U.S. patent application number 16/256436 was filed with the patent office on 2020-01-23 for live bacterial vaccines resistant to carbon dioxide (co2), acidic ph and/or osmolarity for viral infection prophylaxis or treatm.
This patent application is currently assigned to Aviex Technologies LLC. The applicant listed for this patent is David Gordon Bermudes. Invention is credited to David Gordon Bermudes.
Application Number | 20200023053 16/256436 |
Document ID | / |
Family ID | 42040155 |
Filed Date | 2020-01-23 |
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United States Patent
Application |
20200023053 |
Kind Code |
A1 |
Bermudes; David Gordon |
January 23, 2020 |
LIVE BACTERIAL VACCINES RESISTANT TO CARBON DIOXIDE (CO2), ACIDIC
PH AND/OR OSMOLARITY FOR VIRAL INFECTION PROPHYLAXIS OR
TREATMENT
Abstract
Gram-negative bacterial mutants resistant to one or more stress
conditions, including CO.sub.2, acid pH, and high osmolarity, and
more particularly to gram-negative bacterial mutants with reduced
TNF-.alpha. induction having a mutation in one or more lipid
biosynthesis genes, including, but not limited to msbB, that are
rendered stress-resistant by a mutation in the zwf gene.
Compositions are provided comprising one or more stress-resistant
gram-negative bacterial mutants, preferably attenuated
stress-resistant gram-negative bacterial mutants. Methods are
provided for prophylaxis or treatment of a virally induced disease
in a subject comprising administering to a subject a
stress-resistant gram-negative bacterial mutant, preferably
attenuated stress-resistant gram-negative bacterial mutants. The
stress-resistant gram-negative bacterial mutants may serve as
vectors for the delivery of one or more therapeutic molecules to a
host. The methods of the invention provide more efficient delivery
of therapeutic molecules by stress-resistant gram-negative
bacterial mutants engineered to express said therapeutic
molecules.
Inventors: |
Bermudes; David Gordon;
(Kenwood, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bermudes; David Gordon |
Kenwood |
CA |
US |
|
|
Assignee: |
Aviex Technologies LLC
New York
NY
|
Family ID: |
42040155 |
Appl. No.: |
16/256436 |
Filed: |
January 24, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15243904 |
Aug 22, 2016 |
10188722 |
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16256436 |
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14172272 |
Feb 4, 2014 |
9421252 |
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15243904 |
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12560947 |
Sep 16, 2009 |
8647642 |
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14172272 |
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61098174 |
Sep 18, 2008 |
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61165886 |
Apr 1, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/12 20130101;
C12N 2760/16034 20130101; A61K 2039/523 20130101; C12N 1/20
20130101; C12N 7/00 20130101; A61P 31/12 20180101; A61K 2039/522
20130101; C12N 1/36 20130101; C12N 2510/00 20130101; A61K 39/145
20130101; C12N 2760/16134 20130101; C12N 9/1025 20130101; A61P
31/16 20180101 |
International
Class: |
A61K 39/145 20060101
A61K039/145; C12N 7/00 20060101 C12N007/00; A61K 39/12 20060101
A61K039/12; C12N 9/10 20060101 C12N009/10; C12N 1/36 20060101
C12N001/36; C12N 1/20 20060101 C12N001/20 |
Claims
1. A method of treating a human or animal, comprising:
administering to the human or mammal a live genetically engineered
bacteria derived from a wild type species having an MsbB gene and a
zwf gene, the live genetically engineered bacteria having a
knockout mutation of MsbB and a knockout mutation of zwf; allowing
the live genetically engineered bacteria to replicate within and
colonize a tissue of the human or animal having a pH of pH 6.7 or
below, to cause a transient maintenance of the live genetically
engineered bacteria in the tissue; and secreting, by the live
genetically engineered bacteria, within the tissue, a heterologous
protein.
2. The method according to claim 1, further comprising clearing the
live genetically engineered bacteria from the tissue.
3. The method according to claim 1, wherein the heterologous
protein comprises an antigen adapted act as a vaccine.
4. The method according to claim 1, wherein the heterologous
protein comprises a eukaryotic-type antigen adapted act as a
vaccine.
5. The method according to claim 1, wherein the heterologous
protein comprises a fusion of a bacterial-type secretion signal and
an antigenic peptide portion.
6. The method according to claim 1, wherein the live genetically
engineered bacteria are Salmonella.
7. The method according to claim 1, wherein the wild type species
is Salmonella enterica.
8. The method according to claim 1, wherein the live genetically
engineered bacteria are zwf Salmonella YS1646 ATCC Accession No.
202165.
9. The method according to claim 1, wherein the live genetically
engineered bacteria have at least one mutation in a biosynthetic
pathway selected from the group consisting of the isoleucine
biosynthetic pathway, valine biosynthetic pathway, phenylalanine
biosynthetic pathway, tryptophan biosynthetic pathway, tyrosine
biosynthetic pathway, and arginine biosynthetic pathway.
10. A live genetically engineered bacteria derived from a wild type
species having an MsbB gene and a zwf gene, comprising: a knockout
mutation of MsbB; and a knockout mutation of zwf; at least one gene
configured to cause secretion of a heterologous protein; the live
genetically engineered bacteria being adapted to replicate within
and colonize a tissue of a human or animal having a pH of pH 6.7 or
below, to cause a transient maintenance of the live genetically
engineered bacteria in the tissue.
11. The live genetically engineered bacteria according to claim 10,
wherein the wild type species is Salmonella, and live genetically
engineered bacteria is adapted to colonize a gut of a human
recipient of the live genetically engineered bacteria.
12. The live genetically engineered bacteria according to claim 10,
wherein the heterologous protein comprises an antigen adapted to
induce a protective vaccination immune response of the human or
animal.
13. The live genetically engineered bacteria according to claim 10,
wherein the heterologous protein comprises an antigen adapted to
induce a therapeutic immune response of the human or animal.
14. The live genetically engineered bacteria according to claim 10,
wherein the live genetically engineered bacteria, are adapted after
colonization, to produce a therapeutically effective amount of the
heterologous protein.
15. The live genetically engineered bacteria according to claim 10,
wherein the live genetically engineered bacteria, are adapted after
colonization, to produce a therapeutically effective amount of the
heterologous protein to induce an immune response against an
infectious organism.
16. The live genetically engineered bacteria according to claim 10,
wherein the heterologous protein comprises a fusion protein having
at least a bacterial secretion signal and a eukaryotic-type
antigenic peptide.
17. The live genetically engineered bacteria according to claim 10,
in combination with a pharmaceutically acceptable carrier.
18. A live genetically engineered bacterium, comprising: a knockout
mutation of MsbB; and a knockout mutation of zwf; the knockout
mutation of MsbB and the knockout mutation of zwf together causing
the live genetically engineered bacterium to have resistance to
growth suppressive effects of CO.sub.2.gtoreq.5%, pH.ltoreq.6.7,
and osmolarity of .gtoreq.455 milliosmoles; at least one gene
configured to cause secretion from the live genetically engineered
bacterium of a heterologous protein; the live genetically
engineered bacteria being adapted to replicate within and colonize
a tissue of a human or animal having a pH of pH 6.7 or below, to
cause a transient maintenance of the live genetically engineered
bacteria in the tissue.
19. The live genetically engineered bacterium according to claim
18, further comprising: an attenuating mutation of at least one
gene to auxotrophy; and the heterologous protein comprises a fusion
of a therapeutic peptide portion and a secretion signal.
20. The live genetically engineered bacteria according to claim 19,
wherein the therapeutic peptide sequence portion comprises a
eukaryotic protein antigen.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a:
[0002] Continuation of U.S. patent application Ser. No. 15/243,904,
filed Aug. 22, 2016, now U.S. Pat. No. 10,188,722, issued Jan. 29,
2019, which is a
[0003] Continuation of U.S. patent application Ser. No. 14/172,272,
filed Feb. 4, 2014, now U.S. Pat. No. 9,421,252, issued Aug. 23,
2016, which is a
[0004] Continuation of U.S. patent application Ser. No. 12/560,947,
filed Sep. 16, 2009, now U.S. Pat. No. 8,647,642, issued Feb. 11,
2014, which
[0005] Claims benefit of priority from U.S. Provisional Patent
Application No. 61/165,886, filed Apr. 1, 2009, and
[0006] Claims benefit of priority from U.S. Provisional Patent
Application No. 61/098,174, filed Sep. 18, 2008,
[0007] each of which is expressly incorporated herein by
reference.
[0008] This application is also related to U.S. patent application
Ser. No. 12/562,532, filed Sep. 18, 2009, now abandoned.
FIELD OF THE INVENTION
[0009] This invention is generally in the field of live bacterial
vaccines for viral infection prophylaxis or treatment.
BACKGROUND OF THE INVENTION
[0010] Citation or identification of any reference herein, or any
section of this application shall not be construed as an admission
that such reference is available as prior art to the present
application.
[0011] There are three types of influenza viruses Influenza A, B,
and C. Influenza types A or B viruses cause epidemics of disease
almost every winter. In the United States, these winter influenza
epidemics can cause illness in 10% to 20% of people and are
associated with an average of 36,000 deaths and 114,000
hospitalizations per year. Influenza type C infections cause a mild
respiratory illness and are not thought to cause epidemics.
Influenza type A viruses are divided into subtypes based on two
proteins on the surface of the virus. These proteins are termed
hemagglutinin (H) and neuraminidase (N). Influenza A viruses are
divided into subtypes based on these two proteins. There are 16
different hemagglutinin subtypes H1, H2, H3, H4, H6, H7, H8, H9 H10
H11 H12, H13, H14, H15 or H16 and 9 different neuraminidase
subtypes N1 N2 N3 N4 N5 N6 N7 N8 or N9, all of which have been
found among influenza A viruses in wild birds. Wild birds are the
primary natural reservoir for all subtypes of Influenza A viruses
and are thought to be the source of Influenza A viruses in all
other animals. The current subtypes of influenza A viruses found in
people are A(H1N1) and A(H3N2). Influenza B virus is not divided
into subtypes.
[0012] In 1918, a new highly pathogenic influenza H1N1 pandemic
swept the world, killing an estimated 20 and 50 million people. The
H1N1 subtype circulated from 1918 until 1957 which then was
replaced by viruses of the H2N2 subtype, which continued to
circulate until 1968. Since 1968, H3N2 viruses have been found in
the population. Because H1N1 viruses returned in 1977, two
Influenza A viruses are presently co-circulating (Palese and
Garcia-Sarstre J. Clin. Invest., July 2002, Volume 110, Number 1,
9-13). The pathogenicity of the initial 1918 H1N1 has not been
equaled by any of the latter H1N1, H2N2 or H3N2 subtypes, although
infection from some subtypes can be severe and result in death. By
molecular reconstruction, the genome of the 1918 flu including the
amino acid sequences of the H1 and N1 antigens is now known
(Kaiser, Science 310: 28-29, 2005; Tumpey et al., Science 310:
77-81, 2005).
[0013] In 1997, 2003, and again in 2004, antigenically-distinct
avian H5N1 influenza viruses emerged as pandemic threats to human
beings. During each of these outbreaks there was concern that the
avian viruses would adapt to become transmissible from human to
human. Furthermore, oseltamivir (Tamiflu.RTM.) was ineffective in
50% of avian influenza patients in Thailand (Tran et al. N. Engl.
