an adenovirus comprising a short-shafted adenoviral fiber gene
reduces the level or efficiency of adenoviral fiber binding to its
cell-surface receptor and increases adenoviral penton base binding
to its cell-surface receptor, thereby increasing the specificity of
binding of the adenovirus to a given cell. Alternatively, use of an
adenovirus comprising a short-shafted fiber enables targeting of
the adenovirus to a desired cell-surface receptor by the
introduction of a normative amino acid sequence either into the
penton base or the fiber knob.
[0039] The ability of an adenoviral vector to recognize a potential
host cell can be modulated without genetic manipulation of the coat
protein, i.e., through use of a bi-specific molecule. For instance,
complexing an adenovirus with a bispecific molecule comprising a
penton base-binding domain and a domain that selectively binds a
particular cell surface binding site enables the targeting of the
adenoviral vector to a particular cell type. Likewise, an antigen
can be conjugated to the surface of the adenoviral particle through
non-genetic means.
[0040] A non-native amino acid sequence can be conjugated to any of
the adenoviral coat proteins to form a chimeric adenoviral coat
protein. Therefore, for example, a non-native amino acid sequence
can be conjugated to, inserted into, or attached to a fiber
protein, a penton base protein, a hexon protein, proteins IX, VI,
or IIIa, etc. The sequences of such proteins, and methods for
employing them in recombinant proteins, are well known in the art
(see, e.g., U.S. Pat. Nos. 5,543,328; 5,559,099; 5,712,136;
5,731,190; 5,756,086; 5,770,442; 5,846,782; 5,962,311; 5,965,541;
5,846,782; 6,057,155; 6,127,525; 6,153,435; 6,329,190; 6,455,314;
6,465,253; 6,576,456; 6,649,407; 6,740,525, and International
Patent Application Publications WO 96/07734, WO 96/26281, WO
97/20051, WO 98/07877, WO 98/07865, WO 98/40509, WO 98/54346, WO
00/15823, WO 01/58940, and WO 01/92549). The chimeric adenoviral
coat protein can be generated using standard recombinant DNA
techniques known in the art. Preferably, the nucleic acid sequence
encoding the chimeric adenoviral coat protein is located within the
adenoviral genome and is operably linked to a promoter that
regulates expression of the coat protein in a wild-type adenovirus.
Alternatively, the nucleic acid sequence encoding the chimeric
adenoviral coat protein is located within the adenoviral genome and
is part of an expression cassette which comprises genetic elements
required for efficient expression of the chimeric coat protein.
[0041] The coat protein portion of the chimeric adenovirus coat
protein can be a full-length adenoviral coat protein to which the
ligand domain is appended, or it can be truncated, e.g., internally
or at the C- and/or N-terminus. However modified (including the
presence of the non-native amino acid), the chimeric coat protein
preferably is able to incorporate into an adenoviral capsid. Where
the non-native amino acid sequence is attached to the fiber
protein, preferably it does not disturb the interaction between
viral proteins or fiber monomers. Thus, the non-native amino acid
sequence preferably is not itself an oligomerization domain, as
such can adversely interact with the trimerization domain of the
adenovirus fiber. Preferably the non-native amino acid sequence is
added to the virion protein, and is incorporated in such a manner
as to be readily exposed to a substrate, cell surface-receptor, or
immune cell (e.g., at the N- or C-terminus of the adenoviral
protein, attached to a residue facing a substrate, positioned on a
peptide spacer, etc.) to maximally expose the non-native amino acid
sequence. Ideally, the non-native amino acid sequence is
incorporated into an adenoviral fiber protein at the C-terminus of
the fiber protein (and attached via a spacer) or incorporated into
an exposed loop (e.g., the HI loop) of the fiber to create a
chimeric coat protein. Where the non-native amino acid sequence is
attached to or replaces a portion of the penton base, preferably it
is within the hypervariable regions to ensure that it contacts the
substrate, cell surface receptor, or immune cell. Where the
non-native amino acid sequence is attached to the hexon, preferably
it is within a hypervariable region (Miksza et al., J. Virol.,
70(3): 1836-44 (1996)). Where the non-native amino acid is attached
to or replaces a portion of pIX, preferably it is within the
C-terminus of pIX. Use of a spacer sequence to extend the
non-native amino acid sequence away from the surface of the
adenoviral particle can be advantageous in that the non-native
amino acid sequence can be more available for binding to a
receptor, and any steric interactions between the non-native amino
acid sequence and the adenoviral fiber monomers can be reduced.
[0042] Binding affinity of a non-native amino acid sequence to a
cellular receptor can be determined by any suitable assay, a
variety of which assays are known and are useful in selecting a
non-native amino acid sequence for incorporating into an adenoviral
coat protein. Desirably, the transduction levels of host cells are
utilized in determining relative binding efficiency. Thus, for
example, host cells displaying .alpha.v.beta.3 integrin on the cell
surface (e.g., MDAMB435 cells) can be exposed to an adenoviral
vector comprising the chimeric coat protein and the corresponding
adenovirus without the non-native amino acid sequence, and then
transduction efficiencies can be compared to determine relative
binding affinity. Similarly, both host cells displaying
.alpha.v.beta.3 integrin on the cell surface (e.g., MDAMB435 cells)
and host cells displaying predominantly .alpha.v.beta.1 on the cell
surface (e.g., 293 cells) can be exposed to the adenoviral vectors
comprising the chimeric coat protein, and then transduction
efficiencies can be compared to determine binding affinity.
[0043] In other embodiments (e.g., to facilitate purification or
propagation within a specific engineered cell type), a non-native
amino acid (e.g., ligand) can bind a compound other than a
cell-surface protein. Thus, the ligand can bind blood- and/or
lymph-borne proteins (e.g., albumin), synthetic peptide sequences
such as polyamino acids (e.g., polylysine, polyhistidine, etc.),
artificial peptide sequences (e.g., FLAG), and RGD peptide
fragments (Pasqualini et al., J. Cell. Biol., 130: 1189 (1995)). A
ligand can even bind non-peptide substrates, such as plastic (e.g.,
Adey et al., Gene, 156: 27 (1995)), biotin (Saggio et al., Biochem.
J., 293: 613 (1993)), a DNA sequence (Cheng et al., Gene, 171: 1
(1996), and Krook et al., Biochem. Biophys., Res. Commun., 204: 849
(1994)), streptavidin (Geibel et al., Biochemistry, 34: 15430
(1995), and Katz, Biochemistry, 34: 15421 (1995)),
nitrostreptavidin (Balass et al., Anal. Biochem., 243: 264 (1996)),
heparin (Wickham et al., Nature Biotechnol., 14: 1570-73 (1996)),
and other substrates.
[0044] Suitable modifications to an adenoviral vector are described
in U.S. Pat. Nos. 5,543,328, 5,559,099, 5,712,136, 5,731,190,
5,756,086, 5,770,442, 5,846,782, 5,871,727, 5,885,808, 5,922,315,
5,962,311, 5,965,541, 6,057,155, 6,127,525, 6,153,435, 6,329,190,
6,455,314, 6,465,253, 6,576,456, 6,649,407, 6,740,525; U.S. Patent
Application Publications 20010047081, 20020013286, 20020151027
20030022355 20030099619, 20030166286, and 20040161848; and
International Patent Application Publications WO 95/02697, WO
95/16772, WO 95/34671, WO 96/07734, WO 96/22378, WO 96/26281, WO
97/20051, WO 98/07865, WO 98/07877, WO 98/40509, WO 98/54346, WO
00/15823, WO 01/58940, and WO 01/92549. Similarly, it will be
appreciated that numerous adenoviral vectors are available
commercially. Construction of adenoviral vectors is well understood
in the art. Adenoviral vectors can be constructed and/or purified
using methods known in the art (e.g., using complementing cell
lines, such as the 293 cell line, Per.C6 cell line, or 293-ORF6
cell line) and methods set forth, for example, in U.S. Pat. Nos.
