U.S. patent application number 09/010733 was filed with the patent office on 2002-03-14 for polynucleotide tuberculosis vaccine.
Invention is credited to CONTENT, JEAN, HUYGEN, KRIS, LIU, MARGARET A., MONTGOMERY, DONNA, ULMER, JEFFREY.
Application Number | 20020032162 09/010733 |
Document ID | / |
Family ID | 23326996 |
Filed Date | 2002-03-14 |
United States Patent
Application |
20020032162 |
Kind Code |
A1 |
CONTENT, JEAN ; et
al. |
March 14, 2002 |
POLYNUCLEOTIDE TUBERCULOSIS VACCINE
Abstract
Genes encoding Mycobacterium tuberculosis (M.tb) proteins were
cloned into eukaryotic expression vectors to express the encoded
proteins in mammalian muscle cells in vivo. Animals were immunized
by injection of these DNA constructs, termed polynucleotide
vaccines or PNV, into their muscles. Immune antisera was produced
against M.tb antigens. Specific T-cell responses were detected in
spleen cells of vaccinated mice and the profile of cytokine
secretion in response to antigen 85 was indicative of a T.sub.h1
type of helper T-cell response (i.e., high IL-2 and IFN-.gamma.).
Protective efficacy of an M.tb DNA vaccine was demonstrated in mice
after challenge with M.bovis BCG, as measured by a reduction in
mycobacterial multiplication in the spleens and lungs of M.tb
DNA-vaccinated mice compared to control DNA-vaccinated mice or
primary infection in naive mice.
Inventors: |
CONTENT, JEAN;
(RHODE-SAINT-GENESE, BE) ; HUYGEN, KRIS;
(BRUSSELS, BE) ; LIU, MARGARET A.; (ROSEMONT,
PA) ; MONTGOMERY, DONNA; (CHALFONT, PA) ;
ULMER, JEFFREY; (CHALFONT, PA) |
Correspondence
Address: |
JOHN W WALLEN III
MERCK & CO INC
PATENT DEPT
P O BOX 2000
RAHWAY
NJ
070650907
|
Family ID: |
23326996 |
Appl. No.: |
09/010733 |
Filed: |
January 22, 1998 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09010733 |
Jan 22, 1998 |
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08338992 |
Nov 14, 1994 |
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5736524 |
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Current U.S.
Class: |
514/44R |
Current CPC
Class: |
A61K 2039/51 20130101;
A61K 48/00 20130101; A61K 31/711 20130101; A61K 39/00 20130101;
A61P 31/04 20180101; A61P 31/06 20180101; C07K 14/35 20130101; A61P
37/04 20180101 |
Class at
Publication: |
514/44 |
International
Class: |
C07H 021/04; A61K
031/70; A01N 043/04 |
Claims
What is claimed is:
1. A polynucleotide which induces upon introduction into vertebrate
tissue, one or more anti-Mycobacterial immune responses selected
from antibodies, CTL, helper T lymphocyte responses and protective
immune responses, wherein said polynucleotide comprises one or more
genes encoding one or more Mycobacterial proteins or functional
equivalents thereof, said genes being operably linked to a
transcription promoter.
2. The polynucleotide of claim 1, wherein said gene encodes a
Mycobacterium tuberculosis protein, and functional equivalents
thereof.
3. The polynucleotide of claim 2, wherein said gene encodes a
protein selected from a group consisting of antigen 85A, B, and/or
C, and functional equivalents thereof.
4. A method for inducing immune responses in a vertebrate against
Mycobacterial epitopes, comprising introducing between 1 ng and 5
mg of a polynucleotide according to claim 1 into a tissue of a
vertebrate.
5. The method of claim 4, wherein said gene encodes a Mycobacterium
tuberculosis protein, and functional equivalents thereof.
6. The method of claim 5, wherein said gene encodes a protein
selected from a group consisting of antigen 85A, B, and C, and
functional equivalents thereof.
7. A vaccine for inducing immune responses against Mycobacterial
antigens, comprising the polynucleotide of claim 1 and a
pharmaceutically acceptable carrier.
8. The vaccine of claim 7, wherein said antigen is a Mycobacterium
tuberculosis antigen, and functional equivalents thereof.
9. The vaccine of claim 8, wherein said antigen is a protein
selected from a group consisting of antigen 85A, B, and C, and
functional equivalents thereof.
10. A method for inducing immune responses against mycobacterial
antigens, comprising introducing into a tissue of a vertebrate one
or more isolated and purified mycobacterial genes eliciting an
immune response which prevents mycobacterial infection and/or
ameliorates mycobacterial disease.
11. The method of claim 10, wherein said gene encodes a
Mycobacterium tuberculosis protein, and functional equivalents
thereof.
12. The method of claim 11, wherein said gene encodes a protein
selected from a group consisting of antigen 85A, B, and C, and
functional equivalents thereof.
13. A polynucleotide comprising: a) a eukaryotic transcription
promoter; b) an open reading frame operably linked to said promoter
encoding one or more mycobacterial epitopes, and a translation
termination signal; and c) optionally containing one or more
operably linked IRES, one or more open reading frames encoding one
or more additional genes, and one or more transcription termination
signals.
14. The polynucleotide of claim 13 wherein said additional genes of
c) are immunomodulatory or immunostimulatory genes selected from a
group consisting of GM-CSF, IL-12, interferon, and a member of the
B7 family of T-cell costimulatory proteins.
15. The polynucleotide of claim 13 wherein said mycobacterial gene
of a) encodes a Mycobacterium tuberculosis protein, and functional
equivalents thereof.
16. The polynucleotide of claim 15 wherein said mycobacterial gene
of a) encodes a Mycobacterium tuberculosis protein selected from a
group consisting of antigen 85A, B, and C, and functional
equivalents thereof.
17. The polynucleotide of claim 13 wherein said additional genes of
c) are Mycobacterium tuberculosis genes selected from a group
consisting of antigen 85A, B, and C, and functional equivalents
thereof.
18. A method of treating a patient in need of such treatment with a
polynucleotide which induces upon introduction into vertebrate
tissue, one or more anti-mycobacterial immune responses selected
from antibodies, CTL, helper T lymphocyte responses and protective
immune responses, wherein said polynucleotide comprises a gene
encoding one or more mycobacterial proteins or functional
equivalents thereof, said gene being operably linked to a
transcription promoter.
19. The method of claim 18, wherein said gene encodes a
Mycobacterium tuberculosis protein, and functional equivalents
thereof.
20. The method of claim 19 wherein said gene encodes one or more
proteins selected from a group consisting of antigen 85A, B, and C,
and functional equivalents thereof.
21. The method of claim 10 wherein said patient is a domestic
animal or livestock.
22. A vaccine for inducing immune responses against Mycobacterial
infection in domesticated or agricultural animals comprising the
polynucleotide of claim 1 and a pharmaceutically acceptable
carrier.
Description
BACKGROUND OF THE INVENTION
[0001] A major obstacle to the development of vaccines against
viruses and bacteria, particularly those with multiple serotypes or
a high rate of mutation, against which elicitation of neutralizing
antibodies and/or protective cell-mediated immune responses is
desirable, is the diversity of the external proteins among
different isolates or strains. Since cytotoxic T-lymphocytes (CTLs)
in both mice and humans are capable of recognizing epitopes derived
from conserved internal viral proteins [J. W. Yewdell et al., Proc.
Natl. Acad. Sci. (USA) 82, 1785 (1985); A. R. M. Townsend, et al.,
Cell 44, 959 (1986); A. J. McMichael et al., J. Gen. Virol. 67, 719
(1986); J. Bastin et al., J. Exp. Med. 165, 1508 (1987); A. R. M.
Townsend and H. Bodmer, Annu. Rev. Immunol. 7, 601 (1989)], and are
thought to be important in the immune response against viruses [Y.
-L. Lin and B. A. Askonas, J. Exp. Med. 154, 225 (1981); I. Gardner
et al., Eur. J. Immunol. 4, 68 (1974); K. L. Yap and G. L. Ada,
Nature 273, 238 (1978); A. J. McMichael et al., New Engl. J. Med.
309, 13 (1983); P. M. Taylor and B. A. Askonas, Immunol. 58, 417
(1986)], efforts have been directed towards the development of CTL
vaccines capable of providing heterologous protection against
different viral strains.