J. Med 350: 1179, 2004) and a new mutation in the neuraminidase has
been identified which causes resistance to oseltamivir. Sequence
analysis of the neuraminidase gene revealed the substitution of
tyrosine for histidine at amino acid position 274 (H274Y),
associated with high-level resistance to oseltamivir in influenza
(N1) viruses (Gubareva et al., Selection of influenza virus mutants
in experimentally infected volunteers treated with oseltamivir. J
Infect Dis 2001; 183:523-531; de Jong et al., Oseltamivir
Resistance during Treatment of Influenza A (H5N1) Infection. N.
Engl. J. Med. 353:2667-2672, 2005). Such changes may alter the
antigenic nature of the protein and reduce the effectiveness of
vaccines not matched to the new variant. Other avian influenza
strains of potential danger include H1N1, H7N7 and H9N2.
[0014] The optimum way of dealing with a human pandemic virus would
be to provide a clinically approved well-matched vaccine (i.e.,
containing the hemagglutinin and/or neuraminidase antigens of the
emerging human pandemic strain), but this cannot easily be achieved
on an adequate timescale because of the time consuming method of
conventional influenza vaccine production in chicken eggs.
[0015] Live Bacterial Vaccine Vectors
[0016] Live attenuated bacterial vaccine vectors offer an important
alternative to conventional chicken egg based vaccines. Growth on
embryonated hen eggs, followed by purification of viruses from
allantoic fluid, is the method by which influenza virus has
traditionally been grown for vaccine production. More recently,
viruses have been grown on cultured cell lines, which avoids the
need to prepare virus strains that are adapted to growth on eggs
and avoids contamination of the final vaccine with egg proteins.
However, because some of the vaccine virus may be produced in
canine tumor cells (e.g., MDCK), there is concern for contamination
of the vaccine by cancer causing elements. Moreover, both must
undergo a labor intensive and technically challenging purification
process, with a total production time of 3 to 6 months. Because of
the time factors and scale-up, these vaccines are produced in
large, but finite batches. Meeting a world-wide demand requires
stockpiling of multiple batches. Therefore, traditionally produced
vaccine produced before a pandemic, would likely be generated based
upon an avian influenza virus and its antigens more than a year
earlier and therefore may not be well matched to an emerging
variant and could result in only partial protection. Bacterial
vectors self-replicate in simple growth media can be produced
extremely rapidly by virtue of exponential growth and require
minimal purification such as a single centrifugation and
resuspension in a pharmaceutically acceptable excipient.
[0017] Human studies have shown that antibody titers against
hemagglutinin of human influenza virus are correlated with
protection (a serum sample hemagglutination-inhibition titer of
about 30-40 gives around 50% protection from infection by a
homologous virus) (Potter & Oxford (1979) Br Med Bull 35:
69-75). Antibody responses are typically measured by enzyme linked
immunosorbent assay (ELISA), immunoblotting, hemagglutination
inhibition, by microneutralisation, by single radial
immunodiffusion (SRID), and/or by single radial hemolysis (SRH).
These assay techniques are well known in the art.
[0018] Cellular responses to vaccination may also occur which
participate in antiviral immunity. Cells of the immune system are
commonly purified from blood, spleen or lymph nodes. Separate cell
populations (lymphocytes, granulocytes and monocyte/macrophages and
erythrocytes) are usually prepared by density gradient
centrifugation through Ficoll-Hypaque or Percoll solutions.
Separation is based on the buoyant density of each cell
subpopulation at the given osmolality of the solution. Monocytes
and neutrophils are also purified by selective adherence. If known
subpopulations are to be isolated, for example CD4+ or CD8+ T
cells, fluorescence activated cell sorting (FACS) will be employed
or magnetic beads coated with specific anti-CD4 or anti-CD8
monoclonal antibody are used. The beads are mixed with peripheral
blood leukocytes and only CD4+ or CD8+ cells will bind to the
beads, which are then separated out from the non-specific cells
with a magnet. Another method depends on killing the undesired
populations with specific antibodies and complement. In some cases,
a noncytotoxic antibody or other inhibitor can block the activity
of a cell subtype. Characterization of cell types and
subpopulations can be performed using markers such as specific
enzymes, cell surface proteins detected by antibody binding, cell
size or morphological identification. Purified or unseparated
lymphocytes can be activated for proliferation and DNA synthesis is
measured by .sup.3H-thymidine incorporation. Other measures of
activation such as cytokine production, expression of activation
antigens, or increase in cell size are utilized. Activation is
accomplished by incubating cells with nonspecific activators such
as Concanavalin A, phytohemagglutinin (PHA), phorbol myristic
acetate (PMA), an ionophore, an antibody to T cell receptors, or
stimulation with specific antigen to which the cells are
sensitized.
[0019] A key activity of cellular immunity reactions to pathogens
such as viruses is the development of T lymphocytes that
specifically kill target cells. These activated cells develop
during in vivo exposure or by in vitro sensitization. The CTL assay
consists of increasing number of sensitized lymphocytes cultured
with a fixed number of target cells that have been prelabeled with
.sup.51Cr. To prelabel the target cells, the cells are incubated
with the radiolabel. The .sup.51Cr is taken up and reversibly binds
to cytosolic proteins. When these target cells are incubated with
sensitized lymphocytes, the target cells are killed and the
.sup.51Cr is released.
[0020] Natural killer (NK) cells are an essential defense in the
early stage of the immune response to pathogens. NK cells are
active in naive individuals and their numbers can be enhanced in
certain circumstances. The NK assay typically uses a
.sup.51Cr-labeled target and is similar to the CTL assay described
above.
[0021] Specifically activated lymphocytes synthesize and secrete a
number of distinctive cytokines. These are quantitated by various
ELISA methods. Alternatively, induced cytokines are detected by
fluorescence activated flow cytometry (FACS) using fluorescent
antibodies that enter permeabilized cells. Activated cells also
express new cell surface antigens where the number of cells is
quantitated by immunofluorescent microscopy, flow cytometry, or
ELISA. Unique cell surface receptors that distinguish cell
populations are detected by similar immunochemical methods or by
the binding of their specific labeled ligand.
[0022] Salmonella bacteria have been recognized as being
particularly useful as live "host" vectors for orally administered
vaccines because these bacteria are enteric organisms that, when
ingested, can infect and persist in the gut (especially the
intestines) of humans and animals.
[0023] As a variety of Salmonella bacteria are known to be highly
virulent to most hosts, e.g., causing typhoid fever or severe
diarrhea in humans and other mammals, the virulence of Salmonella
bacterial strains toward an individual that is targeted to receive
a vaccine composition must be attenuated. Attenuation of virulence
of a bacterium is not restricted to the elimination or inhibition
of any particular mechanism and may be obtained by mutation of one
or more genes in the Salmonella genome (which may include
chromosomal and non-chromosomal genetic material). Thus, an
"attenuating mutation" may comprise a single site mutation or
multiple mutations that may together provide a phenotype of
attenuated virulence toward a particular host individual who is to
receive a live vaccine composition for avian influenza. In recent
years, a variety of bacteria and, particularly, serovars of
Salmonella enterica, have been developed that are attenuated for
pathogenic virulence in an individual (e.g., humans or other
mammals), and thus proposed as useful for developing various live
bacterial vaccines (see, e.g., U.S. Pat. Nos. 5,389,368; 5,468,485;
5,387,744; 5,424,065; Zhang-Barber et al., Vaccine, 17; 2538-2545
(1999); all expressly incorporated herein by reference). In the
case of strains of Salmonella, mutations at a number of genetic
loci have been shown to attenuate virulence including, but not
limited to, the genetic loci phoP, phoQ, cdt, cya, crp, poxA, rpoS,
htrA, nuoG, pmi, pabA, pts, damA, purA, purB, purI, zwf, aroA,
aroC, gua, cadA, rfc, rjb, rfa, ompR, msbB and combinations
thereof.
[0024] Bacterial flagella are known to be antigenic and subject to
antigenic or phase variation which is believed to help a small
portion of the bacteria in escaping the host immune response. The
bacterial flagellar antigens are referred to as the H1 and H2
antigens. To avoid confusion with the viral hemagglutinin H
antigen, the bacterial flagellar H antigen will be referred to as
fH henceforth. Because the Salmonella-based vaccination of a
heterologous antigen is dependent upon the bacteria's ability to
colonize the gut, which may be reduced do to the initial immune
response, the vaccination ability of the second immunization may be
diminished due to an immune response to the vector. In Salmonella
Hin invertase belongs to the recombinase family, which includes Gin
invertase from phage Mu, Cin invertase from phage P1, and
resolvases from Tn3 and the transposon (Glasgow et al. 1989., p.
637-659. In D. E. Berg, and M. M. Howe (ed.), Mobile DNA. American
Society for Microbiology, Washington, D.C.). Hin promotes the
inversion of a chromosomal DNA segment of 996 bp that is flanked by
the 26-bp DNA sequences of hixL and hixR (Johnson and Simon. 1985.
Cell 41:781-791). Hin-mediated DNA inversion in S. typhimurium
leads to the alternative expression of the fH1 and fH2 flagellin
genes known as phase variation. Hin (21 kDa) exists in solution as
a homodimer and binds to hix sites as a dimer (Glasgow et al. 1989.
J. Biol. Chem. 264:10072-10082). In addition to Hin and the two hix
sites, a cis-acting DNA sequence (recombinational enhancer) and its
binding protein (Fis, 11 KDa) are required for efficient inversion
in vitro (Johnson et al. 1986. Cell 46:531-539). Live Salmonella
vaccines have not had deletions of the hin gene nor defined fH1 or
fH2 antigens, nor have they been constructed such that they lack fH
antigens altogether. Accordingly, live Salmonella vaccines have not
been constructed to maximize a prime-boost strategy which
alternates or eliminates the fH antigen whereby the immune response
of the fH antigen of the first immunization (prime) is not specific
for the antigen of the second immunization (boost). Therefore, the
boost immunization is not diminished by a rapid elimination by the
immune system, and is therefore able to persist longer and more
effectively present the immunizing antigen.
[0025] Introduction of viral genes into bacteria results in
genetically engineered microorganisms (GEMs) for which there may be
concern regarding containment of the introduced gene in the
environment and its ability to reassort. Such genes could in theory
provide virulence factors to non-pathogenic or less pathogenic
viral strains if allowed to recombine under circumstances were the
bacterial vaccine could co-occur at the same time in the same
individual as a wild type viral infection. Thus, methods that
reduce bacterial recombination and increase bacterial genetic
isolation are desirable.
[0026] Insertion sequences (IS) are genetic elements that can
insert copies of themselves into different sites in a genome. These
elements can also mediate various chromosomal rearrangements,
including inversions, deletions and fusion of circular DNA segments
and alter the expression of adjacent genes. IS200 elements are
found in most Salmonella species. S. typhimurium strain LT2 has six
IS200s. Salmonella typhimurium strain 14028 has been described to
possess an additional IS200 element at centisome 17.7 which is
absent in other commonly studied Salmonella strains LT2 and SL1344
(Murray et al., 2004 Journal of Bacteriology, 186: 8516-8523).