5,965,358, 5,994,128, 6,033,908, 6,168,941, 6,329,200, 6,383,795,
6,440,728, 6,447,995, 6,475,757, and 6,908,762; U.S. Patent
Application Publication 2002/0034735 A1; and International Patent
Application Publications WO 98/53087, WO 98/56937, WO 99/15686, WO
99/54441, WO 00/12765, WO 01/77304, and WO 02/29388, as well as the
other references identified herein.
[0045] The term "pathogen," as used herein, refers to any
microorganism that causes disease. Examples of suitable pathogens
include bacteria, viruses, parasites, fungi, protozoa, and prions.
Suitable viruses include, but not limited to, a virus from any of
the following viral families: Arenaviridae, Arterivirus,
Astroviridae, Baculoviridae, Badnavirus, Barnaviridae,
Birnaviridae, Bromoviridae, Bunyaviridae (e.g., hantavirus),
Caliciviridae, Capillovirus, Carlavirus, Caulimovirus,
Circoviridae, Closterovirus, Comoviridae, Coronaviridae (e.g.,
Coronavirus, such as severe acute respiratory syndrome (SARS)
virus), Corticoviridae, Cystoviridae, Deltavirus, Dianthovirus,
Enamovirus, Filoviridae (e.g., Marburg virus and Ebola virus (e.g.,
Zaire, Reston, Ivory Coast, or Sudan strain)), Flaviviridae, (e.g.,
Hepatitis C virus, Dengue virus 1, Dengue virus 2, Dengue virus 3,
Dengue virus 4, tick-borne encephalitis virus (TBEV), and yellow
fever virus), Hepadnaviridae (e.g., Hepatitis B virus),
Herpesviridae (e.g., Human herpesvirus 1, 3, 4, 5, and 6, and
Cytomegalovirus), Hypoviridae, Iridoviridae, Leviviridae,
Lipothrixviridae, Microviridae, Orthomyxoviridae (e.g.,
Influenzavirus A and B), Papovaviridae, Paramyxoviridae (e.g.,
measles, mumps, human respiratory syncytial virus, and Nipah
virus), Parvoviridae, Picornaviridae (e.g., poliovirus, rhinovirus,
hepatovirus, and aphthovirus), Poxviridae (e.g., vaccinia virus,
smallpox), Reoviridae (e.g., rotavirus), Retroviridae (e.g.,
lentivirus, such as human immunodeficiency virus (HIV) 1 and HIV
2), Rhabdoviridae, and Totiviridae. Particularly preferred
picornaviridae include, for example, aphthovirus of any serotype,
including serotypes A, O, C, SAT1, SAT2, SAT3, Asia1, which is the
causative agent of foot and mouth disease (FMD). Particularly
preferred retroviridae (retrovirus) include, for example, HIV of
any Glade, including clades A, B, C, MN, and the like. The virus
also can be a coronavirus, such as a SARS virus.
[0046] Suitable bacteria include, but are not limited to,
Actinomyces, Anabaena, Bacillus (e.g., Bacillus anthracis),
Bacteroides, Bdellovibrio, Brucella, Burkholderia (e.g.,
Burkholderia pseudomallei), Caulobacter, Chlamydia, Chlorobium,
Chromatium, Clostridium (e.g., Clostridium botulinum, Clostridium
perfringens), Coxiella (e.g., Coxiella burnetii), Cytophaga,
Deinococcus, Escherichia, Francisella (e.g., Francisella
tularensis), Halobacterium, Heliobacter, Hyphomicrobium,
Methanobacterium, Micrococcus, Myobacterium, Mycoplasma,
Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron,
Proteus, Pseudomonas, Phodospirillum, Rickettsia, Ricinus (e.g.,
Ricinus communis), Salmonella, Shigella, Spirillum, Spirochaeta,
Staphylococcus, Streptococcus, Streptomyces, Sulfolobus,
Thermoplasma, Thiobacillus, Treponema, Yersinia (e.g., Yersinia
pestis). When the pathogen is a bacterium, the bacterium preferably
is a Mycobacterium species, such as, for example, Mycobacterium
tuberculosis, which is the causative agent for tuberculosis
(TB).
[0047] Preferably, the pathogen is a parasite. Suitable parasites
include, but are not limited to, a parasite of the phylum Sporozoa
(also referred to as phylum Apicomplexa), Ciliophora, Rhizopoda, or
Zoomastigophora. Preferably, the pathogen is a parasite of the
phylum Sporozoa and species Plasmodium. The parasite can be any
suitable Plasmodium species, but preferably is a Plasmodium species
that infects humans and causes malaria. Human-infecting Plasmodium
species include P. malariae, P. ovale, P. vivax, and P. falciparum.
P. vivax and P. falciparum are the most common, and P. falciparum
is the most deadly, species of Plasmodium in human. Alternatively,
the pathogen can be a species of Plasmodium that infects non-human
animals. For example, P. vinckei, P. chabaudi, P. yoelii, and P.
berghei. infect rodents, P. knowlesi, P. cynomolgi, P. simiovale,
P. fieldi, P. inui, and P. brasilianum infect non-human primates.
P. gallinaceum infects birds. In order to advance vaccine
discovery, the genomes of a number of Plasmodium species have been
sequenced. For example, the P. falciparum genome sequence is
disclosed in Gardner et al., Nature, 419: 498-511 (2002). In
addition, the P. yoelii genome sequence is disclosed in Carlton et
al., Nature, 419: 512-519 (2002). Thus, an antigen identified using
the inventive method can be sequenced and located within the
Plasmodium genome using routine methods known in the art.
[0048] One of ordinary skill in the art will appreciate that it is
not possible to correlate in vitro immunogenicity of an antigen
identified by the inventive method with in vivo protection against
pathogen challenge in a human without incurring a long and
expensive preclinical and clinical development process. Thus, in
some embodiments, the inventive method preferably is practiced in
an animal model, most preferably a mouse model. The selection of an
appropriate pathogen, e.g., Plasmodium species, on which the
adenoviral vector array is based will therefore depend on the
species of the animal model used. When the animal model is a mouse,
the Plasmodium species on which the adenoviral vector array is
based preferably is P. yoelii. Based on the similarity between the
P. yoelii and P. falciparum genomes, one of ordinary skill in the
art can identify P. falciparum orthologues of any P. yoelii
antigens identified by the inventive method using routine methods
known in the art.
[0049] In accordance with the inventive method, each of the
adenoviral vectors in the adenoviral vector array comprises a
nucleic acid sequence encoding a different antigen of a pathogen.
An "antigen" is a molecule that induces an immune response in a
mammal. An "immune response" can entail, for example, antibody
production and/or the activation of immune effector cells (e.g., T
cells). An antigen in the context of the invention can comprise any
subunit, fragment, or epitope of any proteinaceous molecule,
including a protein or peptide of viral, bacterial, parasitic,
fungal, protozoan, prion, cellular, or extracellular origin, which
ideally provokes an immune response in mammal, preferably leading
to protective immunity. By "epitope" is meant a sequence on an
antigen that is recognized by an antibody or an antigen receptor.
Epitopes also are referred to in the art as "antigenic
determinants."