[0002] It is known that CTLs kill virally- or bacterially-infected
cells when their T cell receptors recognize foreign peptides
associated with MHC class I and/or class II molecules. These
peptides can be derived from endogenously synthesized foreign
proteins, regardless of the protein's location or function within
the pathogen. By recognition of epitopes from conserved proteins,
CTLs may provide heterologous protection. In the case of
intracellular bacteria, proteins secreted by or released from the
bacteria are processed and presented by MHC class I and II
molecules, thereby generating T-cell responses that may play a role
in reducing or eliminating infection.
[0003] Most efforts to generate CTL responses have either used
replicating vectors to produce the protein antigen within the cell
[J. R. Bennink et al., ibid. 311, 578 (1984); J. R. Bennink and J.
W. Yewdell, Curr. Top. Microbiol. Immunol. 163, 153 (1990); C. K.
Stover et al., Nature 351, 456 (1991); A. Aldovini and R. A. Young,
Nature 351, 479 (1991); R. Schafer et al., J. Immunol. 149, 53
(1992); C. S. Hahn et al., Proc. Natl. Acad. Sci. (USA) 89, 2679
(1992)], or they have focused upon the introduction of peptides
into the cytosol [F. R. Carbone and M. J. Bevan, J. Exp. Med. 169,
603 (1989); K. Deres et al., Nature 342, 561 (1989); H. Takahashi
et al., ibid. 344, 873 (1990); D. S. Collins et al., J. Immunol.
148, 3336 (1992); M. J. Newman et al., ibid. 148, 2357 (1992)].
Both of these approaches have limitations that may reduce their
utility as vaccines. Retroviral vectors have restrictions on the
size and structure of polypeptides that can be expressed as fusion
proteins while maintaining the ability of the recombinant virus to
replicate [A. D. Miller, Curr. Top. Microbiol. Immunol. 158, 1
(1992)], and the effectiveness of vectors such as vaccinia for
subsequent immunizations may be compromised by immune responses
against vaccinia [E. L. Cooney et al., Lancet 337, 567 (1991)].
Also, viral vectors and modified pathogens have inherent risks that
may hinder their use in humans [R. R. Redfield et al., New Engl. J.
Med. 316, 673 (1987); L. Mascola et al., Arch. Intern. Med. 149,
1569 (1989)]. Furthermore, the selection of peptide epitopes to be
presented is dependent upon the structure of an individual's MHC
antigens and, therefore, peptide vaccines may have limited
effectiveness due to the diversity of MHC haplotypes in outbred
populations.
[0004] Benvenisty, N., and Reshef, L. [PNAS 83, 9551-9555, (1986)]
showed that CaCl.sub.2 precipitated DNA introduced into mice
intraperitoneally (i.p.), intravenously (i.v.) or intramuscularly
(i.m.) could be expressed. The intramuscular (i.m.) injection of
DNA expression vectors in mice has been demonstrated to result in
the uptake of DNA by the muscle cells and expression of the protein
encoded by the DNA [J. A. Wolff et al., Science 247, 1465 (1990);
G. Ascadi et al., Nature 352, 815 (1991)]. The plasmids were shown
to be maintained episomally and did not replicate. Subsequently,
persistent expression has been observed after i.m. injection in
skeletal muscle of rats, fish and primates, and cardiac muscle of
rats [H. Lin et al., Circulation 82, 2217 (1990); R. N. Kitsis et
al., Proc. Natl. Acad. Sci. (USA) 88, 4138 (1991); E. Hansen et
al., FEBS Lett. 290, 73 (1991); S. Jiao et al., Hum. Gene Therapy
3, 21 (1992); J. A. Wolff et al., Human Mol. Genet. 1, 363 (1992)].
The technique of using nucleic acids as therapeutic agents was
reported in WO90/11092 (Oct. 4, 1990), in which naked
polynucleotides were used to vaccinate vertebrates.
[0005] Recently, the coordinate roles of B7 and the major
histocompatibility complex (MHC) presentation of epitopes on the
surface of antigen presenting cells in activating CTLs for the
elimination of tumors was reviewed [Edgington, Biotechnology 11,
1117-1119, 1993]. Once the MHC molecule on the surface of an
antigen presenting cell (APC) presents an epitope to a T-cell
receptor (TCR), B7 expressed on the surface of the same APC acts as
a second signal by binding to CTLA-4 or CD28. The result is rapid
division of CD4.sup.+ helper T-cells which signal CD8.sup.+ T-cells
to proliferate and kill the APC.
[0006] It is not necessary for the success of the method that
immunization be intramuscular. Thus, Tang et al., [Nature, 356,
152-154 (1992)] disclosed that introduction of gold
microprojectiles coated with DNA encoding bovine growth hormone
(BGH) into the skin of mice resulted in production of anti-BGH
antibodies in the mice. Furth et al., [Analytical Biochemistry,
205, 365-368, (1992)] showed that a jet injector could be used to
transfect skin, muscle, fat, and mammary tissues of living animals.
Various methods for introducing nucleic acids was recently reviewed
[Friedman, T., Science, 244, 1275-1281 (1989)]. See also Robinson
et al., [Abstracts of Papers Presented at the 1992 meeting on
Modern Approaches to New Vaccines, Including Prevention of AIDS,
Cold Spring Harbor, p92; Vaccine 11, 957 (1993)], where the im, ip,
and iv administration of avian influenza DNA into chickens was
alleged to have provided protection against lethal challenge.
Intravenous injection of a DNA:cationic liposome complex in mice
was shown by Zhu et al., [Science 261, 209-211 (9 July 1993); see
also WO93/24640, Dec. 9, 1993] to result in systemic expression of
a cloned transgene. Recently, Ulmer et al., [Science 259,
1745-1749, (1993)] reported on the heterologous protection against
influenza virus infection by injection of DNA encoding influenza
virus proteins.
[0007] Wang et al., [P.N.A.S. USA 90, 4156-4160 (May, 1993)]
reported on elicitation of immune responses in mice against HIV by
intramuscular inoculation with a cloned, genomic (unspliced) HIV
gene. However, the level of immune responses achieved was very low,
and the system utilized portions of the mouse mammary tumor virus
(MMTV) long terminal repeat (LTR) promoter and portions of the
simian virus 40 (SV40) promoter and terminator. SV40 is known to
transform cells, possibly through integration into host cellular
DNA. Thus, the system described by Wang et al., is wholly
inappropriate for administration to humans, which is one of the
objects of the instant invention.
[0008] WO 93/17706 describes a method for vaccinating an animal
against a virus, wherein carrier particles were coated with a gene
construct and the coated particles are accelerated into cells of an
animal.
[0009] Studies by Wolff et al. (supra) originally demonstrated that
intramuscular injection of plasmid DNA encoding a reporter gene
results in the expression of that gene in myocytes at and near the
site of injection. Recent reports demonstrated the successful
immunization of mice against influenza by the injection of plasmids
encoding influenza A hemagglutinin (Montgomery, D. L. et al., 1993,
Cell Biol., 12, pp.777-783), or nucleoprotein (Montgomery, D. L. et
al., supra; Ulmer, J. B. et al., 1993, Science, 259, pp.1745-1749).
The first use of DNA immunization for a herpes virus has been
reported (Cox et al., 1993, J.Virol., 67, pp.5664-5667). Injection
of a plasmid encoding bovine herpesvirus 1 (BHV-1) glycoprotein g
IV gave rise to anti-g IV antibodies in mice and calves. Upon
intranasal challenge with BHV-1, immunized calves showed reduced
symptoms and shed substantially less virus than controls.
[0010] Tuberculosis (TB) is a chronic infectious disease of the
lung caused by the pathogen Mycobacterium tuberculosis. TB is one
of the most clinically significant infections worldwide, with an
incidence of 3 million deaths and 10 million new cases each year.
It has been estimated that as much as one third of the world's
population may be infected and, in developing countries, 55 million
cases of active TB have been reported. Until the turn of the
century, TB was the leading cause of death in the United States.
But, with improved sanitary conditions and the advent of
antimicrobial drugs, the incidence of mortality steadily declined
to the point where it was predicted that the disease would be
eradicated by the year 2000. However, in most developed countries,
the number of cases of active TB has risen each year since the
mid-1980's. Part of this resurgence has been attributed to
immigration and the growing number of immunocompromised,
HIV-infected individuals. If left unabated, it is predicted that TB
will claim more than 30 million human lives in the next ten years.
As alarming as these figures may seem, it is of even greater
concern that multidrug-resistant (MDR) strains of M. tuberculosis
have arisen. These MDR strains are not tractable by traditional
drug therapy and have been responsible for several recent outbreaks
of TB, particularly in urban centers. Therefore, one of the key
components in the management of TB in the long-term will be an
effective vaccine [for review see Bloom and Murray, 1993, Science
257, 1055].