These authors describe a spontaneous hot spot (high frequency)
deletion of the Cs 17.7 to Cs 19.9 region. Live Salmonella vaccines
have not had deletions of IS200 elements which would limit such
recombination events.
[0027] Salmonella strains are known to possess phage and prophage
elements. Such phage are often capable of excision and infection of
other susceptible strains and are further capable of transferring
genes from one strain by a process known as transduction. Live
Salmonella vaccines have not had deletions in phage elements such
as phage recombinases which exist in Salmonella, such that the
phage are no longer capable of excision and reinfection of other
susceptible strains.
[0028] Salmonella strains are known to be capable of being infected
by bacteria phage. Such phage have the potential to carry genetic
elements from one Salmonella strain to another. Live Salmonella
vaccines have not comprised mechanisms to limit phage infection
such as the implantation and constitutive expression of the P22
phage repressor C2.
[0029] Bacterial expression of the viral hemagglutinin genes was
first described by Heiland and Gething (Nature 292: 581-582, 1981)
and Davis et al., (Proc. Natl. Acad. Sci. USA 78: 5376-5380). These
authors suggest that the recombinant protein could be used as a
vaccine without regard to the fact that the viral genetic loci are
not optimal for bacterial expression. These authors did not suggest
the use of live bacterial vectors as vaccine carriers, such as the
genetically stabilized and isolated vectors of the present
application, nor the use of defined flagellar antigens or no
flagellar antigens. Nor did these authors suggest the use of
secreted proteins.
[0030] Use of secreted proteins in live bacterial vectors has been
demonstrated by several authors. Holland et al. (U.S. Pat. No.
5,143,830, expressly incorporated herein by reference) have
illustrated the use of fusions with the C-terminal portion of the
hemolysin A (hlyA) gene. When co-expressed in the presence of the
hemolysin protein secretion channel (hlyBD) and a functional TolC,
heterologous fusions are readily secreted from the bacteria.
Similarly, Galen et al. (Infection and Immunity 2004 72: 7096-7106)
have shown that heterologous fusions to the ClyA are secreted and
immunogenic. Other heterologous protein secretion systems include
the use of the autotransporter family. For example, Veiga et al.
(2003 Journal of Bacteriology 185: 5585-5590) demonstrated hybrid
proteins containing the .alpha.-autotransporter domain of the
immunoglogulin A (IgA) protease of Nisseria gonorrhea. Fusions to
flagellar proteins have also been shown to be immunogenic. The
antigen, a peptide, usually of 15 to 36 amino acids in length, is
inserted into the central, hypervariable region of the FliC gene
such as that from Salmonella muenchen (Verma et al. 1995 Vaccine
13: 235-24; Wu et al., 1989 Proc. Natl. Acad. Sci. USA 86:
4726-4730; Cuadro et al., 2004 Infect. Immun. 72: 2810-2816; Newton
et al., 1995, Res. Microbiol. 146: 203-216, expressly incorporated
by reference in their entirety herein). Antigenic peptides are
selected by various methods, including epitope mapping (Joys and
Schodel 1991. Infect. Immune. 59: 3330-3332; Hioe et al., 1990 J.
Virol. 64: 6246-6251; Kaverin et al. 2002, J. Gen. Virol. 83:
2497-2505; Hulse et al. 2004, J. Virol. 78: 9954-9964; Kaverin et
al. 2007, J. Virol. 81:12911-12917; Cookson and Bevan 1997, J.
Immunol. 158: 4310-4319, expressly incorporated by reference in
their entirety herein), T-cell epitope determination (Walden, 1996,
Current Opinion in Immunology 8: 68-74) and computer programs such
as Predict? (Carmenes et al. 1989 Biochem. Biophys. Res. Comm 159:
687-693) Pepitope (Mayrose et al., 2007. Bioinformatics 23:
3244-3246). Multihybrid FliC insertions of up to 302 amino acids
have also been prepared and shown to be antigenic (Tanskanen et al.
2000, Appl. Env. Microbiol. 66: 4152-4156, expressly incorporated
by reference in its entirety herein). Modification of the fusion
protein by inclusion of flanking cathepsin cleavage sites has been
used to facilitate release within the endosomal compartment of
antigen presenting cells (Verma et al. 1995 Vaccine 13: 235-244).
Trimerization of antigens has been achieved using the T4 fibritin
foldon trimerization sequence (Wei et al. 2008, J. Virology 82:
6200-6208, expressly incorporated by reference in its entirety
herein).
[0031] Bacterial expression of the viral hemagglutinin genes was
first described by Heiland and Gething (Nature 292: 581-582, 1981)
and Davis et al., (Proc. Natl. Acad. Sci. USA 78: 5376-5380). These
authors teach that the antigens may be purified from the bacteria
in order to be used as vaccines and did not suggest the use of live
attenuated bacterial vectors. Furthermore, the codon usage of the
viral genome is not optimal for bacterial expression. Accordingly,
a gram-negative bacterium of the enterobacteraceae such as E. coli
and Salmonella will have a different codon usage preference
(National Library of Medicine, National Center for Biotechnology
Information, GenBank Release 150.0 [Nov. 25, 2005]) and would not
be codon optimized. Further, these authors used
antibiotic-containing plasmids and did not use stable chromosomal
localization. Nor did these authors suggest heterologous fusions in
order for the bacteria to secrete the antigens.
[0032] Kahn et al. (EP No. 0863211) have suggested use of a live
bacterial vaccine with in vivo induction using the E. coli nitrite
reductase promoter nirB. These authors further suggest that the
antigenic determinant may be an antigenic sequence derived from a
virus, including influenza virus. However, Khan et al. did not
describe a vaccine for avian influenza virus. They did not describe
the appropriate antigens for an avian influenza virus, the
hemagglutinin and neuraminidase, and did not describe how to
genetically match an emerging avian influenza virus. Furthermore,
it has become apparent that certain assumptions, and experimental
designs described by Khan et al. regarding live avian influenza
vaccines would not be genetically isolated or have improved genetic
stability in order to provide a live vaccine for avian influenza
that would be acceptable for use in humans. For example, Khan et
al. state that any of a variety of known strains of bacteria that
have an attenuated virulence may be genetically engineered and
employed as live bacterial carriers (bacterial vectors) that
express antigen polypeptides to elicit an immune response including
attenuated strains of S. typhimurium and, for use in humans,
attenuated strains of S. typhi (i.e., S. enterica serovar Typhi).
In support of such broad teaching, they point to the importance of
"non-reverting" mutations, especially deletion mutations which
provide the attenuation. However, non-reversion only refers to the
particular gene mutated, and not to the genome per se with its
variety of IS200, phage and prophage elements capable of a variety
of genetic recombinations and/or even transductions to other
bacterial strains. Khan et al. did not describe a bacterial strain
with improved genetic stability, nor methods to reduce genetic
recombination, such as deletion of the IS200 elements. Khan et al.
did not describe a bacterial strain with improved genetic stability
by deletion of the bacteria phage and prophage elements nor
limiting their transducing capacity. Neither did Khan et al.
describe methods to minimize bacterial genetic exchange, such as
constitutive expression of the P22 C2 phage repressor.
[0033] The above comments illustrate that Khan et al. have not
provided the field with an effective vaccine against avian
influenza. Clearly, needs remain for a genetically isolated and
genetically stable, orally administered vaccine against avian
influenza which is capable of rapid genetically matching an
emerging pathogenic variant.
[0034] Bermudes (WO/2008/039408), expressly incorporated herein in
its entirety, describes live bacterial vaccines for viral infection
prophylaxis or treatment. The bacteria described are live
attenuated bacterial strains that express one or more immunogenic
polypeptide antigens of a virus. The bacteria useful for the
techniques described include Salmonella, Bordetella, Shigella,
Yersenia, Citrobacter, Enterobacter, Klebsiella, Morganella,
Proteus, Providencia, Serratia, Plesiomonas, and Aeromonas.
Bermudes describes the serovars of Salmonella enterica that may be
used as the attenuated bacterium of the live vaccine compositions
to include, without limitation, Salmonella enterica serovar
Typhimurium ("S. typhimurium"), Salmonella montevideo, Salmonella
enterica serovar Typhi ("S. typhi"), Salmonella enterica serovar
Paratyphi B ("S. paratyphi B"), Salmonella enterica serovar
Paratyphi C ("S. paratyphi C"), Salmonella enterica serovar Hadar
("S. hadar"), Salmonella enterica serovar Enteriditis ("S.
enteriditis"), Salmonella enterica serovar Kentucky ("S.
kentucky"), Salmonella enterica serovar Infantis ("S. infantis"),
Salmonella enterica serovar Pullorurn ("S. pullorum"), Salmonella
enterica serovar Gallinarum ("S. gallinarum"), Salmonella enterica
serovar Muenchen ("S. muenchen"), Salmonella enterica serovar
Anaturn ("S. anatum"), Salmonella enterica serovar Dublin ("S.
dublin"), Salmonella enterica serovar Derby ("S. derby"),
Salmonella enterica serovar Choleraesuis var. kunzendorf ("S.
cholerae kunzendorf"), and Salmonella enterica serovar minnesota
("S. minnesota").
[0035] Bermudes describes attenuating mutations useful in the
Salmonella bacterial strains which may include genetic locus
selected from the group consisting of phoP, phoQ, edt, cya, crp,
poxA, rpoS, htrA, nuoG, pmi, pabA, pts, damA, purA, purB, purI,
zwf, purF, aroA, aroB, aroC, aroD, serC, gua, cadA, rfc, rjb, rfa,
ompR, msbB and combinations thereof.
[0036] Although Bermudes discloses the msbB gene and the zwf gene,
it was not recognized that in Salmonella, the deletion of the msbB
gene confers sensitivity to carbon dioxide (CO.sub.2) and that
deletion of zwf, a member of the pentose phosphate pathway
(Fraenkel, D. G. 1996 Glycolysis, pp 189-198, In Eschericia coli
and Salmonella typhimurium, F. C. Neidehardt (ed), ASM Press,
Washington, D.C.), compensates for that deletion and restores
resistance to carbon dioxide without losing the low degree of lipid
A pyrogenicity (TNF-.alpha. induction) conferred by the msbB
mutation. Furthermore, it was also not known that the msbB.sup.-
Salmonella are also sensitive to acidic pH and osmolarity, and that
the zwf mutation also enhances resistance to acidic pH and
osmolarity. Therefore, the prior art does not teach a specific
combination of these two mutations in order to obtain CO.sub.2
resistant bacteria. Nor would one ordinarily skilled in the arts be
motivated to test for CO.sub.2 resistance in Salmonella deleted in
msbB as there is no teaching that describes the occurrence of
sensitivity or its importance. As described herein, CO.sub.2 and
acidic pH-resistant .DELTA.msbB.sup.- bacteria have improved
survival under physiological conditions advantageous for
penetration into gut mucosal, lymphoidal and dendridic tissues at
lower doses, in order to elicit an immune response to viral
diseases.
SUMMARY OF THE INVENTION
[0037] The present invention provides improved live attenuated
bacterial strains that express one or more immunogenic polypeptide
antigens of a virus, preferably an avian influenza virus, that is
effective in raising an immune response in animals, including
mammals and birds.