[0050] In another embodiment, the inventive method can be used to
identify one or more disease antigens in a mammal. By "disease
antigen" is meant a self antigen whose presence or overexpression
is indicative of a particular disease. The disease antigen can be
any suitable antigen, but is preferably an antigen that is
associated with cancer or an autoimmune disease. A "cancer antigen"
is an antigen that is expressed by tumor cells but not normal
cells, or an antigen that is expressed in normal cells but is
overexpressed in tumor cells. Examples of suitable tumor antigens
include, but are not limited to, .beta.-catenin, BCR-ABL fusion
protein, K-ras, N-ras, PTPRK, NY-ESO-1/LAGE-2, SSX-2, TRP2-INT2,
CEA, gp100, kallikrein 4, prostate specific antigen (PSA),
TRP-1/gp75, TRP-2, tyrosinase, EphA3, HER-2/neu, MUC1, p53, mdm-2,
PSMA, RAGE-1, surviving, telomerase, and WT1. Other tumor antigens
are known in the art and are described in, for example, The Peptide
Database of T-Cell Defined Tumor Antigens, maintained by the Ludwig
Institute for Cancer Research
(http://www.cancerimmunity.org/statics/databases.htm), Van den
Eynde et al., Curr. Opin. Immunol., 9: 684-93 (1997), Houghton et
al., Curr. Opin. Immunol., 13: 134-140 (2001), and van de Bruggen
et al., Immunol. Rev., 188: 51-64 (2002). Antigens that are
associated with autoimmune diseases typically are nuclear antigens,
including, but not limited to, antigens derived from histones,
nonhistone proteins bound to RNA, the nucleolus, and
centromeres.
[0051] The term "array," as used herein, refers to a collection of
molecules, compounds, cells, organisms, etc., that are arranged in
an ordered manner. The array can be one-dimensional or
multi-dimensional (e.g., a "matrix"). Desirably, the array contains
a single type of molecule, compound, organism, etc. For example,
all of the molecules in the array are proteins or nucleic acids. In
the context of the invention, the array comprises two or more
different adenoviral vectors, wherein each adenoviral vector
comprises a nucleic acid sequence encoding a different antigen of a
pathogen. Methods of generating and using DNA and protein arrays
are well-known in the art and are described in, for example, Brown
et al., Nat. Genet., 21: 33-7 (1999), Duggan et al., Nat. Genet.,
21(1 Suppl): 10-4 (1999), Bubendorf, Eur. Urol., 40(2): 231-8
(2001), Sakanyan, J. Chromatogr. B. Analyt. Technol. Biomed. Life
Sci., 815(1-2): 77-95 (2005), and Eickhoff et al., Adv. Biochem.
Eng. Biotechnol., 77: 103-12 (2002).
[0052] The adenoviral vector array can be prepared using any
suitable method. One of ordinary skill in the art will appreciate
that, in order to conduct a high-throughput analysis of hundreds of
potential antigen genes, the adenoviral vector array desirably is
constructed in a manner that is amenable to automation. Adenoviral
vectors typically are produced using a plasmid-based system (see,
e.g., International Patent Application Publication WO 99/15686 and
U.S. Pat. No. 6,329,200). For example, E1/E3/E4-deleted adenoviral
vectors can be generated by a plasmid-based system that utilizes
the site-specific integrative recombination machinery of
bacteriophage lambda, which facilitates in vitro transfer of genes
from a small plasmid into a larger plasmid that contains the entire
adenoviral genome, with the exception of the E1, E3 and E4 regions
(i.e., adenovector plasmids). These adenovector plasmids are
infectious and can be converted into adenovirus particles when
grown in the appropriate complementing cell line. Such methods,
however, enable the production of one only adenoviral vector at a
time. Thus, for the purposes of the invention, such art-recognized
methods of preparing adenoviral vectors must be adapted for
high-throughput adenoviral vector construction, or alternative
methods must be used.
[0053] The adenoviral vector array preferably is generated using a
site-specific recombination-based cloning method which does not
require standard nucleic acid digestion and ligation. An example of
such a method is the Gateway.TM. Technology cloning system
(Invitrogen Life Technologies, Carlsbad, Calif.), which relies on
the well-characterized site-specific recombination process between
bacteriophage .lamda. and E. coli (see, e.g., Hartley et al.,
Genome Res., 10: 1788-1795 (2000), Walhout et al., Methods Enzym.,
328: 575-592 (2000), and U.S. Pat. Nos. 5,888,732, 6,270,969,
6,277,608, and 6,720,140). This recombinant-based cloning
technology provides for highly efficient and accurate directional
cloning, and has been used for the high-throughput cloning of P.
falciparum open reading frames (ORFs) (see Aguiar et al., Genome
Research, 14: 2076-2082, (2004)). The Gateway.TM. technology allows
for the transfer of DNA segments between different cloning vectors
while maintaining orientation and reading frame, without the need
for restriction endonucleases and ligase. Expression vectors are
generated utilizing site-specific recombination between phage
lambda site-specific attachment (att) sites. For example,
site-specific recombination occurs between an attL site on the E.
coli chromosome and an attR site on the lambda chromosome. Upon
lambda integration, recombination occurs between attL and attR
sites to give rise to attB and attP sites, respectively (see, e.g.,
Landy et al., Ann. Rev. Biochem., 58: 913-949 (1989)).
[0054] In the context of the invention, a nucleic acid sequence of
interest (e.g., a Plasmodium nucleic acid sequence) is cloned in a
Gateway.TM. "Entry" vector that is transcriptionally silent,
kanamycin resistant (Km.sup.r), and is flanked by two recombination
sites (attL1 and attL2). The sequence of interest is then
transferred to a Gateway.TM. "Destination" vector, which contains
sequences necessary for gene expression, and is ampicillin
resistant (Ap.sup.r). The Destination vector also contains two
recombination sites (attR1 and attR2) that flank a gene for
negative selection, ccdB. Att1 and att2 sites confer directionality
and specificity for recombination, so that only attL1 will react
with attR1, and attL2 with attR2. The Entry vector and Destination
vector are then combined, such that two recombination events occur:
one between attL1 and attR1 and the other between attL2 and attR2.
The product of these two recombination events is a plasmid
construct comprising the nucleic acid of interest and a by-product
(known as the "Donor" vector). The desired plasmid is under two
forms of selection: antibiotic resistance and negative selection.
Selecting for ampicillin resistance eliminates the Entry vector and
the by-product, and selection against the negative selection marker
eliminates the destination vector and co-integrate molecules.
[0055] In a preferred embodiment of the invention, Gateway.TM.
Entry vectors are constructed which comprise nucleic acid sequences
encoding one or more Plasmodium genes flanked by appropriate att
site-specific recombination sites. Each Entry vector is then
incubated with an adenovector Donor vector in a single well of a
96-well tissue culture plate. Each of the adenovector Donor vectors
comprise a nucleic acid sequence encoding the adenoviral genome,
with the exception of one or more adenoviral genome regions
required for replication (i.e., so as to render the resulting
adenoviral vector replication-deficient). In this regard, the
adenovector Donor vectors preferably do not comprise a nucleic acid
sequence encoding all or part of the E1 region and all or part of
the E4 region. As discussed above, the adenovector Donor vectors
also preferably do not comprise a nucleic acid sequence encoding
the E3 region. Recombination between the Entry vectors and the
adenovector Donor vectors preferably is catalyzed using Gateway.TM.
technology, and products of the recombination reaction preferably
are packaged into lambda phage heads in vitro. Phage lysates are
then used to transduce a host cell strain that is permissive for
growth of the recombinant adenovector plasmid but not the parental
adenovector Donor plasmid. Any suitable host cell strain can be
used. Preferably, the host cell strain is E. coli DH10B.