[0011] M. tuberculosis is an intracellular pathogen that infects
macrophages and is able to survive within the harsh environment of
the phagolysosome in this type of cell. Most inhaled bacilli are
destroyed by activated alveolar macrophages. However, the surviving
bacilli can multiply in macrophages and be released upon cell
death, which signals the infiltration of lymphocytes, monocytes and
macrophages to the site. Lysis of the bacilli-laden macrophages is
mediated by delayed-type hypersensitivity (DTH) and results in the
development of a solid caseous tubercle surrounding the area of
infected cells. Continued DTH causes the tubercle to liquefy,
thereby releasing entrapped bacilli. The large dose of
extracellular bacilli triggers further DTH, causing damage to the
bronchi and dissemination by lymphatic, hematogenous and bronchial
routes, and eventually allowing infectious bacilli to be spread by
respiration.
[0012] Immunity to TB involves several types of effector cells.
Activation of macrophages by cytokines, such as interferon-.gamma.,
is an effective means of minimizing intracellular mycobacterial
multiplication. However, complete eradication of the bacilli by
this means is often not achieved. Acquisition of protection against
TB requires T lymphocytes. Among these, both CD8.sup.+ and
CD4.sup.+ T cells seem to be important [Orme et al, 1993, J.
Infect. Dis. 167, 1481]. These cell types secrete
interferon-.gamma. in response to mycobacteria, indicative of a
T.sub.h1 immune response, and possess cytotoxic activity to
mycobacteria-pulsed target cells. In recent studies using .beta.-2
microglobulin- and CD8-deficient mice, CTL responses have been
shown to be critical in providing protection against M.
tuberculosis [Flynn et al, 1992, Proc. Natl. Acad. Sci. USA 89,
12013; Flynn et al, 1993, J. Exp. Med. 178, 2249; Cooper et al,
1993, J. Exp. Med. 178, 2243]. In contrast, B lymphocytes do not
seem to be involved, and passive transfer of anti-mycobacterial
antibodies does not provide protection. Therefore, effective
vaccines against TB must generate cell-mediated immune
responses.
[0013] Antigenic stimulation of T cells requires presentation by
MHC molecules. In order for mycobacterial antigens to gain access
to the antigen presentation pathway they must be released from the
bacteria. In infected macrophages, this could be accomplished by
secretion or bacterial lysis. Mycobacteria possess many potential
T-cell antigens and several have now been identified [Andersen
1994, Dan. Med. Bull. 41, 205]. Some of these antigens are secreted
by the bacteria. It is generally believed that immunity against TB
is mediated by CD8.sup.+ and CD4.sup.+ T cells directed toward
these secreted antigens. In mouse and guinea pig models of TB,
protection from bacterial challenge, as measured by reduced weight
loss, has been achieved using a mixture of secreted mycobacterial
antigens [Pal and Horowitz, 1992 Infect. Immunity 60, 4781;
Andersen 1994, Infect. Immunity 62, 2536; Collins, 1994, Veterin.
Microbiol. 40, 95].
[0014] Several potentially protective T cell antigens have been
identified in M. tuberculosis and some of these are being
investigated as vaccine targets. Recent work has indicated that the
predominant T-cell antigens are those proteins that are secreted by
mycobacteria during their residence in macrophages, such as: i) the
antigen 85 complex of proteins (85A, 85B, 85C) [Wiker and Harboe,
1992, Microbiol. Rev. 56, 648], ii) a 6 kDa protein termed ESAT-6
[Andersen 1994, Infect. Immunity 62, 2536], iii) a 38 kDa
lipoprotein with homology to PhoS [Young and Garbe, 1991, Res.
Microbiol. 142, 55; Andersen, 1992, J. Infect. Dis. 166, 874], iv)
the 65 kDa GroEL heat-shock protein [Siva and Lowrie, 1994,
Immunol. 82, 244], v) a 55 kDa protein rich in proline and
threonine [Romain et al, 1993, Proc. Natl. Acad. Sci. USA 90,
5322], and vi) a 19 kDa lipoprotein [Faith et al, 1991, Immunol.
74, 1].
[0015] The genes for each of the three antigen 85 proteins (A, B,
and C) have been cloned and sequenced [Borremans et al, 1989,
Infect. Immunity 57, 3123; Content et al, Infect. Immunity 59,
3205; DeWit et al 1994, DNA Seq. 4, 267]. In addition, these
structurally-related proteins are targets for strong T-cell
responses after both infection and vaccination [Huygen et al, 1988,
Scand. J. Immunol. 27, 187; Launois et al, 1991, Clin. Exp.
Immunol. 86, 286; Huygen et al, 1992, Infect. Immunity 60, 2880;
Munk et al, 1994, Infect. Immunity 62, 726; Launois et al, 1994,
Infect. Immunity 62, 3679]. Therefore, the antigen 85 proteins are
considered to be good vaccine targets.
SUMMARY OF THE INVENTION
[0016] To test the efficacy of DNA immunization in the prevention
of M.tb disease, M.tb protein-coding DNA sequences were cloned into
eukaryotic expression vectors. These DNA constructions elicit an
immune response when injected into animals. Immunized animals are
infected with mycobacteria to evaluate whether or not direct DNA
immunization with the gene (or other M.tb genes) could protect them
from disease. Nucleic acids, including DNA constructs and RNA
transcripts, capable of inducing in vivo expression of M.tb
proteins upon direct introduction into animal tissues via injection
or otherwise are therefore disclosed. Injection of these nucleic
acids may elicit immune responses which result in the production of
cytotoxic T lymphocytes (CTLs) specific for M.tb antigens, as well
as the generation of M.tb-specific helper T lymphocyte responses,
which are protective upon subsequent challenge. These nucleic acids
are useful as vaccines for inducing immunity to M.tb, which can
prevent infection and/or ameliorate M.tb-related disease.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1. General principle for cloning M.tb genes into
expression vectors is shown.
[0018] FIG. 2. Vector map of V1Jns.tPA85A.C1 is shown.
[0019] FIG. 3. Vector map of V1Jns.85A.C2 is shown.
[0020] FIG. 4. Vector map of V1Jns.85A.C3 is shown.
[0021] FIG. 5. Vector map of V1Jns.tPA85B.C1 is shown.
[0022] FIG. 6. Vector map of V1Jns.tPA85C.C1 is shown.
[0023] FIG. 7 N-Terminal sequence verification of constructs is
shown.
[0024] FIG. 8 Expression of M.tb proteins in tissue culture is
shown.
[0025] FIG. 9 Production of antigen 85A-specific antibodies in
DNA-vaccinated mice is shown.
[0026] FIG. 10 IL-2 production in BALB/c mice by a Tb DNA vaccine
is shown.
[0027] FIG. 11 IL-2 production in C57BL/6 mice by a Tb DNA vaccine
is shown.
[0028] FIG. 12 IFN-.beta. production in BALB/c mice by a Tb DNA
vaccine is shown.
[0029] FIG. 13 IFN-.gamma. production in C57BL/6 mice by a Tb DNA
vaccine is shown.
[0030] FIG. 14 Lack of IL-4 production in BALB/c mice by a Tb DNA
vaccine is shown.
[0031] FIG. 15 Lack of IL-6 production in mice by a Tb DNA vaccine
is shown.
[0032] FIG. 16 Lack of IL-10 production in mice by a Tb DNA vaccine
is shown.
[0033] FIG. 17 Reduction of BCG multiplication in lungs of C57BL/6
mice vaccinated with a Tb DNA vaccine is shown.
[0034] FIG. 18 Reduction of BCG multiplication in lungs of BALB/c
mice vaccinated with a Tb DNA vaccine is shown.
[0035] FIG. 19 Reduction of BCG multiplication in spleens of BALB/c
mice vaccinated with a Tb DNA vaccine is shown.
[0036] FIG. 20 Reduction of BCG multiplication in spleens of
C57BL/6 mice vaccinated with a Tb DNA vaccine is shown.