[0038] In particular, one aspect of the invention relates to
improved live attenuated bacterial strains which may include
Salmonella vectoring avian influenza antigens that can be
administered orally to an individual to elicit an immune response
to protect the individual from avian influenza. The invention
provides gram-negative bacterial mutants resistant to one or more
stress conditions, including, but not limited to, CO.sub.2, acid
pH, and high osmolarity. In a preferred embodiment, attenuated
gram-negative bacterial mutants are provided which are resistant to
CO.sub.2, acid pH, and/or high osmolarity. In a more preferred
embodiment, attenuated gram-negative bacterial msbB.sup.- mutants
resistant to CO.sub.2, acid pH, and high osmolarity are provided.
In a more preferred embodiment, attenuated gram-negative bacterial
msbB.sup.- mutants resistant to CO.sub.2, acid pH, and high
osmolarity are provided by a mutation in the pentose phosphate
pathway. In a specific embodiment, attenuated gram-negative
bacterial msbB.sup.- mutants resistant to CO.sub.2, acid pH, and
high osmolarity by deletion or disruption of the zwf gene are
provided. However, it should be understood that the scope of the
invention is limited by the claims, and not otherwise constricted
to particular genotypes or phenotypes.
[0039] The preferred bacteria are serovars of Salmonella. The
preferred Salmonella strains of the invention are specifically
attenuated by at least one first mutation at genetic locus which,
alone or in combination, results in increased sensitivity to
CO.sub.2, osmolarity and/or acidic pH combined with at least one
second mutation that compensates for the increased sensitivity to
CO.sub.2, osmolarity and acidic pH and restores resistance to
CO.sub.2, osmolarity and acidic pH. The attenuating mutation
resulting in sensitivity to CO.sub.2, osmolarity and acidic pH may
be those of known lipid biosynthesis genes which exhibit a degree
of safety in animals including but not limited to msbB (also known
as mlt, waaN, lpxM), firA, kdsA, kdsB, kdtA, lpxA, lpxB, lpxC,
lpxD, ssc, pmrA, and htrB. The resistance-conferring gene mutation
can be any member of the pentose phosphate pathway, including zwf,
pgl, gnd, rpe, rpiA, rpiB, tktA, tktB, talA, talB, especially those
genes directly related to gluconate production, including zwf, gnd
and pgl, or related gene products that provide gluconate into the
pentose pathway including gntT and other transporters for gluconate
including but not limited to the homologous gntU, gntP and idnT
transporters. The invention also provides stress-resistant
gram-negative bacterial mutants engineered to contain and/or
express one or more nucleic acid molecules encoding one or more
therapeutic molecules.
[0040] In one embodiment, stress-resistant gram-negative bacterial
mutants are provided which are facultative anaerobes or facultative
aerobes. In another embodiment, stress-resistant gram-negative
bacterial mutants are provided which are facultative anaerobes or
facultative aerobes and that comprise one or more nucleic acid
molecules encoding one or more therapeutic molecules. Examples of
facultative anaerobes or facultative aerobes include, but are not
limited to, Salmonella typhi, Salmonella choleraesuis, or
Salmonella enteritidis.
[0041] In a specific embodiment, the present invention provides
stress-resistant Salmonella mutants. Examples of Salmonella sp.
which may be used in accordance with the invention include, but are
not limited to, Salmonella typhi, Salmonella choleraesuis, or
Salmonella enteritidis. Preferably, the stress-resistant Salmonella
mutants are attenuated by introducing one or more mutations in one
or more genes in the lipopolysaccharide (LPS) biosynthetic pathway,
and optionally one or more mutations to auxotrophy for one or more
nutrients or metabolites. In a preferred embodiment, attenuated
stress-resistant Salmonella mutants comprise a genetically modified
msbB gene, express an altered lipid A molecule compared to
wild-type Salmonella sp., and induce TNF-.alpha. expression at a
level less than that induced by a wild-type Salmonella sp. The
growth of attenuated stress-resistant Salmonella mutants used in
accordance with the invention may be sensitive to a chelating agent
such as, e.g., Ethylenediaminetetraacetic Acid (EDTA), Ethylene
Glycol-bis (.beta.-aminoethyl Ether) N, N, N', N'-Tetraacetic Acid
(EGTA), or sodium citrate. For example, a chelating agent may
inhibit the growth of attenuated stress-resistant Salmonella
mutants by about 25%, 50%, 80%, or 99.5% compared to the growth of
a wild-type Salmonella sp. Preferably, the attenuated
stress-resistant Salmonella mutants used in accordance with the
invention survive in macrophages up to about 1% of the level of
survival of a wild-type Salmonella sp, preferably up to about 10%,
more preferably from about 10% up to about 30%, even more
preferably from about 30% up to about 50%, and most preferably up
to about 90% or even higher.
[0042] In one embodiment, the present invention provides
stress-resistant Salmonella mutants comprising one or more nucleic
acid molecules encoding one or more therapeutic molecules. In a
preferred embodiment, the present invention provides attenuated
stress-resistant Salmonella mutants, wherein the attenuation of the
stress-resistant Salmonella mutants is due, at least in part, to
one or more mutations in the msbB gene. In another preferred
embodiment, the present invention provides attenuated
stress-resistant Salmonella mutants, wherein the attenuated
stress-resistant Salmonella mutants comprise one or more nucleic
acid molecules encoding one or more therapeutic molecules and the
attenuation of the stress-resistant Salmonella mutants is due, at
least in part, to one or more mutations in the msbB gene.
[0043] A therapeutic molecule may be, for example, a molecule which
directly reduces the cause of a pathological condition, one which
enhances host response to a condition or reduces an adverse host
response due to the condition, one which reduces the incidence of
superinfection or improves host health or immune response, or the
like.
[0044] In one embodiment, the present invention provides mutant
Salmonella sp. comprising a genetically modified msbB gene and a
mutation characterized by increased growth when grown under
CO.sub.2 conditions compared to the msbff mutant Salmonella
designated YS1646 having ATCC Accession No. 202165 (Low, et. al.,
1999, Nature Biotechnology 17: 37-41; Low et al., 2004 Methods Mol.
Med. 90: 47-60). In another embodiment, the present invention
provides a mutant Salmonella sp. comprising a genetically modified
msbB gene and a mutation characterized by increased growth when
grown in acidified media compared to the msbB.sup.- mutant
Salmonella designated YS1646 having ATCC Accession No. 202165. In
yet another embodiment, the present invention provides mutant
Salmonella sp. comprising a genetically modified msbB gene and a
mutation characterized by increased growth in media with high
osmolarity compared to the msbff mutant Salmonella designated
YS1646 having ATCC Accession No. 202165. In accordance with these
embodiments, the mutant Salmonella sp. may further comprise one or
more genetically modified genes to auxotrophy and/or one or more
nucleic acid molecules encoding one or more therapeutic
molecules.
[0045] In another preferred embodiment, the present invention
provides a mutant Salmonella sp. comprising a genetically modified
msbB gene and a genetically modified zwf gene.
[0046] According to various embodiments, the invention provides
pharmaceutical compositions comprising pharmaceutically acceptable
carriers and one or more stress-resistant gram-negative bacterial
mutants. The invention also provides pharmaceutical compositions
comprising pharmaceutically acceptable carriers and one or more
stress-resistant gram-negative bacterial mutants comprising
nucleotide sequences encoding one or more therapeutic molecules.
The pharmaceutical compositions of the invention may be used in
accordance with the methods of the invention for the prophylaxis or
treatment of virally induced disease. Preferably, the
stress-resistant gram-negative bacterial mutants are attenuated by
introducing one or more mutations in one or more genes in the
lipopolysaccharide (LPS) biosynthetic pathway, and optionally one
or more mutations to auxotrophy for one or more nutrients or
metabolites.
[0047] In one embodiment, a pharmaceutical composition comprises a
pharmaceutically acceptable carrier and one or more attenuated
stress-resistant gram-negative bacterial mutants, wherein said
attenuated stress-resistant gram-negative bacterial mutants are
facultative anaerobes or facultative aerobes. In another
embodiment, a pharmaceutical composition comprises a
pharmaceutically acceptable carrier and one or more attenuated
stress-resistant gram-negative bacterial mutants, wherein said
attenuated stress-resistant gram-negative bacterial mutants are
facultative anaerobes or facultative aerobes and comprise one or
more nucleic acid molecules encoding one or more therapeutic
molecules where the therapeutic molecule is a viral antigen.
[0048] In a specific embodiment, a pharmaceutical composition
comprises a pharmaceutically acceptable carrier and one or more
attenuated stress-resistant gram-negative bacterial mutants,
wherein the attenuated stress-resistant gram-negative bacterial
mutants are a Salmonella sp. In another specific embodiment, a
pharmaceutical composition comprises a pharmaceutically acceptable
carrier and one or more attenuated stress-resistant gram-negative
bacterial mutants, wherein the attenuated stress-resistant
gram-negative bacterial mutants are a Salmonella sp., and the
attenuated stress-resistant gram-negative bacterial mutants
comprise one or more nucleic acid molecules encoding one or more
therapeutic molecules.
[0049] In a preferred embodiment, a pharmaceutical composition
comprises a pharmaceutically acceptable carrier and one or more
attenuated stress-resistant Salmonella mutants. In another
preferred embodiment, a pharmaceutical composition comprises a
pharmaceutically acceptable carrier and one or more attenuated
stress-resistant Salmonella mutants, wherein said attenuated
stress-resistant Salmonella mutants comprise one or more nucleic
acid molecules encoding one or more therapeutic molecules.
[0050] The present invention encompasses treatment protocols that
provide a better therapeutic effect than current existing vaccines.
In particular, the present invention provides methods for
prophylaxis or treatment of virally induced disease in a subject
comprising administering to said subject and one or more
stress-resistant gram-negative bacterial mutants, preferably
attenuated stress-resistant gram-negative bacterial mutants. The
present invention also provides methods for the prophylaxis or
treatment of virally induced disease in a subject comprising
administering to said subject one or more stress-resistant
gram-negative bacterial mutants, preferably attenuated
stress-resistant gram-negative bacterial mutants, wherein said
stress-resistant gram-negative bacterial mutants comprise one or
more nucleic acid molecules encoding one or more therapeutic
molecules.
[0051] The present invention provides methods for the enhanced
delivery of one or more therapeutic molecules in a subject
comprising administering to said subject one or more
stress-resistant gram-negative bacterial mutants, preferably
attenuated stress-resistant gram-negative bacterial mutants,
comprising nucleic acid molecules encoding one or more therapeutic
molecules. The methods of the present invention permit lower
dosages and/or less frequent dosing of stress-resistant
gram-negative bacterial mutants (preferably attenuated
stress-resistant gram-negative bacterial mutants) to be
administered to a subject for prophylaxis or treatment of virally
induced disease to achieve a therapeutically effective amount of
one or more therapeutic molecules.
[0052] In a specific embodiment, the present invention provides a
method of prophylaxis or treatment of virally induced disease in a
subject, said method comprising administering to said subject an
effective amount of a mutant Salmonella sp. comprising a
genetically modified msbB gene and a mutation characterized by
increased growth when grown under CO.sub.2 conditions compared to
the msbB.sup.- mutant Salmonella designated YS1646 having ATCC
Accession No. 202165. In another embodiment, the present invention
provides a method for viral prophylaxis or treatment in a subject,
said method comprising administering to said subject an effective
amount of a mutant Salmonella sp. comprising a genetically modified
msbB gene and a mutation characterized by increased growth when
grown in acidified media compared to the msbB.sup.- mutant
Salmonella designated YS1646 having ATCC Accession No. 202165. In
accordance with these embodiments, the mutant Salmonella sp.
further comprise one or more genetically modified genes to
auxotrophy and/or one or more nucleic acid molecules encoding one
or more therapeutic molecules.