Recombinant adenovector plasmids can be purified and isolated using
any suitable method known in the art, such as those described in
Sambrook et al., Molecular Cloning, a Laboratory Manual, 3.sup.rd
edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001),
and Ausubel et al., Current Protocols in Molecular Biology, Greene
Publishing Associates and John Wiley & Sons, New York, N.Y.
(1994).
[0056] Once isolated, the recombinant adenovector plasmids
generated as described above must be converted into infectious
adenovirus particles and expanded to a scale suitable for antigen
screening. Conversion preferably is accomplished by transfecting
the recombinant adenovector plasmids into complementing cell lines
used for propagation of replication-deficient adenoviral vectors.
In this regard, any suitable complementing cell line can be used to
convert the recombinant adenovector plasmids into recombinant
adenovirus particles. Suitable complementing cell lines are known
in the art and are described herein. Titers of viral particles
generated by this method preferably are expanded in parallel in
96-well plates by successive passaging of the complementing cells
until a cytopathic effect (CPE) is observed. Virus yields are then
determined using any suitable method known in the art, such as the
HPLC-based particle determination assay described in U.S. Pat. No.
6,447,995.
[0057] Once sufficient titers of recombinant adenoviral vector
particles in the adenoviral vector array are produced, the
inventive method further comprises contacting antigen presenting
cells (APCs) with the adenoviral vector array. Antigen presenting
cells are known in the art as highly specialized cells that can
process antigens and display their peptide fragments on the cell
surface together with molecules required for T cell activation.
Antigen presenting cells can be "professional," meaning that they
have both antigen-presenting and accessory (i.e., costimulatory)
functions. In addition to processing antigens to peptides that are
presented on MHC products (i.e., signal 1), professional APCs
express additional "second signals" that mediate T-cell binding and
costimulation. Examples of such second signals include
intracellular adhesion molecules (e.g., ICAMs, CD50, CD54, and
CD102), lymphocyte function associated antigens (e.g., CD2, CD11a,
and CD58), and B7 molecules (e.g., CD80 and CD86). Any suitable
primary, cultured, long-term, or immortalized APCs can be contacted
with the adenoviral vector array. Suitable APCs include, for
example, dendritic cells (DCs) (e.g., Langerhans' cells,
interdigitating cells, follicular dendritic cells, and veiled
cells), macrophages, B cells, fibroblasts, dendritic-like cells,
and artificial APCs. APCs may or may not be transfected with
specific immune molecules, such as human leukocyte antigen (HLA) or
costimulatory molecules (e.g., HLA-A2-transfected jurkat cells or
EBV-immortalized B cell lines).
[0058] Dendritic cells are found primarily in the skin and mucosal
epithelium, continuously express high levels of the co-stimulatory
B7 molecule, and function to present antigen to T cells. Upon
recognition of infectious particles, DCs migrate through the
lymphatics to the nearest lymph node, where they come into close
contact with naive T cells. Unlike macrophages, DCs can recognize
viral particles as non-self. In addition, DCs can present antigen
via both MHC I and MHC II. Thus, DCs can activate both CD8+ and
CD4+ T cells. Macrophages are part of the innate immune system and
continuously phagocytose self-proteins and cells in their vicinity
during normal tissue repair and aging (e.g., old red blood cells).
Phagocytosed proteins are degraded and presented in the context of
MHC II. In the case of infection, macrophages posses certain types
of receptors that recognize differential carbohydrate patterns on
foreign cells. Macrophages also have receptors for specific
bacterial products such as lipopolysaccharide (LPS) (endotoxin).
When these molecules bind their bacterial ligands, they stimulate
the macrophages to up regulate MHC II and co-stimulatory B7,
providing macrophages with strong antigen presentation properties.
Stimulated macrophages produce cytokines, such as IL-1, IL-6, IL-8,
IL-12, and TNF-.alpha., that aid in antigen presentation. Unlike
DCs and macrophages, B cells are uniquely adapted to bind specific
soluble molecules through their cell-surface immunoglobulin. B
cells ingest soluble proteins by pinocytosis and present antigen in
the context of MHC-II. B cells, however, do not express
co-stimulatory molecules, unless activated by helper T cells.
Preferably, the antigen presenting cells are dendritic cells.
Antigen presenting cells are further described in, for example,
Janeway et al., eds., Immunobiology, 5.sup.th ed., Garland
Publishing, New York, (2001).
[0059] The APCs can be contacted with the adenoviral vector array
using any suitable method for transducing animal cells known in the
art, such as those described in Sambrook et al., supra, and Ausubel
et al., supra. Desirably, cultured APCs are added directly to the
adenoviral vector array. More preferably, the adenoviral vector
array is contained in a 96-well tissue culture plate, and cultured
APCs can be added to each well of the 96-well plate. Whatever
method is used, the APCs preferably are contacted with the
adenoviral vector array under conditions wherein each different
adenoviral vector transduces an APC so that the nucleic acid
sequences of the different adenoviral vectors are expressed and the
different antigens are produced in the APCs.
[0060] One of ordinary skill in the art will appreciate that every
protein or peptide encoded by a particular pathogen does not
necessarily elicit an immune response in an infected host. Thus,
the invention comprises assaying the immunogenicity of the antigen
produced in each APC. To this end, the APCs preferably are
incubated with effector T lymphocytes obtained from a mammal
immunized against the pathogen from which the nucleic acid
sequences are derived. Unlike naive T lymphocytes, effector T
lymphocytes can mediate the removal of pathogens from a host
without the need for further differentiation or costimulation.
Effector T lymphocytes are often referred to in the art at "armed"
effector T lymphocytes, because their effector function can be
triggered by antigen binding alone. The three types of effector T
lymphocytes, CD8, CD4 Th1, and CD4 Th2 have specificity for
different kinds of pathogens. CD8 T lymphocytes (also referred to
in the art as cytotoxic T lymphocytes (CTL)) kill infected cells
displaying cytosolic pathogen peptides on MHC Class 1 molecules.
CD4 Th1 cells activate macrophages with persistent vesicular
pathogens whose peptides are displayed on MHC Class II molecules.
CD4 Th1 cells also activate B cells to produce opsonizing
antibodies. CD4 Th2 cells activate B cells that have internalized
specific antigens and display peptides on MHC Class II molecules.
Effector T lymphocytes are further described in, for example,
Janeway et al., supra.
[0061] The effector T lymphocytes preferably are obtained from a
mammal immunized with the pathogen from which the nucleic acid
sequences encoded by the adenoviral vector array are derived. In
this regard, the effector T lymphocytes are obtained from a mammal
that previously has been infected with any of the pathogens
described herein, such that the mammal has mounted an immune
response against the pathogen. Thus, a mammal is "immunized"
against a pathogen if the mammal has developed humoral immunity
(i.e., antibodies) and/or cellular immunity (e.g. effector T
lymphocytes) against one or more antigens of a pathogen. The
effector T lymphocytes can be isolated from a mammal using any
suitable method known in the art. For example, T lymphocytes can be
isolated from peripheral blood from an immunized mammal (e.g., a
human) by using density centrifugation over a step gradient
consisting of a mixture of the carbohydrate Ficoll.TM. and the
dense iodine-containing compound metrizamide. This results in a
population of mononuclear cells, called peripheral blood
mononuclear cells (PBMCs), that have been depleted of red blood
cells and most polymorphonuclear leukocytes or granulocytes, and
consists mainly of lymphocytes and monocytes. T lymphocytes can be
isolated from PBMCs by binding a sample to antibody-coated plastic
surfaces, which is known in the art as "panning," or by killing
unwanted cells by treatment with a specific antibody and
complement. Alternatively, PBMCs can be passed over columns of
antibody- and nylon-coated steel wool, and different populations
differentially eluted. Preferably, T lymphocytes are isolated from
PBMCs using flow cytometry or fluorescence-activated cell sorting
(FACS). Methods for isolating lymphocytes from mammals,
particularly humans, are further described in Janeway et al.,
supra.