DETAILED DESCRIPTION OF THE INVENTION
[0037] This invention provides polynucleotides which, when directly
introduced into a vertebrate in vivo, including mammals such as
humans, induces the expression of encoded proteins within the
animal. As used herein, a polynucleotide is a nucleic acid which
contains essential regulatory elements such that upon introduction
into a living vertebrate cell, and is able to direct the cellular
machinery to produce translation products encoded by the genes
comprising the polynucleotide. In one embodiment of the invention,
the polynucleotide is a polydeoxyribonucleic acid comprising
Mycobacterium tuberculosis (M.tb) genes operatively linked to a
transcriptional promoter. In another embodiment of the invention
the polynucleotide vaccine comprises polyribonucleic acid encoding
M.tb genes which are amenable to translation by the eukaryotic
cellular machinery (ribosomes, tRNAs, and other translation
factors). Where the protein encoded by the polynucleotide is one
which does not normally occur in that animal except in pathological
conditions, (i.e. an heterologous protein) such as proteins
associated with M.tb, the animals' immune system is activated to
launch a protective immune response. Because these exogenous
proteins are produced by the animals' own tissues, the expressed
proteins are processed by the major histocompatibility system (MHC)
in a fashion analogous to when an actual M.tb infection occurs. The
result, as shown in this disclosure, is induction of immune
responses against M.tb. Polynucleotides for the purpose of
generating immune responses to an encoded protein are referred to
herein as polynucleotide vaccines or PNV.
[0038] There are many embodiments of the instant invention which
those skilled in the art can appreciate from the specification.
Thus, different transcriptional promoters, terminators, carrier
vectors or specific gene sequences may be used successfully.
[0039] The instant invention provides a method for using a
polynucleotide which, upon introduction into mammalian tissue,
induces the expression, in vivo, of the polynucleotide thereby
producing the encoded protein. It is readily apparent to those
skilled in the art that variations or derivatives of the nucleotide
sequence encoding a protein can be produced which alter the amino
acid sequence of the encoded protein. The altered expressed protein
may have an altered amino acid sequence, yet still elicits immune
responses which react with the mycobacterial protein, and are
considered functional equivalents. In addition, fragments of the
full length genes which encode portions of the full length protein
may also be constructed. These fragments may encode a protein or
peptide which elicits antibodies which react with the mycobacterial
protein, and are considered functional equivalents.
[0040] In one embodiment of this invention, a gene encoding an M.tb
gene product is incorporated in an expression vector. The vector
contains a transcriptional promoter recognized by eukaryotic RNA
polymerase, and a transcriptional terminator at the end of the M.tb
gene coding sequence. In a preferred embodiment, the promoter is
the cytomegalovirus promoter with the intron A sequence (CMV-intA),
although those skilled in the art will recognize that any of a
number of other known promoters such as the strong immunoglobulin,
or other eukaryotic gene promoters may be used. A preferred
transcriptional terminator is the bovine growth hormone terminator.
The combination of CMVintA-BGH terminator is preferred. In
addition, to assist in preparation of the polynucleotides in
prokaryotic cells, an antibiotic resistance marker is also
optionally included in the expression vector under transcriptional
control of a suitable prokaryotic promoter. Ampicillin resistance
genes, neomycin resistance genes or any other suitable antibiotic
resistance marker may be used. In a preferred embodiment of this
invention, the antibiotic resistance gene encodes a gene product
for neomycin/kanamycin resistance. Further, to aid in the high
level production of the polynucleotide by growth in prokaryotic
organisms, it is advantageous for the vector to contain a
prokaryotic origin of replication and be of high copy number. Any
of a number of commercially available prokaryotic cloning vectors
provide these elements. In a preferred embodiment of this
invention, these functionalities are provided by the commercially
available vectors known as the pUC series. It may be desirable,
however, to remove non-essential DNA sequences. Thus, the lacZ and
lacI coding sequences of pUC may be removed. It is also desirable
that the vectors are not able to replicate in eukaryotic cells.
This minimizes the risk of integration of polynucleotide vaccine
sequences into the recipients' genome.
[0041] In another embodiment, the expression vector pnRSV is used,
wherein the Rous sarcoma virus (RSV) long terminal repeat (LTR) is
used as the promoter. In yet another embodiment, V1, a mutated
pBR322 vector into which the CMV promoter and the BGH
transcriptional terminator were cloned is used. In a preferred
embodiment of this invention, the elements of V1 and pUC19 have
been been combined to produce an expression vector named V1J.
[0042] Into V1J, V1JtPA or another desirable expression vector is
cloned an M.tb gene, such as one of the antigen 85 complex genes,
or any other M.tb gene which can induce anti-M.tb immune responses
(CTLs, helper T lymphocytes and antibodies). In another embodiment,
the ampicillin resistance gene is removed from V1J and replaced
with a neomycin resistance gene, to generate V1J-neo, into which
any of a number of different M.tb genes may be cloned for use
according to this invention. In yet another embodiment, the vector
is V1Jns, which is the same as V1Jneo except that a unique Sfil
restriction site has been engineered into the single Kpn1 site at
position 2114 of V1J-neo. The incidence of Sfi1 sites in human
genomic DNA is very low (approximately 1 site per 100,000 bases).
Thus, this vector allows careful monitoring for expression vector
integration into host DNA, simply by Sfi1 digestion of extracted
genomic DNA. In a further embodiment, the vector is V1R. In this
vector, as much non-essential DNA as possible is "trimmed" to
produce a highly compact vector. This vector allows larger inserts
to be used, with less concern that undesirable sequences are
encoded and optimizes uptake by cells when the construct encoding
specific virus genes is introduced into surrounding tissue. The
methods used in producing the foregoing vector modifications and
development procedures may be accomplished according to methods
known by those skilled in the art.
[0043] From this work those skilled in the art will recognize that
one of the utilities of the instant invention is to provide a
system for in vivo as well as in vitro testing and analysis so that
a correlation of M.tb sequence diversity with CTL and T-cell
proliferative responses, as well as other parameters can be made.
The isolation and cloning of these various genes may be
accomplished according to methods known to those skilled in the
art. This invention further provides a method for systematic
identification of M.tb strains and sequences for vaccine
production. Incorporation of genes from primary isolates of M.tb
strains provides an immunogen which induces immune responses
against clinical isolates of the organism and thus meets a need as
yet unmet in the field. Furthermore, if the virulent isolates
change, the immunogen may be modified to reflect new sequences as
necessary.
[0044] In one embodiment of this invention, a gene encoding an M.tb
protein is directly linked to a transcriptional promoter. The use
of tissue-specific promoters or enhancers, for example the muscle
creatine kinase (MCK) enhancer element may be desirable to limit
expression of the polynucleotide to a particular tissue type. For
example, myocytes are terminally differentiated cells which do not
divide. Integration of foreign DNA into chromosomes appears to
require both cell division and protein synthesis. Thus, limiting
protein expression to non-dividing cells such as myocytes may be
preferable. However, use of the CMV promoter is adequate for
achieving expression in many tissues into which the PNV is
introduced.
[0045] M.tb and other genes are preferably ligated into an
expression vector which has been specifically optimized for
polynucleotide vaccinations. Elements include a transcriptional
promoter, immunogenic epitopes, and additional cistrons encoding
immunoenhancing or immunomodulatory genes, with their own
promoters, transcriptional terminator, bacterial origin of
replication and antibiotic resistance gene, as described herein.
Optionally, the vector may contain internal ribosome entry sites
(IRES) for the expression of polycistronic mRNA. Those skilled in
the art will appreciate that RNA which has been transcribed in
vitro to produce multi-cistronic mRNAs encoded by the DNA
counterparts is within the scope of this invention. For this
purpose, it is desirable to use as the transcriptional promoter
such powerful RNA polymerase promoters as the T7 or SP6 promoters,
and performing in vitro run-on transcription with a linearized DNA
template. These methods are well known in the art.
[0046] The protective efficacy of polynucleotide M.tb immunogens
against subsequent challenge is demonstrated by immunization with
the DNA of this invention. This is advantageous since no infectious
agent is involved, no assembly/replication of bacteria is required,
and determinant selection is permitted. Furthermore, because the
sequence of mycobacterial gene products may be conserved among
various strains of M.tb, protection against subsequent challenge by
another strain of M.tb is obtained.
[0047] The injection of a DNA expression vector encoding antigen
85A, B or C may result in the generation of significant protective
immunity against subsequent challenge. In particular, specific CTLs
and helper T lymphocyte responses may be produced.
[0048] Because each of the M.tb gene products exhibit a high degree
of conservation among the various strains of M.tb and because
immune responses may be generated in response to intracellular
expression and MHC processing, it is expected that many different
M.tb PNV constructs may give rise to cross reactive immune
responses.
[0049] The invention offers a means to induce heterologous
protective immunity without the need for self-replicating agents or
adjuvants. The generation of high titer antibodies against
expressed proteins after injection of viral protein and human
growth hormone DNA, [Tang et al., Nature 356, 152, 1992], indicates
this is a facile and highly effective means of making
antibody-based vaccines, either separately or in combination with
cytotoxic T-lymphocyte and helper T lymphocyte vaccines targeted
towards conserved antigens.