[0053] In a preferred embodiment, the present invention provides a
method of prophylaxis or treatment of virally induced disease in a
subject, said method comprising administering to said subject an
effective amount of a mutant Salmonella sp. comprising a
genetically modified msbB gene and a genetically modified zwf gene.
In accordance with this embodiment, the mutant Salmonella sp. may
further comprise one or more genetically modified genes to
auxotrophy and/or one or more nucleic acid molecules encoding one
or more therapeutic molecules.
[0054] In a preferred embodiment of the invention, the bacteria
have genetic modifications which result in the expression of at
least one hemagglutinin and/or one neuraminidase, where each gene
is optimized for bacterial expression in at least one codon. In a
most preferred embodiment, the hemagglutinin and/or neuraminidase
genes are further modified to be secreted by the bacteria as
heterologous fusion proteins. In a most preferred embodiment, the
neuraminidase and hemagglutinin heterologous fusion proteins are
integrated into the chromosome in delta IS200 sites.
[0055] In a preferred embodiment, the bacterial strains are
genetically stabilized by deletion of IS200 elements, which reduces
their genetic recombination potential.
[0056] In another embodiment, the bacterial strains are genetically
stabilized by deletion of phage and prophage elements, which
reduces their genetic recombination and transduction potential.
[0057] In another embodiment, the bacterial strains are genetically
isolated from phage infection by constitutive expression of the P22
C2 repressor, which reduces their ability to be infected by phage
and the subsequent transduction of genes by such phage.
[0058] In another embodiment, the bacterial strains have
genetically defined flagellar antigens, or no flagellar antigens,
which reduces the immune system elimination of the vector,
enhancing its immunization potential in second immunizations.
[0059] In a preferred embodiment, the genetically modified bacteria
are used in animals, including humans, birds, dogs and pigs, for
protection against avian influenza and highly pathogenic
derivatives.
[0060] In another embodiment, a kit allows for rapid construction
of a bacterial vaccine which is closely matched to an emerging
avian influenza or its highly pathogenic derivative.
[0061] In another embodiment, the invention provides a bacterium
capable of having its growth inhibited by gluconate and a method of
controlling bacterial growth by means of administering gluconate.
In a preferred embodiment, the bacterium capable of having its
growth inhibited by gluconate is deficient in both the msbB and zwf
genes.
[0062] The live attenuated bacteria described by Bermudes
WO/2008/039408 are designed to achieve a close antigenic match
between the vaccine strain and the target strain. Bermudes targets
viruses for vaccine strains based on their emerging pathogenicity,
and produces an effective vaccine more closely matched to the
antigen profile of the emerging pathogen. As Bermudes requires
detailed knowledge of the antigenic profile of an emerging strain,
such a vaccine can be produced at the time of need in order to
reduce the risk of an unmatched vaccine and potential effects of
partial protection in a human pandemic outbreak. Thus Bermudes
provides vaccines for protecting a human patient against infection
by an emerging avian influenza virus strain.
[0063] Accordingly, when orally or nasally administered to an
individual, a live Salmonella bacterial vaccine, in accordance with
the present invention, that is genetically engineered to express
one or more avian influenza antigens as described herein and having
a first attenuating mutation that reduces TNF-.alpha. induction and
confers sensitivity to CO.sub.2, osmolarity and/or acidic pH and a
second mutation that confers resistance to CO.sub.2, osmolarity
and/or acidic pH and restores their ability to grow therein without
increasing TNF-.alpha. induction and have improved ability to
establish a population (infection) in the nasopharyngeal and/or
bronchial/pulmonary or gut tissues and, if properly modified they
could provide a desirable source of immunogenic avian influenza
antigen polypeptide(s) to elicit an immune response in the mucosal
tissue of the individual.
[0064] The antigen(s) can invoke an antibody and/or cellular immune
responses in the patient that are capable of neutralizing the
emerging avian influenza vaccine strains with high efficiency, as
well as emerging heterologous avian influenza vaccine strains, with
moderate efficiency. Preferably, the emerging avian influenza
vaccine will be within the same hemagglutinin and or neuraminidase
type (i.e., H1, H5, H5 (H274Y), H7 or H9 and/or N1, N2 or N7) as
are the current pathogenic avian influenza strains.
[0065] The live vaccine compositions are suitable for oral
administration to an individual to provide protection from avian
influenza. Preferably, a vaccine composition comprises a suspension
of a live bacterial strain described herein in a physiologically
accepted buffer or saline solution that can be swallowed from the
mouth of an individual. However, oral administration of a vaccine
composition to an individual may also include, without limitation,
administering a suspension of a bacterial vaccine strain described
herein through a nasojejunal or gastrostomy tube and administration
of a suppository that releases a live bacterial vaccine strain to
the lower intestinal tract of an individual. Vaccines of the
invention may be formulated for delivery by other various routes
e.g. by intramuscular injection, subcutaneous delivery, by
intranasal delivery (e.g. WO 00/47222, U.S. Pat. No. 6,635,246),
intradermal delivery (e.g. WO02/074336, WO02/067983, WO02/087494,
WO02/0832149 WO04/016281) by transdermal delivery, by
transcutaneous delivery, by topical routes, etc. Injection may
involve a needle (including a microneedle), or may be
needle-free.
[0066] Vaccines of the invention use one or more avian antigens to
protect patients against infection by an influenza virus strain
that is capable of human-to-human transmission i.e., a strain that
will spread geometrically or exponentially within a given human
population without necessarily requiring physical contact. The
patient may also be protected against strains that infect and cause
disease in humans, but that are caught from birds rather than from
other humans (i.e., bird to human transmission). The invention is
particularly useful for protecting against infection by pandemic,
emerging pandemic and future pandering human strains e.g. for
protecting against H5 and N1 influenza subtypes. Depending on the
particular season and on the nature of the antigen included in the
vaccine, however, the invention may protect against any
hemagglutinin subtypes, including H1, H2, H3, H4, H5, H6, H7, H8,
H9, H10, H11, H12, H13, H14, H15 or H16 or various neuraminidase
subtypes, including N1, N2, N3, N4, N5, N6, N7, N8 or N9.
[0067] The characteristics of an influenza strain that give it the
potential to cause a pandemic outbreak may include: (a) it contains
a new or antigenically altered hemagglutinin compared to the
hemagglutinins in currently-circulating human strains i.e., one
that has not been evident in the human population for over a decade
(e.g. H2), or has not previously been seen at all in the human
population (e.g. H5, H6 or H9, that have generally been found only
in bird populations), such that the human population will be
immunologically naive to the strain's hemagglutinin or that is a
subtype which is antigenically altered by changes in amino acid
sequence or glycosylation; (b) it is capable of being transmitted
horizontally in the human population; (c) is capable of being
transmitted from animals (including birds, dogs, pigs) to humans;
and/or (d) it is pathogenic to humans.
[0068] As a preferred embodiment of the invention protects against
a strain that is capable of human-to-human or bird-to-human or
bird-to-bird transmission, one embodiment of the invention in
accordance with that aspect will generally include at least one
gene that originated in a mammalian (e.g. in a human) influenza
virus and one gene which originated in a bird or non-human
vertebrate. Vaccines in accordance with various aspects of the
invention may therefore include an antigen from an avian influenza
virus strain. This strain is typically one that is capable of
causing highly pathogenic avian influenza (HPAI). HPAI is a
well-defined condition (Alexander Avian Dis (2003) 47(3
Suppl):976-81) that is characterized by sudden onset, severe
illness and rapid death of affected birds/flocks, with a mortality
rate that can approach 100%. Low pathogenicity (LPAI) and high
pathogenicity strains are easily distinguished e.g. van der Goot et
al. (Epidemiol Infect (2003) 131(2):1003-13) presented a
comparative study of the transmission characteristics of low and
high pathogenicity H5N2 avian strains. For the 2004 season,
examples of HPAI strains are H5N1 Influenza A viruses e.g. A/Viet
Nam/I 196/04 strain (also known as A Vietnam/3028/2004 or
A/Vietnam/3028/04). The skilled person will thus be able to
identify or predict future HPAI strains and the DNA sequence and
amino acid compositions of the H and N antigens as and when they
emerge. The avian influenza strain may be of any suitable
hemagglutinin subtype, including H1, H2, H3, H4, H5, H6, H7, H8,
H9, H10, H11, H12, H13, H14, H15 or H16. The avian influenza strain
may further be of any suitable neuraminidase subtype N1, N2, N3,
N4, N5, N6, N7, N8, or N9. The vaccines of the invention may
comprise two or more (i.e., two, three, four, or five) avian
influenza hemagglutinin and neuraminidase antigens. Such avian
influenza strains may comprise the same or different hemagglutinin
subtypes and the same or different neuraminidase subtypes.
[0069] A preferred vaccine composition will contain a sufficient
amount of live bacteria expressing the antigen(s) to produce an
immunological response in the patient. Accordingly, the attenuated
stress-resistant Salmonella strains described herein are both safe
and useful as live bacterial vaccines that can be orally
administered to an individual to provide immunity to avian
influenza and, thereby, protection from avian influenza.
[0070] Although not wishing to be bound by any particular
mechanism, an effective mucosal immune response to avian influenza
antigen(s) in humans by oral administration of genetically
engineered, attenuated strains of Salmonella strains as described
herein may be due to the ability of such mutant strains to pass
through the acidic environment of the stomach and persist in the
intestinal tract which is known to contain high levels of CO.sub.2
and to exhibit acidic pH (Jensen and Jorgensen, Applied and
Environmental Microbiology 60: 1897-1904) before accessing the gut
mucosa, gut lymphoidal cells and/or gut dendridic cells. Each
bacterial strain useful in the invention carries an
antigen-expressing plasmid or chromosomally integrated cassette
that encodes and directs expression of one or more avian influenza
antigens of avian influenza virus when resident in an attenuated
Salmonella strain described herein. As noted above, avian influenza
antigens that are particularly useful in the invention include an
H1, H5, H5 (H274Y), H7 or H9 antigen polypeptide (or immunogenic
portion thereof), a N1, N2 or N7 antigen polypeptide (or
immunogenic portion thereof), and a fusion polypeptide comprising a
heterologous secretion peptide linked in-frame to the antigenic
peptide.
[0071] The serovars of S. enterica that may be used as the
attenuated bacterium of the live vaccine compositions described
herein include, without limitation, Salmonella enterica serovar
Typhimurium ("S. typhimurium"), Salmonella montevideo, Salmonella
enterica serovar Typhi ("S. typhi"), Salmonella enterica serovar
Paratyphi B ("S. paratyphi B"), Salmonella enterica serovar
Paratyphi C ("S. paratyphi C"), Salmonella enterica serovar Hadar
("S. Hadar"), Salmonella enterica serovar Enteriditis ("S.
enteriditis"), Salmonella enterica serovar Kentucky ("S.
kentucky"), Salmonella enterica serovar Infantis ("S. infantis"),
Salmonella enterica serovar Pullorurn ("S. pullorum"), Salmonella
enterica serovar Gallinarum ("S. gallinarum"), Salmonella enterica
serovar Muenchen ("S. muenchen"), Salmonella enterica serovar
Anaturn ("S. anatum"), Salmonella enterica serovar Dublin ("S.
dublin"), Salmonella enterica serovar Derby ("S. derby"),
Salmonella enterica serovar Choleraesuis var. kunzendorf ("S.
cholerae kunzendorf"), and Salmonella enterica serovar minnesota
("S. Minnesota").