[0062] In nature, malaria parasites are spread by successively
infecting two types of hosts: humans and female Anopheles
mosquitoes. In this respect, malaria parasites are present as
"sporozoites" in the salivary glands of the female Anopheles
mosquito. When the Anopheles mosquito takes a blood meal on another
human, the sporozoites are injected with the mosquito's saliva,
enter the circulatory system, and within minutes of inoculation
will invade a human liver cell (hepatocyte). After invading
hepatocytes, the parasite undergoes asexual replication. The stage
of the parasite life cycle encompassing sporozoite and liver stages
typically is referred to in the art as the "pre-erythrocytic
stage," the "liver stage," or "the exo-erythrocytic stage." The
progeny, called "merozoites," are released into the circulatory
system following rupture of the host hepatocyte. Antigens expressed
during the pre-erythrocytic stage of infection include, but are not
limited to, circumsporozoite protein (CSP), sporozoite surface
protein 2 (SSP2), liver-stage antigen 1 (LSA-1), Pf exported
protein 1 (PfExp-1)/Py hepatocyte erythrocyte protein 17 (PyHEP17),
and Pf antigen 1.
[0063] Merozoites released from the infected liver cells invade
erythrocytes (red blood cells). The merozoites recognize specific
proteins on the surface of the erythrocyte and actively invade the
cell in a manner similar to other mosquito-borne parasites. After
entering the erythrocyte, the parasite undergoes a trophic period
followed by asexual replication to produce successive broods of
merozoites. The progeny merozoites grow inside the erythrocytes and
destroy them, and are then released to initiate another round of
infection. This stage of infection typically is referred to in the
art as the "blood-stage" or "erythrocytic stage." Blood-stage
parasites are those that cause the symptoms of malaria. When
certain forms of blood-stage parasites (i.e., "gametocytes") are
picked up by a female Anopheles mosquito during a blood meal, they
start another, different cycle of growth and multiplication in the
mosquito. Antigens expressed during the blood-stage of infection
include, but are not limited to, merozoite surface protein 1
(MSP-1), merozoite surface protein 2 (MSP-2), erythrocyte binding
antigen 175 (EBA-175), ring-infected erythrocyte surface antigen
(RESA), serine repeat antigen (SERA), glycophorin binding protein
(GBP-130), histidine rich protein 2 (HRP-2), rhoptry-associated
proteins 1 and 2 (RAP-1 and RAP-2), erythrocyte membrane protein 1
(PfEMP1), and apical membrane antigen 1 (AMA-1). The Plasmodium
life cycle is described in, for example, Ramasamy et al., Med. Vet.
Entomol., 11(3): 290-96 (1997), Hall et al., Science, 307(5706):
82-86 (2005), and I. W. Sherman, ed., Malaria: Parasite Biology,
Pathogenesis, and Protection, American Society of Microbiology
(1998).
[0064] In a preferred embodiment of the invention, the effector T
lymphocytes are obtained from a mammal that has been immunized with
Plasmodium sporozoites. More preferably, the effector T lymphocytes
are obtained from a mammal that has been immunized with Plasmodium
sporozoites that have been attenuated via radiation so that the
sporozoite infects the liver and undergoes partial development but
does not develop to the blood-stage form. The mammal can be
immunized with irradiated sporozoites obtained from any Plasmodium
species described herein. Most preferably, the mammal has been
immunized with irradiated P. yoelii sporozoites.
[0065] Following incubation of the APCs with effector T lymphocytes
obtained from a mammal immunized against the pathogen, the
inventive method comprises screening for an immune response from
the effector T lymphocytes. The immune response can be any suitable
effector T lymphocyte immune response known in the art, including,
but not limited to, cytokine secretion, effector T cell
cytotoxicity, and immune activation of effector T cells.
Preferably, the inventive method comprises screening for secretion
by the effector T lymphocytes. In this regard, cytokine secretion
from an effector T lymphocyte contacting an APC indicates that the
effector T lymphocyte recognizes the antigen produced and displayed
by the APC. Furthermore, it is well known in the art that effector
T lymphocytes, such as effector helper T effector lymphocytes,
secrete cytokines upon antigen recognition which promote different
activities. In this regard, inflammatory or Th1 CD4 T cells produce
interleukin-2 (IL-2), interferon gamma (IFN.gamma.), and tumor
necrosis factor beta (TNF.beta.), which activate CTLs and
macrophages to stimulate cellular immunity and inflammation. Th1
CD4 T cells also secrete interleukin-3 (IL-3) and
granulocyte-macrophage colony-stimulating factor (GM-CSF) to
stimulate bone marrow to produce more leukocytes and signal B cells
to produce opsonizing antibodies (e.g., IgG1 and IgG3 in humans and
IgG2a and IgG2b in the mouse). Helper or Th2 CD4 T cells activate
naive B cells to divide and secrete IgM. Th2 CD4 cells also secrete
IL-4, IL-5, and IL-6, which stimulate neutralizing antibody
production by B cells. Thus, in the context of the inventive
method, the effector T lymphocytes are screened for secretion of
any suitable cytokine. Suitable cytokines include, but are not
limited to, IFN-.gamma., TNF-.beta., TNF-.alpha., GM-CSF, CD40
ligand, Fas ligand, and interleukins (e.g., IL-1, IL-2, IL-3, IL-4,
IL-5, IL-6, and IL-10). Most preferably, the effector T lymphocytes
are screened for secretion of IFN-.gamma.. Cytokine secretion can
be detected and measured using any suitable method known in the
art, such as, for example, ELISPOT assays, intracellular cytokine
staining assays, flow cytometry, or fluorescence-activated cell
sorting (FACS) assays. In addition, effector T lymphocyte
cytotoxicity can be measured using CTL assays, such as a chromium
release assay (see, e.g., Walker et al., Nature, 328: 345-48
(1987)), and immune activation can be measured using multiparameter
flow cytometry (see, e.g., Picker et al., Blood, 86: 1408-1419
(1995)).
[0066] The invention also provides a method of identifying an
antigen from a pathogen comprising: (a) providing an adenoviral
vector array comprising two or more different adenoviral vectors,
wherein each adenoviral vector comprises a nucleic acid sequence
encoding a different antigen of a pathogen, (b) administering each
of the adenoviral vectors of the adenoviral vector array to a
mammal, such that the nucleic acid sequence is expressed and the
antigen is produced in the mammal, (c) infecting each mammal with
the pathogen, and (d) screening the infected mammal for onset of a
disease caused by the pathogen, wherein the absence in the infected
mammal of a disease caused by the pathogen indicates that the
adenoviral vector encodes an antigen of the pathogen, whereupon the
antigen is identified. Descriptions of the adenoviral vector array
and pathogen, and components thereof, set forth above in connection
with other embodiments of the invention also are applicable to
those same aspects of the aforesaid method.