[0050] The ease of producing and purifying DNA constructs compares
favorably with traditional protein purification, facilitating the
generation of combination vaccines. Thus, multiple constructs, for
example encoding antigen 85 complex genes and any other M.tb gene
also including non-M.tb genes may be prepared, mixed and
co-administered. Additionally, protein expression is maintained
following DNA injection [H. Lin et al., Circulation 82, 2217
(1990); R. N. Kitsis et al., Proc. Natl. Acad. Sci. (USA) 88, 4138
(1991); E. Hansen et al., FEBS Lett. 290, 73 (1991); S. Jiao et
al., Hum. Gene Therapy 3, 21 (1992); J. A. Wolff et al., Human Mol.
Genet. 1, 363 (1992)], the persistence of B- and T-cell memory may
be enhanced [D. Gray and P. Matzinger, J. Exp. Med. 174, 969
(1991); S. Oehen et al., ibid. 176, 1273 (1992)], thereby
engendering long-lived humoral and cell-mediated immunity.
[0051] The amount of expressible DNA or transcribed RNA to be
introduced into a vaccine recipient will have a very broad dosage
range and may depend on the strength of the transcriptional and
translational promoters used. In addition, the magnitude of the
immune response may depend on the level of protein expression and
on the immunogenicity of the expressed gene product. In general, an
effective dose ranges of about 1 ng to 5 mg, 100 ng to 2.5 mg, 1
.mu.g to 750 .mu.g, and preferably about 10 .mu.g to 300 .mu.g of
DNA is administered directly into muscle tissue. Subcutaneous
injection, intradermal introduction, impression through the skin,
and other modes of administration such as intraperitoneal,
intravenous, or inhalation delivery are also suitable. It is also
contemplated that booster vaccinations may be provided. Following
vaccination with M.tb polynucleotide immunogen, boosting with M.tb
protein immunogens such as the antigen 85 complex gene products is
also contemplated. Parenteral administration, such as intravenous,
intramuscular, subcutaneous or other means of administration of
interleukin-12 protein (or other cytokines, e.g. GM-CSF),
concurrently with or subsequent to parenteral introduction of the
PNV of this invention may be advantageous.
[0052] The polynucleotide may be naked, that is, unassociated with
any proteins, adjuvants or other agents which affect the
recipients' immune system. In this case, it is desirable for the
polycucleotide to be in a physiologically acceptable solution, such
as, but not limited to, sterile saline or sterile buffered saline.
Alternatively, the DNA may be associated with liposomes, such as
lecithin liposomes or other liposomes known in the art, as a
DNA-liposome mixture, or the DNA may be associated with an adjuvant
known in the art to boost immune responses, such as a protein or
other carrier. Agents which assist in the cellular uptake of DNA,
such as, but not limited to, calcium ions, may also be used. These
agents are generally referred to herein as transfection
facilitating reagents and pharmaceutically acceptable carriers.
Techniques for coating microprojectiles coated with polynucleotide
are known in the art and are also useful in connection with this
invention. For DNA intended for human use it may be useful to have
the final DNA product in a pharmaceutically acceptable carrier or
buffer solution. Pharmaceutically acceptable carriers or buffer
solutions are known in the art and include those described in a
variety of texts such as Remington's Pharmaceutical Sciences.
[0053] In another embodiment, the invention is a polynucleotide
which comprises contiguous nucleic acid sequences capable of being
expressed to produce a gene product upon introduction of said
polynucleotide into eukaryotic tissues in vivo. The encoded gene
product preferably either acts as an immunostimulant or as an
antigen capable of generating an immune response. Thus, the nucleic
acid sequences in this embodiment encode an M.tb immunogenic
epitope, and optionally a cytokine or a T-cell costimulatory
element, such as a member of the B7 family of proteins.
[0054] There are several advantages of immunization with a gene
rather than its gene product. The first is the relative simplicity
with which native or nearly native antigen can be presented to the
immune system. Mammalian proteins expressed recombinantly in
bacteria, yeast, or even mammalian cells often require extensive
treatment to insure appropriate antigenicity. A second advantage of
DNA immunization is the potential for the immunogen to enter the
MHC class I pathway and evoke a cytotoxic T cell response.
Immunization of mice with DNA encoding the influenza A
nucleoprotein (NP) elicited a CD8.sup.+ response to NP that
protected mice against challenge with heterologous strains of flu.
(Montgomery, D. L. et al., supra; Ulmer, J. et al., supra)
[0055] There is strong evidence that cell-mediated immunity is
important in controlling M.tb infection [Orme et al, 1993, J.
Infect. Dis. 167, 1481; Cooper et al 1993, J. Exp. Med. 178, 2243;
Flynn et al, 1993, J. Exp. Med. 178, 2249; Orme et al, 1993, J.
Immunol. 151, 518]. Since DNA immunization can evoke both humoral
and cell-mediated immune responses, its greatest advantage may be
that it provides a relatively simple method to survey a large
number of M.tb genes for their vaccine potential.
[0056] Immunization by DNA injection also allows, as discussed
above, the ready assembly of multicomponent subunit vaccines.
Simultaneous immunization with multiple influenza genes has
recently been reported. (Donnelly, J. et al., 1994, Vaccines, pp
55-59). The inclusion in an M.tb vaccine of genes whose products
activate different arms of the immune system may also provide
thorough protection from subsequent challenge.
[0057] The vaccines of the present invention are useful for
administration to domesticated or agricultural animals, as well as
humans. Vaccines of the present invention may be used to prevent
and/or combat infection of any agricultural animals, including but
not limited to, dairy cattle, which are susceptible to
Mycobacterial infection. The techniques for administering these
vaccines to animals and humans are known to those skilled in the
veterinary and human health fields, respectively.
[0058] The following examples are provided to illustrate the
present invention without, however, limiting the same thereto.
EXAMPLE 1
[0059] Vectors for Vaccine Production
[0060] A) V1 Expression Vector
[0061] The expression vector V1 was constructed from
pCMVIE-AKI-DHFR [Y. Whang et al., J. Virol. 61, 1796 (1987)]. The
AKI and DHFR genes were removed by cutting the vector with EcoR I
and self-ligating. This vector does not contain intron A in the CMV
promoter, so it was added as a PCR fragment that had a deleted
internal Sac I site [at 1855 as numbered in B. S. Chapman et al.,
Nuc. Acids Res. 19, 3979 (1991)]. The template used for the PCR
reactions was pCMVintA-Lux, made by ligating the Hind III and Nhe I
fragment from pCMV6a12O [see B. S. Chapman et al., ibid.,] which
includes hCMV-IE1 enhancer/promoter and intron A, into the Hind III
and Xba I sites of pBL3 to generate pCMVIntBL. The 1881 base pair
luciferase gene fragment (Hind III-Sma I Klenow filled-in) from
RSV-Lux [J. R. de Wet et al., Mol. Cell Biol. 7, 725, 1987] was
cloned into the Sal I site of pCMVIntBL, which was Klenow filled-in
and phosphatase treated.
[0062] The primers that spanned intron A are:
[0063] 5' primer, SEQ. ID:1: 5'-CTATATAAGCAGAG CTCGTTTAG-3'; The 3'
primer, SEQ ID:2: 5'-GTAGCAAAGATCTAAGGACGGTGA CTGCAG-3'.
[0064] The primers used to remove the Sac I site are:
[0065] sense primer, SEQ ID:3:
5-GTATGTGTCTGAAAATGAGCGTGGAGATTGGGCTCGCAC-3- ' and the antisense
primer, SEQ ID:4: 5'-GTGCGAGCCCAATCTCCACGCTCATTTTCAGAC- ACA
TAC-3'.
[0066] The PCR fragment was cut with Sac I and Bgl II and inserted
into the vector which had been cut with the same enzymes.
[0067] B) V1J Expression Vector
[0068] The purpose in creating V1J was to remove the promoter and
transcription termination elements from vector V1 in order to place
them within a more defined context, create a more compact vector,
and to improve plasmid purification yields.
[0069] V1J is derived from vectors V1 and pUC18, a commercially
available plasmid. V1 was digested with SspI and EcoRI restriction
enzymes producing two fragments of DNA. The smaller of these
fragments, containing the CMVintA promoter and Bovine Growth
Hormone (BGH) transcription termination elements which control the
expression of heterologous genes, was purified from an agarose
electrophoresis gel. The ends of this DNA fragment were then
"blunted" using the T4 DNA polymerase enzyme in order to facilitate
its ligation to another "blunt-ended" DNA fragment.