[0072] By way of example, live avian influenza vaccines in
accordance with aspects of the invention include known strains of
S. enterica serovar Typhimurium ("S. typhimurium") and S. enterica
serovar Typhi ("S. typhi") which are further modified as provided
by the invention to form suitable vaccines for the prevention and
treatment of avian influenza. Such Strains include Ty21a, CMV906,
CMV908, CMV906-htr, CMV908-htr, Ty800, aroA-/serC-, holavax,
M01ZH09, VNP20009.
[0073] Novel strains are also encompassed that are attenuated in
virulence by mutations in a variety of metabolic and structural
genes. The invention therefore may provide a live vaccine
composition for protecting against avian influenza comprising a
live attenuated bacterium that is a serovar of Salmonella enterica
comprising, an attenuating mutation in a genetic locus of the
chromosome of said bacterium that attenuates virulence of said
bacterium and wherein said attenuating mutation is the Suwwan
deletion (Murray et al., 2004, Journal of Bacteriology 186:
8516-8523) or combinations with other known attenuating mutations.
Other attenuating mutation useful in the Salmonella bacterial
strains described herein may be in a genetic locus selected from
the group consisting of phoP, phoQ, edt, cya, crp, poxA, rpoS,
htrA, nuoG, pmi, pabA, pts, damA, purA, purB, purI, purF, aroA,
aroB, aroC, aroD, serC, gua, cadA, rfc, rjb, rfa, ompR, msbB and
combinations thereof.
[0074] The invention may also be incorporated into a process for
preparing genetically stable bacterial vaccines for protecting a
human patient against infection by an avian influenza virus strain,
comprising genetically engineering the avian antigen from an avian
influenza virus strain that can cause highly pathogenic avian
influenza to comprise a bacterially codon optimized expression
sequence within a bacterial plasmid expression vector or
chromosomal localization expression vector and further containing
engineered restriction endonuclease sites such that the bacterially
codon optimized expression gene contains subcomponents which are
easily and rapidly exchangeable in order to facilitate rapid
exchange of the genetic subcomponents to achieve a well matched
antigen to the emerging avian influenza pathogen. The plasmid
and/or chromosomal expression constructs may be further modified to
result in the secretion of the viral antigens. Administration of
the vaccine to the patient invokes an antibody and/or cellular
immune response that is capable of neutralizing said avian
influenza virus strain.
[0075] The invention may also be incorporated into methods and
compositions for producing a bacterial vector expressing one or
more avian influenza antigens where said bacterial vector has one
or more deletions in IS200 elements which results in enhance
genetic stability. The composition and methods comprise a bacterial
strain with a deletion in the IS200 elements, such that the
bacteria are no longer capable of genetic rearrangement using IS200
elements. Such a deletion is generated in any one or more IS200
element, which is then confirmed using standard genetic
techniques.
[0076] The invention may also be incorporated into methods and
compositions for producing a genetically stabilized bacterial
vector expressing one or more avian influenza antigens where said
bacterial vector has one or more deletions in bacteria phage or
prophage elements which enhanced genetic stability and prevent
phage excision. The composition and methods comprise a bacterial
strain with one or more deletions in bacteria phage or prophage
elements, such that the bacteria are no longer capable of genetic
rearrangement using bacteria phage or prophage elements. Such a
deletion is generated in any bacteria phage or prophage elements,
which is then confirmed using standard genetic techniques. Such
strains have phage with reduced capacity for transduction of genes
to other strains.
[0077] The invention may also be incorporated into methods and
compositions for producing a bacterial vector expressing one or
more avian influenza antigens where said bacterial vector
constitutively expresses the P22 phage C2 repressor, thereby
preventing new infections by bacteria phage and further preventing
subsequent phage transductions by these phage.
[0078] The invention may also be incorporated into live Salmonella
vaccines having had deletions of the hin gene and/or defined fH1 or
fH2 antigens, or may have been constructed such that they lack fH
antigens altogether. The invention may also make use of Salmonella
strains expressing non-overlapping O-antigens, such as those of S.
typhimurium (O-1, 4, 5, 12) S. typhi is (Vi), or S. montevideo
(O-6, 7). Changing of the outer coat may be accomplished by genetic
manipulations known to those skilled in the art. Both antigenic
changes may be used together. Accordingly, the invention may also
be incorporated into live Salmonella vaccines constructed to
maximize a prime-boost strategy which alternates or eliminates the
fH antigen whereby the immune response of the fH antigen of the
first immunization (prime) is not specific for the antigen of the
second immunization (boost) and likewise, the 0 antigen profile of
the first immunization is not the same for the second immunization.
Therefore, the boost immunization is not diminished by a rapid
elimination by the immune system, and is therefore able to persist
longer and more effectively present the immunizing heterologous
avian influenza antigen.
[0079] An embodiment of the present invention therefore may also be
incorporated into methods and compositions for producing a
bacterial vector expressing one or more avian influenza antigens
where said bacterial vector has a defined flagellar H antigen (fH).
The composition and methods comprise a bacterial strain with a
deletion in the Hin recombinase gene, such that the bacteria are no
longer capable of alternating between fH1 and fH2 antigens. Such a
deletion is generated in either an fH1 or fH2 serologically defined
strain, which is then reconfirmed following deletion or disruption
of the hin recombinase gene. The invention may also be incorporated
into methods and compositions for producing a bacterial vector
which lacks flagellar antigens generated by deletion of the fliBC
genes (i.e., fH0). Therefore, an improved composition for a
prime/boost strategy is provided where the second vaccination
comprises administration of a vaccine where the fH antigen
composition is different from the first vaccination. In the case
where the antigen is presented as a fusion with the fliC gene,
preferably only fH1 and fH2 forms are utilized; fH0 is preferably
not used.
[0080] The invention may also may also be incorporated into a
method for protecting a human patient against infection by an avian
influenza virus strain with an improved prime/boost strategy,
comprising the step of administering to the patient a vaccine that
comprises an antigen from an avian influenza virus strain that can
cause highly pathogenic avian influenza or 1918 influenza within a
bacterial vector expressing one or more avian influenza antigens
where said bacterial vector has a defined fH antigen or no fH
antigen (i.e., fH1, fH2, or fH0) and/or various non-overlapping
O-antigens. The invention may further may also be incorporated into
a method of administering a second bacterial vector expressing one
or more avian influenza antigens comprising a second step where the
second administration where said bacterial vector has a defined fH
antigen which is different fH antigen composition than the fH
antigen of the first administration or no fH antigen. The second
administration includes a bacterial vaccine where the first vaccine
administration is a bacterial vaccine of the present invention or
is another vaccine not encompassed by the present application,
e.g., another bacterial vaccine or an egg-based vaccine.
[0081] Similarly, the invention may also may also be incorporated
into a kit comprising (a) a first container comprising a bacterial
expression codon optimized antigen from a pathogenic avian
influenza virus strain containing unique genetically engineered
restriction sites contained within either a bacterial protein
expression plasmid or a bacterial chromosomal protein expression
vector and (b) a second container comprising bacterial vector(s)
with one or more (e.g., fH1, fH2 or fH0) flagellar antigen(s)
and/or various non-overlapping O-antigens. Component (a) will be
modifiable to genetically match an emerging avian influenza virus
using standard in vitro molecular techniques and can be combined
with component (b) to generate one or more bacterial strains with
defined flagellar antigens which constitute a live vaccine. The
variation(s) in flagellar antigens provided by the kit provide for
more than one live vaccine strain in which a first immunization
(prime) using one strain may be followed at an appropriate time
such as 2 to 4 weeks by a second immunization (boost) using a
second strain with a different fH antigen or no fH antigen. The
live vaccine compositions are suitable for oral or nasal
administration to an individual to provide protection from avian
influenza.
[0082] Preferably, the invention may also be incorporated into a
vaccine composition comprising a suspension of a live bacterial
strain described herein in a physiologically accepted buffer or
saline solution that can be swallowed from the mouth of an
individual. However, oral administration of a vaccine composition
to an individual may also include, without limitation,
administering a suspension of a bacterial vaccine strain described
herein through a nasojejunal or gastrostomy tube and administration
of a suppository that releases a live bacterial vaccine strain to
the lower intestinal tract of an individual.
Definitions
[0083] In order that the invention may be more fully understood,
the following terms are defined:
[0084] CO.sub.2 conditions: As used herein, the term "CO.sub.2
conditions" refers to CO.sub.2 levels above ambient air. In
particular, the term "CO.sub.2 conditions" refers to CO.sub.2
levels above 0.3%, 0.4%, 0.45%, 0.5%, 0.75%, 1%, 1.5%, 2%, 2.5%,
3%, 3.5%, 4%, 4.5%, 5% or higher.
[0085] High osmolarity: As used herein, the term "high osmolarity"
refers to osmolarity above the growth-permissive osmolarity level
of LB-O and or MSB media described in the examples of approximately
100 milliosmoles, normal physiological osmolarity found in a
subject (approximately 300 milliosmoles), particular, to the
osmolarity found in an organ or tissue of a subject. In certain
embodiments, the term "high osmolarity" refers to osmolarity above
approximately 340 millosmoles, 450 millosmoles, 475 millosmoles,
500 millosmoles, 525 millosmoles, 550 millosmoles, 575 millosmoles,
600 millosmoles or more. In certain embodiments, the term the
osmolarity-resistant gram-negative mutants are sensitive to normal
physiological osmolarity found in a subject.
[0086] Pentose Phosphate Pathway: The pentose phosphate pathway
(also called Phosphogluconate Pathway, or Hexose Monophosphate
Shunt [HMP shunt]: see Fraenkel 1996, Glycolysis, In Neidhardt (ed)
Escherichia coli and Salmonella, Second Ed., ASM Press, Washington,
D.C., pp. 189-198, expressly incorporated by reference herein) is a
process that serves to generate NADPH and the synthesis of pentose
(5-carbon) sugars. There are two distinct phases in the pathway.
The first is the oxidative phase, in which NADPH is generated, and
the second is the non-oxidative synthesis of 5-carbon sugars. This
pathway is an alternative to glycolysis. While it does involve
oxidation of glucose, its primary role is anabolic rather than
catabolic. The primary functions of the pathway are: 1) To generate
reducing equivalents, in the form of NADPH, for reductive
biosynthesis reactions within cells; 2) to provide the cell with
ribose-5-phosphate (R5P) for the synthesis of the nucleotides and
nucleic acids; and 3) to metabolize pentose sugars derived from the
digestion of nucleic acids as well as to rearrange the carbon
skeletons of carbohydrates into glycolytic/gluconeogenic
intermediates.