[0067] In this embodiment, each adenoviral vector of the adenoviral
vector array preferably is administered to a mammal (e.g., a
human), wherein the nucleic acid sequence encoding the antigen is
expressed and the antigen is produced in the mammal. In other
words, only one adenoviral vector of the adenoviral vector array is
administered to the mammal. Thus, in this embodiment, the analysis
of a complete adenoviral vector array requires the use of multiple
mammals. Desirably, each adenoviral vector is administered as part
of a pharmaceutical composition comprising the adenoviral vector.
Alternatively, the adenoviral vector can be administered as part of
an unpurified cell lysate comprising the adenoviral vector. In this
manner, adenoviral vectors are not purified from the complementing
cell lines in which they are produced, but rather, an unpurified
lysate of the adenovirus-infected complementing cells is
administered to the mammal.
[0068] Following administration of the adenoviral vectors to the
mammals, each mammal is infected with a suitable pathogen, such as
those described herein. Preferably, the mammal is infected with a
Plasmodium species, most preferably P. falciparum or P. yoelii.
Infection of the mammal can be accomplished using any suitable
method know in the art, such as by, for example, administration of
a pharmaceutical composition comprising the pathogen itself (killed
or attenuated), a nucleic acid molecule encoding the genome of the
pathogen, or a live but latent form of the pathogen (e.g.,
Plasmodium sporozoites). Preferably, the mammal is infected with a
live, disease-causing pathogen. Preferred administration routes
include, but are not limited to, intramuscular, intravenous,
intraarterial, oral, and inhalation, as described elsewhere
herein.
[0069] As described herein, infection with a pathogen typically
results in the onset of disease, unless the infected host has
acquired protective immunity against the pathogen. Because the
adenoviral vectors desirably encode at least one antigen of the
pathogen that provides protective immunity against further
challenge from the pathogen, the method comprises screening for
onset of a disease caused by the pathogen. It will be appreciated
that the absence of a disease caused by the pathogen in the mammal
indicates that a particular adenoviral vector of the adenoviral
vector array encodes an antigen of the pathogen which provides
protective immunity to the mammal. On the other hand, the
development of a disease characteristic of pathogen infection
indicates that the adenoviral vector administered to the diseased
mammal does not encode an antigen that contributes to protective
immunity.
[0070] In accordance with the invention, once it is determined that
a particular adenoviral vector of the adenoviral vector array
encodes an antigen of the pathogen, as evidenced by cytokine
secretion or protection from pathogen challenge, the antigen
preferably is identified, e.g., by being recovered from the
adenoviral vector. For example, both the nucleic acid sequence
encoding the antigen and the amino acid sequence of the antigen can
be determined using methods known in the art, such as those
described in Sambrook et al., supra, and Ausbel et al., supra.
[0071] The invention also provides a method of inducing an immune
response against a pathogen in a mammal utilizing the antigens
identified as described above. The method can comprise
administering to the mammal an antigen identified as described
above. Alternatively, and preferably, the method comprises (a)
preparing an adenoviral vector comprising a nucleic acid sequence
encoding an antigen of a pathogen identified by the methods
described above, and (b) administering the adenoviral vector to a
mammal infected by the pathogen, wherein the antigen is expressed
in the mammal to induce an immune response. Descriptions of the
adenoviral vector and pathogen, and components thereof, set forth
above in connection with other embodiments of the invention also
are applicable to those same aspects of the aforesaid method of
inducing an immune response.
[0072] In the method of the invention, the adenoviral vector
preferably is administered to a mammal (e.g., a human), wherein the
nucleic acid sequence encoding the antigen is expressed to induce
an immune response against the antigen. The adenoviral vector
comprises at least one nucleic acid sequence that encodes at least
one antigen. In this respect, the adenoviral vector can encode one
nucleic acid sequence that encodes multiple different antigens
(e.g., 2, 3, 4, or 5, antigens), or the adenoviral vector can
encode multiple nucleic acid sequences, each of which encodes a
different antigen. The immune response can be a humoral immune
response, a cell-mediated immune response, or, desirably, a
combination of humoral and cell-mediated immunity. Ideally, the
immune response provides protection upon subsequent challenge with
the pathogen comprising the antigen. However, protective immunity
is not required in the context of the invention. The inventive
method further can be used for antibody production and
harvesting.
[0073] Administering the adenoviral vector encoding the antigens
can be one component of a multistep regimen for inducing an immune
response in a mammal. In particular, the inventive method can
represent one arm of a prime and boost immunization regimen. The
inventive method, therefore, can comprise administering to the
mammal a priming gene transfer vector comprising a nucleic acid
sequence encoding at least one antigen prior to administering the
adenoviral vector. The antigen encoded by the priming gene transfer
vector can be the same or different from the antigens of the
adenoviral vector. The adenoviral vector is then administered to
boost the immune response to a given pathogen. More than one
boosting composition comprising the adenoviral vector can be
provided in any suitable timeframe (e.g., at least about 1 week, 2
weeks, 4 weeks, 8 weeks, 12 weeks, 16 weeks, or more following
priming) to maintain immunity.
[0074] Any gene transfer vector can be employed as a priming gene
transfer vector, including, but not limited to, a plasmid, a
retrovirus, an adeno-associated virus, a vaccinia virus, a
herpesvirus, an alphavirus, or an adenovirus. Ideally, the priming
gene transfer vector is a plasmid, an alphavirus, or an adenoviral
vector. To maximize the effect of the priming regimen, the priming
gene transfer vector can comprise more than one nucleic acid
sequence encoding an antigen of the pathogen. Preferably, the
priming gene transfer vector comprises two or more (e.g., 2, 3, 5,
or more) nucleic acid sequences each encoding an antigen of the
pathogen. Alternatively, an immune response can be primed or
boosted by administration of the antigen itself, e.g., an antigenic
protein, intact pathogen (e.g., Plasmodium sporozoites),
parasitized erythrocytes, inactivated pathogen, and the like.
[0075] Any route of administration can be used to deliver the
adenoviral vector to the mammal. Indeed, although more than one
route can be used to administer the adenoviral vector, a particular
route can provide a more immediate and more effective reaction than
another route. Preferably, the adenoviral vector is administered
via intramuscular injection. A dose of adenoviral vector also can
be applied or instilled into body cavities, absorbed through the
skin (e.g., via a transdermal patch), inhaled, ingested, topically
applied to tissue, or administered parenterally via, for instance,
intravenous, peritoneal, or intraarterial administration.
[0076] The adenoviral vector can be administered in or on a device
that allows controlled or sustained release, such as a sponge,
biocompatible meshwork, mechanical reservoir, or mechanical
implant. Implants (see, e.g., U.S. Pat. No. 5,443,505), devices
(see, e.g., U.S. Pat. No. 4,863,457), such as an implantable
device, e.g., a mechanical reservoir or an implant or a device
comprised of a polymeric composition, are particularly useful for
administration of the adenoviral vector. The adenoviral vector also
can be administered in the form of sustained-release formulations
(see, e.g., U.S. Pat. No. 5,378,475) comprising, for example, gel
foam, hyaluronic acid, gelatin, chondroitin sulfate, a
polyphosphoester, such as bis-2-hydroxyethyl-terephthalate (BHET),
and/or a polylactic-glycolic acid.
[0077] The dose of adenoviral vector administered to the mammal
will depend on a number of factors, including the size of a target
tissue, the extent of any side-effects, the particular route of
administration, and the like. The dose ideally comprises an
"effective amount" of adenoviral vector, i.e., a dose of adenoviral
vector which provokes a desired immune response in the mammal. The
desired immune response can entail production of antibodies,
protection upon subsequent challenge, immune tolerance, immune cell
activation, and the like. Desirably, a single dose of adenoviral
vector comprises at least about 1.times.10.sup.5 particles (which
also is referred to as particle units) of the adenoviral vector.