[0070] pUC18 was chosen to provide the "backbone" of the expression
vector. It is known to produce high yields of plasmid, is
well-characterized by sequence and function, and is of small size.
The entire lac operon was removed from this vector by partial
digestion with the HaeII restriction enzyme. The remaining plasmid
was purified from an agarose electrophoresis gel, blunt-ended with
the T4 DNA polymerase treated with calf intestinal alkaline
phosphatase, and ligated to the CMVintA/BGH element described
above. Plasmids exhibiting either of two possible orientations of
the promoter elements within the pUC backbone were obtained. One of
these plasmids gave much higher yields of DNA in E. coli and was
designated V1J. This vector's structure was verified by sequence
analysis of the junction regions and was subsequently demonstrated
to give comparable or higher expression of heterologous genes
compared with V1.
[0071] C) V1Jneo Expression Vector
[0072] It was necessary to remove the amp.sup.r gene used for
antibiotic selection of bacteria harboring V1J because ampicillin
may not be desirable in large-scale fermenters. The amp.sup.r gene
from the pUC backbone of V1J was removed by digestion with SspI and
Eam1 1051 restriction enzymes. The remaining plasmid was purified
by agarose gel electrophoresis, blunt-ended with T4 DNA polymerase,
and then treated with calf intestinal alkaline phosphatase. The
commercially available kan.sup.r gene, derived from transposon 903
and contained within the pUC4K plasmid, was excised using the PstI
restriction enzyme, purified by agarose gel electrophoresis, and
blunt-ended with T4 DNA polymerase. This fragment was ligated with
the V1J backbone and plasmids with the kan.sup.r gene in either
orientation were derived which were designated as V1Jneo #'s 1 and
3. Each of these plasmids was confirmed by restriction enzyme
digestion analysis, DNA sequencing of the junction regions, and was
shown to produce similar quantities of plasmid as V1J. Expression
of heterologous gene products was also comparable to V1J for these
V1Jneo vectors. V1Jneo#3, referred to as V1Jneo hereafter, was
selected which contains the kan.sup.r gene in the same orientation
as the amp.sup.r gene in V1J as the expression construct.
[0073] D) V1Jns Expression Vector
[0074] An Sfi I site was added to V1Jneo to facilitate integration
studies. A commercially available 13 base pair Sfi I linker (New
England BioLabs) was added at the Kpn I site within the BGH
sequence of the vector. V1Jneo was linearized with Kpn I, gel
purified, blunted by T4 DNA polymerase, and ligated to the blunt
Sfi I linker. Clonal isolates were chosen by restriction mapping
and verified by sequencing through the linker. The new vector was
designated V1Jns. Expression of heterologous genes in V1Jns (with
Sfi I) was comparable to expression of the same genes in V1Jneo
(with Kpn I).
[0075] E) V1Jns-tPA
[0076] In order to provide an heterologous leader peptide sequence
to secreted and/or membrane proteins, V1Jns was modified to include
the human tissue-specific plasminogen activator (tPA) leader. Two
synthetic complementary oligomers were annealed and then ligated
into V1Jn which had been BglII digested. The sense and antisense
oligomers were 5'-GATC ACC ATG GAT GCA ATG AAG AGA GGG CTC TGC TGT
GTG CTG CTG CTG TGT GGA GCA GTC TTC GTT TCG CCC AGC GA-3', SEQ.
ID:5:, and 5'-GAT CTC GCT GGG CGA AAC GAA GAC TGC TCC ACA CAG CAG
CAG CAC ACA GCA GAG CCC TCT CTT CAT TGC ATC CAT GGT-3', SEQ. ID:6.
The Kozak sequence is underlined in the sense oligomer. These
oligomers have overhanging bases compatible for ligation to
BglII-cleaved sequences. After ligation the upstream BglII site is
destroyed while the downstream BglII is retained for subsequent
ligations. Both the junction sites as well as the entire tPA leader
sequence were verified by DNA sequencing. Additionally, in order to
conform with the consensus optimized vector V1Jns (=V1Jneo with an
SfiI site), an SfiI restriction site was placed at the KpnI site
within the BGH terminator region of V1Jn-tPA by blunting the KpnI
site with T4 DNA polymerase followed by ligation with an SfiI
linker (catalogue #1138, New England Biolabs). This modification
was verified by restriction digestion and agarose gel
electrophoresis.
[0077] F) pGEM-3-X-IRES-B7
[0078] (where X=any antigenic gene) As an example of a dicistronic
vaccine construct which provides coordinate expression of a gene
encoding an immunogen and a gene encoding an immuno-stimulatory
protein, the murine B7 gene was PCR amplified from the B lymphoma
cell line CH1 (obtained from the ATCC). B7 is a member of a family
of proteins which provide essential costimulation T cell activation
by antigen in the context of major histocompatibility complexes I
and II. CH1 cells provide a good source of B7 mRNA because they
have the phenotype of being constitutively activated and B7 is
expressed primarily by activated antigen presenting cells such as B
cells and macrophages. These cells were further stimulated in vitro
using cAMP or IL-4 and mRNA prepared using standard guanidinium
thiocyanate procedures. cDNA synthesis was performed using this
mRNA using the GeneAmp RNA PCR kit (Perkin-Elmer Cetus) and a
priming oligomer (5'-GTA CCT CAT GAG CCA CAT AAT ACC ATG-3', SEQ.
ID:7: ) specific for B7 located downstream of the B7 translational
open reading frame. B7 was amplified by PCR using the following
sense and antisense PCR oligomers: 5'-GGT ACA AGA TCT ACC ATG GCT
TGC AAT TGT CAG TTG ATG C-3', SEQ. ID:8:, and 5'-CCA CAT AGA TCT
CCA TGG GAA CTA AAG GAA GAC GGT CTG TTC-3', SEQ. ID:9:,
respectively. These oligomers provide BglII restriction enzyme
sites at the ends of the insert as well as a Kozak translation
initiation sequence containing an NcoI restriction site and an
additional NcoI site located immediately prior to the 3'-terminal
BglII site. NcoI digestion yielded a fragment suitable for cloning
into pGEM-3-IRES which had been digested with NcoI. The resulting
vector, pGEM-3-IRES-B7, contains an IRES-B7 cassette which can
easily be transferred to V1Jns-X, where X represents an
antigen-encoding gene.
[0079] G) pGEM-3-X-IRES-GM-CSF
[0080] (where X=any antigenic gene) This vector contains a cassette
analogous to that described in item C above except that the gene
for the immunostimulatory cytokine, GM-CSF, is used rather than B7.
GM-CSF is a macrophage differentiation and stimulation cytokine
which has been shown to elicit potent anti-tumor T cell activities
in vivo [G. Dranoff et al., Proc. Natl. Acad. Sci. USA, 90, 3539
(1993).
[0081] H) pGEM-3-X-IRES-IL-12
[0082] (where X=any antigenic gene) This vector contains a cassette
analogous to that described in item C above except that the gene
for the immunostimulatory cytokine, IL-12, is used rather than B7.
IL-12 has been demonstrated to have an influential role in shifting
immune responses towards cellular, T cell-dominated pathways as
opposed to humoral responses [L. Alfonso et al., Science, 263, 235,
1994].
EXAMPLE 2
[0083] Vector V1R Preparation
[0084] In an effort to continue to optimize the basic vaccination
vector, a derivative of V1Jns, designated V1R, was prepared. The
purpose for this vector construction was to obtain a minimum-sized
vaccine vector without unneeded DNA sequences, which still retained
the overall optimized heterologous gene expression characteristics
and high plasmid yields that V1J and V1Jns afford. It was
determined from the literature as well as by experiment that (1)
regions within the pUC backbone comprising the E. coli origin of
replication could be removed without affecting plasmid yield from
bacteria; (2) the 3'-region of the kan.sup.r gene following the
kanamycin open reading frame could be removed if a bacterial
terminator was inserted in its place; and, (3) .about.300 bp from
the 3'-half of the BGH terminator could be removed without
affecting its regulatory function (following the original KpnI
restriction enzyme site within the BGH element).
[0085] V1R was constructed by using PCR to synthesize three
segments of DNA from V1Jns representing the CMVintA promoter/BGH
terminator, origin of replication, and kanamycin resistance
elements, respectively. Restriction enzymes unique for each segment
were added to each segment end using the PCR oligomers: SspI and
XhoI for CMVintA/BGH; EcoRV and BamHI for the kan.sup.r gene; and,
BclI and SalI for the ori.sup.r. These enzyme sites were chosen
because they allow directional ligation of each of the PCR-derived
DNA segments with subsequent loss of each site: EcoRV and SspI
leave blunt-ended DNAs which are compatible for ligation while
BamHI and BclI leave complementary overhangs as do SalI and XhoI.