[0087] Gene comprising the pentose phosphate pathway include zwf
(glucose 6-phosphate dehydrogenase, EC 1.1.1.49), pgl
(6-phosphogluconolactonase, EC 3.1.1.31), gnd (6-phosphogluconate
dehydogenase, EC 1.1.1.4), rpe (ribulose phosphate 3-epimerase, EC
5.1.3.1), rpiA/rpiB (ribose-5-phosphate isomerase A & B, EC
5.3.1.6), tktA/tktB (transkeolase A & B. EC 2.2.1.1), and
talA/talB (transaldolase A & B, EC 2.2.1.2). Additionally,
related gene products that provide gluconate into the pentose
pathway, gntT and other transporters for gluconate including the
homologous gntU, gntP and idnT transporters are encompassed.
[0088] Stress-resistant gram-negative bacterial mutants: As used
herein, the "stress-resistant gram-negative bacterial mutants" and
variations thereof refer to gram-negative bacteria with the ability
to grow under one or more environmental stresses such as may exist
in the body of an animal (mammal, bird, reptile). Examples of
environmental stresses include, but are not limited to, CO.sub.2
concentration, temperature, pH, and osmolarity. Stress-resistant
gram-negative bacterial mutants include, but are not limited to,
gram-negative bacteria that are resistant to CO.sub.2 and/or acid
pH. In a preferred embodiment, stress-resistant gram-negative
mutants are attenuated. In another preferred embodiment,
stress-resistant gram-negative mutants have one or more mutations
in lipid metabolism, in particular, LPS biosynthesis. In a specific
embodiment, stress-resistant gram-negative mutants are
stress-resistant Salmonella sp. In a preferred embodiment,
stress-resistant gram-negative mutants are attenuated
stress-resistant Salmonella sp.
[0089] As used herein, "attenuated", "attenuation", and similar
terms refer to elimination or reduction of the natural virulence of
a bacterium in a particular host organism, such as a mammal.
"Virulence" is the degree or ability of a pathogenic microorganism
to produce disease in a host organism. A bacterium may be virulent
for one species of host organism (e.g., a mouse) and not virulent
for another species of host organism (e.g., a human). Hence,
broadly, an "attenuated" bacterium or strain of bacteria is
attenuated in virulence toward at least one species of host
organism that is susceptible to infection and disease by a virulent
form of the bacterium or strain of the bacterium. As used herein,
the term "genetic locus" is a broad term and comprises any
designated site in the genome (the total genetic content of an
organism) or in a particular nucleotide sequence of a chromosome or
replicating nucleic acid molecule (e.g., a plasmid), including but
not limited to a gene, nucleotide coding sequence (for a protein or
RNA), operon, regulon, promoter, regulatory site (including
transcriptional terminator sites, ribosome binding sites,
transcriptional inhibitor binding sites, transcriptional activator
binding sites), origin of replication, intercistronic region, and
portions therein. A genetic locus may be identified and
characterized by any of a variety of in vivo and/or in vitro
methods available in the art, including but not limited to,
conjugation studies, crossover frequencies, transformation
analysis, transfection analysis, restriction enzyme mapping
protocols, nucleic acid hybridization analyses, polymerase chain
reaction (PCR) protocols, nuclease protection assays, and direct
nucleic acid sequence analysis. As used herein, the term
"infection" has the meaning generally used and understood by
persons skilled in the art and includes the invasion and
multiplication of a microorganism in or on a host organism ("host",
"individual", "patient") with or without a manifestation of a
disease (see, "virulence" above). Infectious microorganisms include
pathogenic viruses, such as avian influenza, that can cause serious
diseases when infecting an unprotected individual. An infection may
occur at one or more sites in or on an individual. An infection may
be unintentional (e.g., unintended ingestion, inhalation,
contamination of wounds, etc.) or intentional (e.g., administration
of a live vaccine strain, experimental challenge with a pathogenic
vaccine strain). In a vertebrate host organism, such as a mammal, a
site of infection includes, but is not limited to, the respiratory
system, the alimentary canal (gut), the circulatory system, the
skin, the endocrine system, the neural system, and intercellular
spaces. Some degree and form of replication or multiplication of an
infective microorganism is required for the microorganism to
persist at a site of infection. However, replication may vary
widely among infecting microorganisms. Accordingly, replication of
infecting microorganisms comprises, but is not limited to,
persistent and continuous multiplication of the microorganisms and
transient or temporary maintenance of microorganisms at a specific
location. Whereas "infection" of a host organism by a pathogenic
microorganism is undesirable owing to the potential for causing
disease in the host, an "infection" of a host individual with a
live vaccine comprising genetically altered, attenuated Salmonella
bacterial strain as described herein is desirable because of the
ability of the bacterial strain to elicit a protective immune
response to antigens of avian influenza virus that cause avian
influenza in humans and other mammals.
[0090] As used herein, the terms "disease" and "disorder" have the
meaning generally known and understood in the art and comprise any
abnormal condition in the function or well-being of a host
individual. A diagnosis of a particular disease or disorder, such
as avian influenza, by a healthcare professional may be made by
direct examination and/or consideration of results of one or more
diagnostic tests.
[0091] A "live vaccine composition", "live vaccine", "live
bacterial vaccine", and similar terms refer to a composition
comprising a strain of live Salmonella bacteria that expresses at
least one antigen of avian influenza, e.g., the H antigen, the N
antigen, or a combination thereof, such that when administered to
an individual, the bacteria will elicit an immune response in the
individual against the avian influenza antigen(s) expressed in the
Salmonella bacteria and, thereby, provide at least partial
protective immunity against avian influenza. Such protective
immunity may be evidenced by any of a variety of observable or
detectable conditions, including but not limited to, diminution of
one or more disease symptoms (e.g., respiratory distress, fever,
pain, diarrhea, bleeding, inflammation of lymph nodes, weakness,
malaise), shorter duration of illness, diminution of tissue damage,
regeneration of healthy tissue, clearance of pathogenic
microorganisms from the individual, and increased sense of
well-being by the individual. Although highly desired, it is
understood by persons skilled in the art that no vaccine is
expected to induce complete protection from a disease in every
individual that is administered the vaccine or that protective
immunity is expected to last throughout the lifetime of an
individual without periodic "booster" administrations of a vaccine
composition. It is also understood that a live vaccine comprising a
bacterium described herein may be, at the discretion of a
healthcare professional, administered to an individual who has not
presented symptoms of avian influenza but is considered to be at
risk of infection or is known to already have been exposed to avian
influenza virus, e.g., by proximity or contact with avian influenza
patients or virally contaminated air, liquids, or surfaces.
[0092] The terms "oral", "enteral", "enterally", "orally",
"non-parenteral", "non-parenterally", and the like, refer to
administration of a compound or composition to an individual by a
route or mode along the alimentary canal. Examples of "oral" routes
of administration of a vaccine composition include, without
limitation, swallowing liquid or solid forms of a vaccine
composition from the mouth, administration of a vaccine composition
through a nasojejunal or gastrostomy tube, intraduodenal
administration of a vaccine composition, and rectal administration,
e.g., using suppositories that release a live bacterial vaccine
strain described herein to the lower intestinal tract of the
alimentary canal.
[0093] The term "recombinant" is used to describe non-naturally
altered or manipulated nucleic acids, cells transformed,
electroporated, or transfected with exogenous nucleic acids, and
polypeptides expressed non-naturally, e.g., through manipulation of
isolated nucleic acids and transformation of cells. The term
"recombinant" specifically encompasses nucleic acid molecules that
have been constructed, at least in part, in vitro using genetic
engineering techniques, and use of the term "recombinant" as an
adjective to describe a molecule, construct, vector, cell,
polypeptide, or polynucleotide specifically excludes naturally
existing forms of such molecules, constructs, vectors, cells,
polypeptides, or polynucleotides.
[0094] Cassette, or expression cassette is used to describe a
nucleic acid sequence comprising (i) a nucleotide sequence encoding
a promoter, (ii) a first unique restriction enzyme cleavage site
located 5' of the nucleotide sequence encoding the promoter, and
(iii) a second unique restriction enzyme cleavage site located 3'
of the nucleotide sequence encoding the promoter. The cassette may
also contain a multiple cloning site (MCS) and transcriptional
terminator within the 5' and 3' restriction endonuclease cleavage
sites. The cassette may also contain cloned genes of interest.
[0095] As used herein, the term "salmonella" (plural,
"salmonellae") and "Salmonella" refers to a bacterium that is a
serovar of Salmonella enterica. A number of serovars of S. enterica
are known. Particularly preferred salmonella bacteria useful in the
invention are attenuated strains of Salmonella enterica serovar
Typhimurium ("S. typhimurium") and serovar Typhi ("S. typhi") as
described herein. As used herein, the terms "strain" and "isolate"
are synonymous and refer to a particular isolated bacterium and its
genetically identical progeny. Actual examples of particular
strains of bacteria developed or isolated by human effort are
indicated herein by specific letter and numerical designations
(e.g. strains Ty21a, CMV906, CMV908, CMV906-htr, CMV908-htr, Ty800,
holavax, M01ZH09, VNP20009).
[0096] The definitions of other terms used herein are those
understood and used by persons skilled in the art and/or will be
evident to persons skilled in the art from usage in the text. This
invention provides live vaccine compositions for protecting against
avian influenza comprising live Salmonella enterica serovars that
are genetically engineered to express one or more avian influenza
antigen polypeptides, such as the H1, H5, H5 (H274Y), H7 or H9 and
N1, N2 and N7 antigens of avian influenza virus.
BRIEF DESCRIPTION OF THE DRAWINGS
[0097] FIG. 1 shows a flow chart depicting the selection scheme for
isolation of transposon insertions used to isolate
CO.sub.2-resistant mutants.
[0098] FIG. 2A shows CO.sub.2-sensitivity of an msbB- strain
derived from Salmonella ATCC 14028.
[0099] FIG. 2B shows CO.sub.2-resistance of a zwf, msbB.sup.-
strain, each derived from Salmonella ATCC 14028.
[0100] FIGS. 3A, 3B, 3C and 3D show msbB.sup.- confers
growth-sensitivity in liquid media under CO.sub.2 conditions
containing physiological amounts of salt and is suppressed by
zwf.
[0101] FIG. 4 shows a .beta.-galactosidase release assays confirm
cell lysis of msbB.sup.- Salmonella in LB in the presence of 5%
CO.sub.2 and that zwf confers resistance.
[0102] FIGS. 5A, 5B, 5C, and 5D show that zwf suppresses growth
sensitivity to acidic pH in LB broth in both ambient air and 5%
CO.sub.2.
[0103] FIGS. 6A and 6B show a .beta.-galactosidase release assays
confirm cell lysis in LB broth, pH 6.6 and that zwf confers
resistance.
[0104] FIG. 7 shows a series of replica plate results for different
strains on different media showing zwf mutation suppresses both
msbB-induced CO.sub.2 sensitivity and osmotic defects.
[0105] FIG. 8 shows a flow chart depicting the selection scheme for
isolation of transposon insertions used to isolate acidic
pH-resistant mutants.
[0106] FIG. 9 shows a flow chart depicting the selection scheme for
isolation of transposon insertions used to isolate
osmolarity-resistant mutants.