The dose preferably is at least about 1.times.10.sup.6 particles
(e.g., about 1.times.10.sup.6-1.times.10.sup.12 particles), more
preferably at least about 1.times.10.sup.7 particles, more
preferably at least about 1.times.10.sup.8 particles (e.g., about
1.times.10.sup.8-1.times.10.sup.11 particles), and most preferably
at least about 1.times.10.sup.9 particles (e.g., about
1.times.10.sup.9-1.times.10.sup.10 particles) of the adenoviral
vector. The dose desirably comprises no more than about
1.times.10.sup.14 particles, preferably no more than about
1.times.10.sup.13 particles, even more preferably no more than
about 1.times.10.sup.12 particles, even more preferably no more
than about 1.times.10.sup.11 particles, and most preferably no more
than about 1.times.10.sup.10 particles (e.g., no more than about
1.times.10.sup.9 particles). In other words, a single dose of
adenoviral vector can comprise, for example, about 1.times.10.sup.6
particle units (pu), 2.times.10.sup.6 pu, 4.times.10.sup.6 pu,
1.times.10.sup.7 pu, 2.times.10.sup.7 pu, 4.times.10.sup.7 pu,
1.times.10.sup.8 pu, 2.times.10.sup.8 pu, 4.times.10.sup.8 pu,
1.times.10.sup.9 pu, 2.times.10.sup.9 pu, 4.times.10.sup.9 pu,
1.times.10.sup.10 pu, 2.times.10.sup.10 pu, 4.times.10.sup.10 pu,
1.times.10.sup.11 pu, 2.times.10.sup.11 pu, 4.times.10.sup.11 pu,
1.times.10.sup.12 pu, 2.times.10.sup.12 pu, or 4.times.10.sup.12 pu
of the adenoviral vector.
[0078] The adenoviral vector desirably is administered in a
composition, preferably a pharmaceutically acceptable (e.g.,
physiologically acceptable) composition, which comprises a carrier,
preferably a pharmaceutically (e.g., physiologically) acceptable
carrier and the adenoviral vector(s). Any suitable carrier can be
used within the context of the invention, and such carriers are
well known in the art. The choice of carrier will be determined, in
part, by the particular site to which the composition is to be
administered and the particular method used to administer the
composition. Ideally, in the context of adenoviral vectors, the
composition preferably is free of replication-competent adenovirus.
The composition can optionally be sterile or sterile with the
exception of the inventive adenoviral vector.
[0079] Suitable formulations for the composition include aqueous
and non-aqueous solutions, isotonic sterile solutions, which can
contain anti-oxidants, buffers, and bacteriostats, and aqueous and
non-aqueous sterile suspensions that can include suspending agents,
solubilizers, thickening agents, stabilizers, and preservatives.
The formulations can be presented in unit-dose or multi-dose sealed
containers, such as ampules and vials, and can be stored in a
freeze-dried (lyophilized) condition requiring only the addition of
the sterile liquid carrier, for example, water, immediately prior
to use. Extemporaneous solutions and suspensions can be prepared
from sterile powders, granules, and tablets of the kind previously
described. Preferably, the carrier is a buffered saline solution.
More preferably, the adenoviral vector for use in the inventive
method is administered in a composition formulated to protect the
expression vector from damage prior to administration. For example,
the composition can be formulated to reduce loss of the adenoviral
vector on devices used to prepare, store, or administer the
expression vector, such as glassware, syringes, or needles. The
composition can be formulated to decrease the light sensitivity
and/or temperature sensitivity of the expression vector. To this
end, the composition preferably comprises a pharmaceutically
acceptable liquid carrier, such as, for example, those described
above, and a stabilizing agent selected from the group consisting
of polysorbate 80, L-arginine, polyvinylpyrrolidone, trehalose, and
combinations thereof. Use of such a composition will extend the
shelf life of the vector, facilitate administration, and increase
the efficiency of the inventive method. Formulations for adenoviral
vector-containing compositions are further described in, for
example, U.S. Pat. No. 6,225,289, U.S. Pat. No. 6,514,943, U.S.
Patent Application Publication 2003/0153065 A1, and International
Patent Application Publication WO 00/34444. A composition also can
be formulated to enhance transduction efficiency. In addition, one
of ordinary skill in the art will appreciate that the adenoviral
vector can be present in a composition with other therapeutic or
biologically-active agents. For example, factors that control
inflammation, such as ibuprofen or steroids, can be part of the
composition to reduce swelling and inflammation associated with in
vivo administration of the viral vector. In addition, immune system
stimulators can be administered to enhance any immune response to
the antigen. Antibiotics, i.e., microbicides and fungicides, can be
present to treat existing infection and/or reduce the risk of
future infection, such as infection associated with gene transfer
procedures.
[0080] The following examples further illustrate the invention but,
of course, should not be construed as in any way limiting its
scope.
Example 1
[0081] This example demonstrates the preparation of an adenoviral
vector array wherein each adenoviral vector comprises a nucleic
acid sequence encoding a different P. yoelii antigen.
[0082] Plasmids encoding an adenoviral vector genome deficient in
the E1, E3, and E4 regions were constructed using AdFlex technology
(GenVec Inc., Gaithersburg, Md.), which utilizes phage lambda
site-specific recombination for in vitro transfer of genes from
smaller plasmid vectors to large plasmid vectors that contain the
entire adenoviral genome. Several different AdFlex plasmids have
been generated, all of which comprise (i) the attR1-CmR-ccB-attR2
expression cassette (Invitrogen, Inc., Carlsbad, Calif.) inserted
into an E1 deletion site in between a CMV promoter and an SV40
polyadenylation site, (ii) a lambda cos site, and (iii) a LacI
restriction site. Certain AdFlex plasmids also contain the p15
origin of replication, the colE1 origin of replication, and/or
genes encoding resistance to kanamycin, ampicillin, or
tetracycline.
[0083] The resulting adenovector plasmids were transfected into
293-ORF6 complementing cells (GenVec Inc., Gaithersburg, Md.), and
infectious adenovector plasmids were rescued. Samples of infectious
adenovector plasmids were added to six wells of a 96-well tissue
culture plate. Gateway.TM. donor plasmids (pDONR) containing the
nucleic acid sequences encoding P. yoelii antigens flanked by att
site-specific recombination sites were generated, and incubated
with the adenovector plasmids in the 96-well plate. Recombination
between each adenovector plasmid and the pDONR plasmids was
catalyzed via an LR reaction using Gateway.TM. technology
(Invitrogen Life Technologies, Carlsbad, Calif.).
[0084] E. coli strain DH10B was transformed with 2 .mu.l of the LR
reaction product and grown in the presence of kanamycin. Cultures
were incubated overnight at 30.degree. C. The following day, two
colonies were chosen from each culture dish for expansion. Plasmid
DNA was purified from E. coli using the QIA well 8 plasmid
purification kit (Qiagen, Inc., Valencia, Calif.) and linearized
with PacI. Restriction digestion patters from each of the six
recombinant adenovector plasmids indicated that each colony
contained only the desired recombinant adenovector plasmid, which
verified that adenovector plasmids could be generated with 100%
efficiency using the Gateway.TM. system.
[0085] The results of this example demonstrate the construction of
multiple infectious recombinant adenovector plasmids in parallel
format, which is suitable for automation.
Example 2
[0086] This example demonstrates the conversion of arrayed
adenovector plasmids into adenovirus particles.