After obtaining these segments by PCR each segment was digested
with the appropriate restriction enzymes indicated above and then
ligated together in a single reaction mixture containing all three
DNA segments. The 5'-end of the ori.sup.r was designed to include
the T2 rho independent terminator sequence that is normally found
in this region so that it could provide termination information for
the kanamycin resistance gene. The ligated product was confirmed by
restriction enzyme digestion (>8 enzymes) as well as by DNA
sequencing of the ligation junctions. DNA plasmid yields and
heterologous expression using viral genes within V1R appear similar
to V1Jns. The net reduction in vector size achieved was 1346 bp
(V1Jns=4.86 kb; V1R=3.52 kb).
[0086] PCR oligomer sequences used to synthesize V1R (restriction
enzyme sites are underlined and identified in brackets following
sequence):
[0087] (1) 5'-GGT ACA AAT ATT GG CTA TTG GCC ATT GCA TAC G-3'
[SspI], SEQ.ID:10:,
[0088] (2) 5'-CCA CAT CTC GAG GAA CCG GGT CAA TTC TTC AGC ACC-3'
[XhoI], SEQ.ID:11:
[0089] (for CMVintA/BGH segment)
[0090] (3) 5'-GGT ACA GAT ATC GGA AAG CCA CGT TGT GTC TCA AAA
TC-3'[EcoRV], SEQ.ID:12:
[0091] (4) 5'-CCA CAT GGA TCC G TAA TGC TCT GCC AGT GTT ACA ACC-3'
[BamHI], SEQ.ID:13:
[0092] (for kanamycin resistance gene segment)
[0093] (5) 5'-GGT ACA TGA TCA CGT AGA AAA GAT CAA AGG ATC TTC
TTG-3'[BclI], SEQ.ID:14:,
[0094] (6) 5'-CCA CAT GTC GAC CC GTA AAA AGG CCG CGT TGC TGG-3'
[SalI], SEQ.ID:15:
[0095] (for E. coli origin of replication)
EXAMPLE 3
[0096] Cell Culture and Transfection
[0097] For preparation of stably transfected cell lines expressing
M.tb antigens RD cells (human rhabdomyosarcoma ATCC CCL 136) were
grown at 37.degree. C., 5% CO.sub.2 in Dulbecco's modified Eagle's
medium (DMEM) supplemented with 10% heat inactivated fetal bovine
serum, 20 mM HEPES, 4 mM L-glutamine, and 100 .mu.g/mL each of
penicillin and streptomycin. Cells were seeded at
1.5.times.10.sup.6 cells/100 mm.sup.2 plate and grown for 18 hours.
Cell were transfected with 10 .mu.g/plate of the TB construct and
10 .mu.g of co-transfected Cat construct using the CellPhect kit
(Pharmacia), and glycerol shocked (15% glycerol in PBS, pH 7.2 for
2.5 min) 5 hours after DNA was added to the cells. Cultures were
harvested 72 hours after transfection by washing the plates
2.times.-10 mL of cold PBS, pH 7.2, adding 5 mL of cold TEN buffer
(40 mM TRIS-Cl, pH 7.5, 1 mM EDTA, 150 mM NaCl) and scraping. For
analysis of protein expression, cell pellets were lysed in 50 .mu.L
of Single Detergent Lysis Buffer (50 mM Tris-Cl, pH 8.0, 150 mM
NaCl, 0.02% NaN3, 1%Nonidet P-40, 100 mM PMSF, 2 .mu.g/mL
aprotinin, 2 .mu.g/mL leupeptin, and 1 .mu.g/mL Pepstatin A) and
sonicated on ice (2-15 second bursts). Lysates were centrifuged at
13,000.times. g, 4.degree. C., for 10 minutes. Protein
concentration was determined by the Bradford method and 20 .mu.g of
cell extract protein per lane was applied to a 10% TRIS-glycine
polyacrylamide gel (Novex), then transferred to inmobilon P
(Millipore) membrane. Immunoblots were reacted overnight with a
1:20 dilution of the mouse monoclonal antibody TD 17-4 [Huygen et
al, 1994, Infect. Immunity 62, 363], followed by a 1.5 hours
reaction with a 1:1000 dilution of goat anti-mouse IgGFc peroxidase
(Jackson). The blots were developed using the ECL kit
(Amersham).
EXAMPLE 4
[0098] Cloning and DNA preparation
[0099] 1. Construction of V1Jns-tPA-85A (contains mature Ag85A with
tPA signal sequence) was done using the following primers:
[0100] sense 85A.C1 primer [SEQ.ID.NO.:16]
[0101] GG AAG ATC TTT TCC CGG CCG GGC TTG CCG
[0102] Bgl II
[0103] antisense 85A primer [SEQ.ID.NO.:17]
[0104] GGAAGATCTTGTCTGTTCGGAGCTAGGC.
[0105] The Ag85A from M. tuberculosis was amplified from plasmid
p85A.tub, which was prepared by ligating an 800 bp HindIII fragment
to a 1600 bp HindIII-SphII fragment from FIG. 2 of Borremans et al,
1989 [Infect. Immunity 57, 3123]. The resulting 2400 bp insert was
subcloned in the HindIII and SphI sites of the BlueScribe Ml
3.sup.+. The entire coding sequence and flanking regions in
BlueScribe M13+ (VCS/Stratagene) were amplified by PCR with the
indicated primers in the following conditions. Each 100 .mu.l
reaction contains 2.5 Units Cloned Pfu DNA Polymerase (Stratagene),
200 mM dNTP, 0.5 .mu.g of each primer and 250 ng of template DNA in
the reaction buffer supplied with the enzyme (Stratagene). The
Hybaid Thermal Reactor was programmed as follows: 5 minutes
denaturation at 94.degree. C. followed by 25 cycles (1 minute at
94.degree. C., 2 minutes at 55.degree. C. and 3 minutes at
72.degree. C.) ending with 10 minutes extension at 72.degree.
C.
[0106] Amplified DNA was digested with 50 .mu.g/ml Proteinase K
(Boehringer Mannheim) for 30 minutes at 37.degree. C., heated 10
minutes at 95.degree. C. followed by 2 phenol (Chloroform-Isoamyl
alcohol) extractions and precipitated with 1 volume of isopropanol,
washed twice with 70% ethanol, dried and dissolved in 20 .mu.l
H.sub.2O. 3 .mu.g of amplified DNA was digested with 40 Units of
Bgl II (Boehringer Mannheim) and the 907 bp fragment (in the case
of 85A-C1) was isolated on a 1% agarose gel and extracted on "Prep
a Gene" (BioRad) following the manufacturer's instructions.
[0107] Fifty ng of this fragment was ligated to 20 ng of the Bgl II
digested and dephosphorylated V1Jns.tPA vector in a 10 .mu.l
reaction containing 2.5 Units T4 DNA ligase (Amersham) in ligation
buffer for 16 hours at 14.degree. C., transformed into competent
DH5 E. coli (BRL) and plated on Kanamycin (50 .mu.g/ml) containing
LB Agar medium. Transformants were picked up and their plasmidic
DNA was restricted with Bgl II (to confirm the presence of insert)
and with Pvu II to define its orientation.
[0108] 2. Construction of V1Jns-85A [C2] (contains mature Ag85A
with no signal sequence) was done using the following primers:
[0109] Sense 85A C2 [SEQ.ID.NO.:18]
[0110] GGAAGATCTACC ATG GGC TTT TCC CGG CCG GGC TTG C
[0111] Antisense 85A [SEQ.ID.NO.:17]
[0112] GGAAGATCTTGCTGTTCGGAGCTAGGC.
[0113] The same procedure as 1 above was followed, except that
cloning was in V1Jns.
[0114] 3. Construction of V1Jns-85A [C3] (contains Ag85A with its
own signal sequence) was done using the primers:
[0115] Sense 85A C3 [SEQ.ID.NO.:19]
[0116] GGAAGATCTACC ATG GCA CAG CTT GTT GAC AGG GTT
[0117] Antisense 85A [SEQ.ID.NO.:17]
[0118] GGAAGATCTTGCTGTTCGGAGCTAGGC.
[0119] The same procedure as 1 above was followed, except that
cloning was in V1Jns.