[0087] Based on published reports indicating that a dose of
1.times.10.sup.4 pu/cell of adenoviral vector is necessary to
infect 90% of dendritic cells in vitro (Wan et al., Hum. Gene
Ther., 8: 1355-63 (1997), and Gahn et al., Int. J. Cancer, 93:
706-13 (2001)), it is estimated that 5.times.10.sup.8 pu of
adenovector will be necessary to practice the inventive method. The
burst size for E1/E3/E4-deficient adenovectors in 293-ORF6 cells is
approximately 5.times.10.sup.4 pu/cell. As 2.times.10.sup.4 cells
can easily be seeded in a single well of a 96-well plate, it is
expected that 1.times.10.sup.9 particles of adenovector can be
produced from a single well. This quantity of vector likely will be
sufficient for the inventive APC cell-based assays for antigen
identification.
[0088] 293-ORF6 complementing cells are transfected using Polyfect
Transfection Reagent (Qiagen, Inc., Gaithersburg, Md.) in 96-well
plates. Based on extrapolations from larger scale rescue, less than
1.times.10.sup.4 particles of adenovector can be rescued using this
approach, as typically less than 1.times.10.sup.6 particles are
rescued in 293-ORF6 cells grown in 60 mm plates. Plates are
incubated at 37.degree. C. for approximately 10 days, or until
complete cytopathic effect (CPE) is observed.
[0089] In the event that the above method does not yield
1.times.10.sup.9 pu of adenovector required for the T cell screen,
a passaging step will be performed to generate the high titer
stock. In this regard, adenovector will be liberated from the
293-ORF6 cells at 3 days post-transfection by three freeze-thaw
cycles, and the cell lysates will be used to infect new cells in
96-well plates. The vector titers are expanded in parallel in
96-well plates by successive passaging until CPE is observed.
Vector yields will be determined by a HPLC-based particle
determination assay, and active virus particles will be quantified
by using the focal forming unit assay described in, for example,
Cleghon et al., Virology, 197: 564-575 (1993).
[0090] The protocol of this example can be used to demonstrate the
conversion of arrayed recombinant adenovector plasmids into
adenovirus particles at a scale suitable for antigen screening.
Example 3
[0091] This example demonstrates the ability of antigen presenting
cells transduced with an adenoviral vector encoding an antigen of a
pathogen to recall cellular immune responses from mice immunized
with the pathogen.
[0092] Cells from the A2/20J (A20) dendritic cell line were
transduced with an E1/E3/E4-deficient Ad5 vector encoding the P.
yoelii CSP protein (AdPyCSP) at a multiplicity of infection (MOI)
of 10, 1, or 0.1 using methods known in the art. As a positive
control, A20 cells were transfected with a plasmid encoding PyCSP,
or a recombinant pox vector encoding PyCSP (PyCSP-COPAC) at an MOI
of 10. Negative controls included A20 cells transfected with an
empty plasmid or parental vaccinia virus at an MOI of 10, and A20
cells transduced with an adenoviral vector encoding green
fluorescent protein (GFP) at an MOI of 10, 1, or 0.1.
[0093] Transduced A20 cells were plated at 1.times.10.sup.5 cells
per well in 96-well plates. Splenocytes from BALB/c mice immunized
with irradiated P. yoelii sporozoites were incubated with the
transduced A20 cells in the 96-well plates. After a 36 hour
incubation, IFN-.gamma. levels were measured by ELISpot, as it is
widely considered that IFN-.gamma. is the most appropriate in vitro
marker of pre-erythrocytic stage protection (Doolan et al., J.
Immunol., 163: 884-92 (1999), and Doolan et al., J. Immunol., 165:
1453-62 (2000)). Spots were read using a CTL automated reader.
[0094] A20 cells transduced with AdPyCSP efficiently recalled PyCSP
antigen-specific T cell responses in vitro following infection at
an MOI of 10. At an MOI of 1 there was a decrease in the number of
T cells stimulated to secrete IFN-.gamma., and at an MOI of 0.1 no
PyCSP specific T cell responses were observed. Greater than 400
cells per million spleen cells produced IFN-.gamma. in response to
A20 cells infected with PyCSP-COPAC, or A20 cells transfected with
PyCSP plasmid. A20 cells infected at an MOI of 10 with Ad5 PyCSP
stimulated more than 300 cells per million responder spleen cells
to produce IFN-.gamma.. At lower MOIs, there was a rapid drop in
the number of cells producing IFN-.gamma..
[0095] The results of this example demonstrate that APCs transduced
with adenovectors encoding Plasmodium antigens can recall cellular
immune responses in Plasmodium-immunized mice.
Example 4
[0096] This example demonstrates the ability of antigen presenting
cells infected with the adenoviral vector array of the invention to
induce a protective immune response in vitro.
[0097] Primary dendritic cells (DC) and the A2/20J (A20) dendritic
cell line are transformed with the adenoviral vectors produced in
Example 1 by incubating separate 96-well plates containing the six
adenoviral vectors produced in Example 1 with either primary DC or
A20 cells using methods known in the art. Effector T lymphocytes
are obtained by harvesting spleens from BALB/c mice immunized with
irradiated P. yoelii sporozoites. These splenocytes are considered
to contain sporozoite-immune effector T cell populations, which
recognize protective target antigens.
[0098] The adenovector-transduced APCs are co-cultured with
autologous lymphocytes for defined periods of time from 24 to 96
hours after transduction to assay for cellular immune responses.
IFN-.gamma. levels are measured using ELlspot, flow cytometry, or
FACS-based assays.
[0099] The protocol set out in this example can be used to confirm
the ability of the inventive method to identify antigens that
induce protective immunity against Plasmodium infection.
Example 5
[0100] This example demonstrates the ability of antigen presenting
cells infected with the adenoviral vector array of the invention to
induce a protective immune response in vivo.
[0101] Mice are immunized with E1/E3/E4-deficient adenoviral
vectors encoding PyCSP, PyHEP17, or three novel Py antigens twice
at 6 week intervals. Alternatively, a DNA prime/adenovector boost
immunization schedule is used. Mice are then challenged with
infectious P. yoelii sporozoites, and the capacity of the antigen
to confer complete or partial protection against parasite challenge
is assessed. Sterile protection is indicated by complete absence of
blood-stage parasitemia (see, e.g., Doolan et al., J. Immunol.,
163: 884-92 (1999), Sedegah et al., Proc. Natl. Acad. Sci. USA, 91:
9866-70 (1994), and Doolan et al., J. Exp. Med., 184: 1739-46
(1996)). Partial protection is indicated by reduction in
liver-stage parasite burden, as evaluated by qRT-PCR (see Witney et
al., Mol. Biochem. Parasitol., 118: 233-45 (2001)). In vitro immune
reactivity, as assayed by IFN-.gamma., ELISpot, or FACS-based
assays (described in Example 4), then is correlated with in vivo
protective capacity, as evaluated by a decrease in liver-stage
and/or blood-stage parasite burden.
[0102] The protocol of this example can be used to demonstrate that
antigens identified using the inventive method are capable of
protecting against parasite challenge in vivo.
[0103] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0104] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0105] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
Sequence CWU 1
1
315PRTArtificialSynthetic 1Cys Arg Gly Asp Cys 1 5
29PRTArtificialSynthetic 2Cys Xaa Cys Arg Gly Asp Cys Xaa Cys 1 5
39PRTArtificialSynthetic 3Cys Asp Cys Arg Gly Asp Cys Phe Cys 1
5
* * * * *
References