[0120] 4. Construction of V1Jns-tPA-85B [C1] (contains Ag85B with
tPA signal sequence) was done using the following primers:
[0121] Sense 85B [C1] [SEQ.ID.NO.:20]
[0122] GGAAG ATC TCC TTC TCC CGG CCG GGG CTG CCG GTC GAG
[0123] Antisense 85B [SEQ.ID.NO.:21]
[0124] GGAAGATCTAACCTTCGGTTGATCCCGTCAGCC.
[0125] The same procedure as 1 above was followed, except that the
template for PCR was p85B.tub.
[0126] 5. Construction of V1Jns-tPA-85C [C1] (contains Ag85C with
tPA signal sequence) was done using the following primers:
[0127] Sense 85C [C1] [SEQ.ID.NO.:22]
[0128] GGAAG ATC TCC TTC TCT AGG CCC GGT CTT CCA
[0129] Antisense 85C [SEQ.ID.NO.:23]
[0130] GGAAGATCTTGCCGATGCTGGCTTGCTGGCTCAGGC.
[0131] The same procedure as 1 above was followed, except that the
template for PCR was p85C.tub.
[0132] 6. Construction of V1Jns-85B [C2] (contains Ag85B with no
signal sequence) is done using the following primers:
[0133] Sense 85B [C2] [SEQ.ID.NO.:24]
[0134] GGA AGA TCT ACC ATG GGC TTC TCC CGG CCG GGG CTG C
[0135] Antisense 85B [SEQ.ID.NO.:21]
[0136] GGAAGATCTAACCTCGGTTGATCCCGTCAGCC.
[0137] The same procedure as 1 above is followed, except that
template for PCR is p85B.tub and that cloning is in V1Jns.
[0138] 7. Construction of V1Jns-85C [C2] (contains Ag85C with no
signal sequence) is done using the following primers:
[0139] Sense 85C [C2] [SEQ.ID.NO.:25]
[0140] GGA AGA TCT ACC ATG GGC TTC TCT AGG CCC GGT CTT C
[0141] Antisense 85C [SEQ.ID.NO.:23]
[0142] GGAAGATCTTGCCGATGCTGGCTTGCTGGCTCAGGC.
[0143] The same procedure as 1 above is followed, except that
template for PCR is p85C.tub and that cloning is in V1Jns.
[0144] After restriction analysis all of the constructions are
partially sequenced across the vector junctions. Large scale DNA
preparation was essentially as described (Montgomery, D. L. et al.,
supra).
[0145] The plasmid constructions were characterized by restriction
mapping and sequence analysis of the vector-insert junctions (see
FIGS. 1-6). Results were consistent with published M.tb sequence
data and showed that the initiation codon was intact for each
construct (FIG. 7). Also shown are the various additional amino
acid residues unrelated to M.tb Ag85 that were inserted as a result
of cloning.
EXAMPLE 5
[0146] Expression of M.tb Proteins from V1Jns.tPA Plasmids
[0147] Rhabdomyosarcoma cells (ATCC CCL136) were planted one day
before use at a density of 1.2.times.10.sup.6 cells per 9.5
cm.sup.2 well in six-well tissue culture clusters in high glucose
DMEM supplemented with 10% heat-inactivated fetal calf serum, 2 mM
L-glutamine, 25 mM HEPES, 50 U/ml penicillin and 50 .mu.g/ml
streptomycin. (All from BRL-Gibco) Phenol chloroform extracted
cesium chloride purified plasmid DNA was precipitated with calcium
phosphate using Pharmacia CellPhect reagents according to the kit
instructions except that 5-15 .mu.g is used for each 9.5 cm.sup.2
well of RD cells. Cultures were glycerol shocked six hours post
addition of calcium phosphate-DNA precipate; after refeeding,
cultures were incubated for two days prior to harvest.
[0148] Lysates of transfected cultures were prepared in 1.times.
RIPA (0.5% SDS, 1.0% TRITON X-100, 1% sodium deoxycholate, 1 mM
EDTA, 150 mM NaCl, 25 mM TRIS-HCl pH 7.4) supplemented with 1 .mu.M
leupeptin, 1 .mu.M pepstatin, 300 nM aprotinin, and 10 .mu.M TLCK,
and sonicated briefly to reduce viscosity. Lysates were resolved by
electrophoresis on 10% Tricine gels (Novex) and then transferred to
nitrocellulose membranes. Immunoblots were processed with M.tb
monoclonal antibodies 17/4 and 32/15 [Huygen et al, 1994, Infect.
Immunity 62, 363] and developed with the ECL detection kit
(Amersham).
[0149] Expression of M.tb antigen 85 complex genes was demonstrated
by transient transfection of RD cells. Lysates of transfected or
mock transfected cells were fractionated by SDS PAGE and analyzed
by immunoblotting. FIG. 8 shows that V1Jns.tPA-85A(C1),
V1Jns.tPA-85A(C2), V1Jns.tPA-85A(C3), and V1Jns.tPA-85B(C1)
transfected RD cells express an immunoreactive protein with an
apparent molecular weight of approximately 30-32 kDa.
EXAMPLE 6
[0150] Immunization with PNV and Expression of Antigen 85 Proteins
in Vivo
[0151] Five- to six-week-old female BALB/c and C57BL/6 mice were
anesthetized by intraperitoneal (i.p.) injection of a mixture of 5
mg ketamine HCl (Aveco, Fort Dodge, Iowa) and 0.5 mg xylazine
(Mobley Corp., Shawnee, Kans.) in saline. The hind legs were washed
with 70% ethanol. Animals were injected three times with 100 .mu.l
of DNA (2 mg/ml) suspended in saline: 50 .mu.l each leg. At 17-18
days after immunization, serum samples were collected and analyzed
for the presence of anti-Ag85 antibodies. FIG. 9 shows specific
immunoblot reactivity of sera from Ag85 DNA-injected mice (C1) but
not from mice that received a control DNA not containing a gene
insert (V1J). Reactivity was detected to a serum dilution of at
least 1:160 against 300 ng of purified antigen 85A (FIG. 9b). This
demonstrates that injection of Ag85 DNA resulted in Ag85 expression
in vivo such that it was available for the generation of antibody
responses in both BALB/c and C57BL/6 (B6) mice.
EXAMPLE 7
[0152] Antigen 85-Specific T-Cell Responses
[0153] Spleen cells from vaccinated mice were analyzed for cytokine
secretion in response to specific antigen restimulation as
described in Huygen et al, 1992 [Infect. Immunity 60, 2880].
Specifically, spleen cells were incubated with culture filtrate
(CF) proteins from M. bovis BCG purified antigen 85A or a 20-mer
peptide (p25) corresponding to a known T-cell epitope for C57BL/6
mice (amino acids 241-260). Mice were immunized with V1Jns.tPA85A
(C1) (100 .mu.g) three times with three week intervals and analyzed
17 days after the final injection. Cytokines were assayed using
bio-assays for IL-2, interferon-.gamma. (IFN-.gamma.) and IL-6, and
by ELISA for IL-4 and IL-10. Substantial IL-2 and IFN-.gamma.
production was observed in both BALB/c and C57BL/6 mice vaccinated
with V1Jns.tPA85A (Cl) (FIGS. 10-13). Furthermore, C57BL/6 mice
also reacted to the H-2b-restricted T-cell epitope (FIG. 13). IL-4,
IL-6 and IL-10 levels were not increased in V1Jns.tPA85A-vaccinated
mice (FIGS. 14-16). These results indicate that a T.sub.h1 type of
helper T-cell response was generated by the DNA vaccine.
EXAMPLE 8
[0154] Protection from Mycobacterial Challenge
[0155] To test the efficacy of an M.tb DNA vaccine, mice were
challenged with an intravenous injection of live M. bovis BCG (0.5
mg) and BCG multiplication was analyzed in the spleens and lungs.
As controls, BCG multiplication was measured in challenged naive
mice (primary infection) and challenged mice that were vaccinated
with BCG at the time of DNA injection (secondary infection). The
number of colony-forming units (CFU) in lungs of V1Jns.tPA85A
(C1)-vaccinated mice was substantially reduced compared to mice
with primary infection or mice vaccinated with control DNA V1J. In
C57BL/6 mice, CFU were reduced by 83% on day 8 after challenge
(FIG. 17) and in BALB/c mice CFU was reduced by 65% on day 20 (FIG.
18). In spleen, CFU was reduced by approximately 40% at day 20
after challenge in BALB/c mice (FIG. 19) and day 8 in C57BL/6 mice
(FIG. 20). Therefore, the immune responses observed after injection
of an M.tb DNA vaccine provided protection in a live M. bovis
challenge model.
Sequence CWU 1
1
* * * * *