DETAILED DESCRIPTION OF THE INVENTION
[0107] The invention provides gram-negative bacterial mutants
resistant to one or more stress conditions, including, but not
limited to, CO.sub.2, acid pH, and/or high osmolarity. In one
embodiment, the present invention provides gram-negative bacterial
mutants resistant to CO.sub.2, acid pH, and/or high osmolarity. In
a more preferred embodiment, the present invention provides
attenuated gram-negative bacterial mutants resistant to CO.sub.2,
acid pH, and/or high osmolarity. Preferably, the stress-resistant
gram-negative bacterial mutants are attenuated by introducing one
or more mutations in one or more genes in the lipopolysaccharide
(LPS) biosynthetic pathway that reduces the induction of
TNF-.alpha., and optionally, one or more mutations to auxotrophy
for one or more nutrients or metabolites.
[0108] The invention also provides stress-resistant gram-negative
bacterial mutants engineered to contain and/or express one or more
nucleic acid molecules encoding one or more therapeutic molecules.
In a specific embodiment, the present invention provides
stress-resistant gram-negative mutants engineered to contain and/or
express one or more nucleic acid molecules encoding one or more
therapeutic molecules. In another embodiment, the present invention
provides attenuated stress-resistant gram-negative mutants
engineered to contain and/or express one or more nucleic acid
molecules encoding one or more therapeutic molecules. In yet
another preferred embodiment, the present invention provides
attenuated stress-resistant gram-negative mutants engineered to
contain and/or express one or more nucleic acid molecules encoding
one or more therapeutic molecules.
[0109] The invention also provides pharmaceutical compositions
comprising pharmaceutically acceptable carriers and one or more
stress-resistant gram-negative bacterial mutants, preferably one or
more stress-resistant gram-negative bacterial mutants. The
invention also provides pharmaceutical compositions comprising
pharmaceutically acceptable carriers and one or more
stress-resistant gram-negative bacterial mutants, comprising
nucleotide sequences encoding one or more therapeutic molecules.
The pharmaceutical compositions of the invention may be used in
accordance with the methods of the invention for prophylaxis or
treatment of virally-induced disease. Preferably, the
stress-resistant gram-negative bacterial mutants are attenuated by
introducing one or more mutations in one or more genes in the
lipopolysaccharide (LPS) biosynthetic pathway, and optionally one
or more mutations to auxotrophy for one or more nutrients or
metabolites.
[0110] The present invention encompasses treatment protocols that
provide a better therapeutic effect than current existing vaccines.
In particular, the present invention provides methods for
prevention or treatment of virally-induced disease in a subject
comprising administering to said subject and one or more
stress-resistant gram-negative bacterial mutants. The present
invention also provides methods for the for viral infection
prophylaxis or treatment in a subject comprising administering to
said subject one or more stress-resistant gram-negative bacterial
mutants, preferably attenuated stress-resistant gram-negative
bacterial mutants, wherein said stress-resistant gram-negative
bacterial mutants comprise one or more nucleic acid molecules
encoding one or more therapeutic molecules.
[0111] The present invention provides methods for the enhanced
delivery of one or more therapeutic molecules for prophylaxis and
treatment of virally-induced disease comprising administering to
said subject one or more stress-resistant gram-negative bacterial
mutants, comprising nucleic acid molecules encoding one or more
therapeutic molecules. The methods of the present invention permit
lower dosages and/or less frequent dosing of stress-resistant
gram-negative bacterial mutants (preferably attenuated
stress-resistant gram-negative bacterial mutants) to be
administered to a subject for prophylaxis or treatment of
virally-induced disease to achieve a therapeutically effective
amount of one or more therapeutic molecules.
[0112] The invention also provides a pharmaceutical pack or kit
comprising one or more containers with one or more of the
components of the pharmaceutical compositions of the invention. The
kit further comprises instructions for use of the composition(s).
In certain embodiments of the invention, the kit comprises a
document providing instructions for the use of the composition(s)
of the invention in, e.g., written and/or electronic form. Said
instructions provide information relating to, e.g., dosage, methods
of administration, and duration of treatment. Optionally associated
with such container(s) can be a notice in the form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products which notice reflects
approval by the agency of manufacture, use or sale for human
administration.
[0113] For reasons of clarity, the detailed description is divided
into the following subsections: Stress-Resistant Gram-Negative
Bacterial Mutants; Production of Stress-Resistant Gram-Negative
Bacterial Mutants; Identification and Selection of Stress-Resistant
Gram-Negative Bacterial Mutants; Genetic Modifications to
Stress-Resistant Mutants with Transposon Insertions or Multicopy
Plasmids; Therapeutic Molecules; Expression Vehicles Methods and
Compositions for Delivery; Methods of Determining the Therapeutic
Utility; and Kits.
[0114] Stress-Resistant Gram-Negative Bacterial Mutants
[0115] Any gram-negative bacterial with the ability to grow under
one or more environmental stresses such as those that exist in
animals (i.e., stress-resistant gram-negative bacterial mutants)
may be used in the compositions and methods of the invention.
Examples of environmental stresses include, but are not limited to,
CO.sub.2 resistant, acid pH resistant, and/or osmolarity resistant
gram-negative bacterial mutants (methods for identifying,
isolating, and producing such gram-negative bacterial are described
infra). In a specific embodiment, the gram-negative bacteria used
in the compositions and methods of the invention are CO.sub.2
resistant and/or acid pH resistant gram-negative bacterial
mutants.
[0116] In a preferred embodiment, the stress-resistant
gram-negative bacterial mutants used in the compositions and
methods of the invention are attenuated. Any technique well-known
to one of skill in the art may be used to attenuate the
stress-resistant gram-negative bacterial mutants. Preferably, the
stress-resistant gram-negative bacterial mutants used in the
compositions and methods of the invention are attenuated by the
introduction of one or more mutations in one or more genes in the
lipopolysaccharide (LPS) biosynthetic pathway, and optionally, one
or more mutations to auxotrophy for one or more nutrients or
metabolites. Examples of genes in the LPS biosynthetic pathway
which may be attenuated include, but are not limited to, htrB,
msbB, kdsA, kdsB, kdtA, lpxB, lpxC, and lpxD. Mutations to
auxotrophy can be produced by the introduction of one or more
mutations in a gene in a biosynthetic pathway such as the leucine,
isoleucine, valine, phenylalanine, tryptophan, tyrosine, arginine,
uracil, or purine biosynthetic pathway. In particular, a mutation
in the AroA gene can result in auxotrophy. The attenuated
stress-resistant gram-negative bacterial mutants induce lower
levels of tumor necrosis factor-.alpha. (TNF-.alpha.) than their
wild-type counterpart (i.e., about 5% to about 40%, about 5% to
about 35%, about 5% to about 25%, about 5% to about 15%, or about
5% to about 10% of TNF-.alpha. induced by wild-type) as measured by
techniques well-known in the art (e.g., immunoassays such as
ELISAs), and thus, avoid or reduce the risk of inducing septic
shock in a subject administered a mutant bacterium for viral
infection prophylaxis or treatment to said subject in accordance
with the methods of the invention.
[0117] In a preferred embodiment, the stress-resistant
gram-negative bacterial mutants used in the compositions and
methods of the invention induce an immune response to avian
influenza. The present invention encompasses compositions and
methods for prophylaxis and treatment of virally-induced disease
using stress-resistant gram-negative bacterial mutant which
replicates at physiological temperatures (i.e., 35.degree. C. to
44.degree. C.) and induce an immune response in vitro or in vivo.
Preferably, such bacteria inhibit or reduce viral burden in vivo.
In accordance with the invention, such a gram-negative bacterial
mutant may be engineered to contain or express one or more
therapeutic molecules which have an anti-viral immunostimulatory
activity in vivo.
[0118] While the teachings in sections of this application may
refer specifically to Salmonella, the compositions and methods of
the invention are in no way meant to be restricted to Salmonella
but encompass any other gram-negative bacterium to which the
teachings apply. Suitable bacteria which may be used in accordance
with the invention include, but are not limited to, Escherichia
coli including enteroinvasive Escherichia coli (e.g.,
enteroinvasive Escherichia coli), Shigella sp., and Yersinia
enterocohtica. Thus, the reference to Salmonella in this
application is intended to serve as an illustration and the
invention is not limited in scope to Salmonella.
[0119] The present invention encompasses the use of Salmonella with
the ability to grow under one or more environmental stresses such
as those that exist within the body of an animal (i.e.,
stress-resistant Salmonella mutants) in the compositions and
methods of the invention. Examples of environmental stresses
include, but are not limited to, CO.sub.2 concentration,
temperature, pH, and osmolarity. Preferably, the Salmonella used in
the compositions and methods of the invention are CO.sub.2
resistant, acid pH resistant, and/or osmolarity resistant
gram-negative bacterial mutants (methods for identifying,
isolating, and producing such Salmonella are described infra). In a
specific embodiment, the Salmonella used in the compositions and
methods of the invention are CO.sub.2 resistant and/or acid pH
resistant gram-negative bacterial mutants.
[0120] In a preferred embodiment, the stress-resistant Salmonella
used in the methods and compositions of the invention are
attenuated. Preferably, the attenuated stress-resistant Salmonella
mutants used in the methods and compositions of the invention have
one or more mutations in one or more genes which reduce the
virulence and toxicity of Salmonella. In a preferred embodiment,
the attenuated stress-resistant Salmonella used in the compositions
and methods of the invention have mutation(s) in one or more genes
in the lipopolysaccharide (LPS) biosynthetic pathway (preferably in
the msbB gene) and optionally, have one or more mutations to
auxotrophy for one or more nutrients or metabolites, such as uracil
biosynthesis, purine biosynthesis, tyrosine biosynthesis, leucine,
isoleucine biosynthesis, arginine biosynthesis, valine
biosynthesis, tryptophan biosynthesis and arginine
biosynthesis.
[0121] The growth of an attenuated stress-resistant Salmonella used
in accordance with the invention may be sensitive to a chelating
agent such as, e.g., Ethylenediaminetetraacetic Acid (EDTA),
Ethylene Glycol-bis (.beta.-aminoethyl Ether) N, N, N',
N'-Tetraacetic Acid (EGTA), or sodium citrate. For example, a
chelating agent may inhibit the growth of an attenuated Salmonella
for viral infection prophylaxis or treatment by about 90%, 95%,
99%, or 99.5% compared to the growth of a wild-type Salmonella used
in accordance with the invention survive in macrophages at about
50% to about 30%, about 0% to about 10%, or about 10% to about 1%
of the level of survival of a wild-type Salmonella sp.
[0122] The present invention provides a mutant Salmonella sp. for
viral infection prophylaxis or treatment comprising a genetically
modified msbB gene and a mutation characterized by increased growth
when grown under CO.sub.2 conditions compared to the msbB- mutant
Salmonella designated YS1646 having ATCC Accession No. 202165. The
present invention also provides a mutant Salmonella sp. for viral
infection prophylaxis or treatment comprising a genetically
modified msbB gene and a mutation characterized by increased growth
when grown in acidified media compared to the msbff mutant
Salmonella designed YS1646 having ATCC Accession No. 202165. The
present invention further provides a mutant Salmonella sp. for
viral infection prophylaxis or treatment comprising a genetically
modified msbB gene and a mutation characterized by increased growth
in media with high osmolarity compared to the msbff mutant
Salmonella designed YS1646 having ATCC Accession No. 202165. Such
mutant Salmonella sp. may further comprise one or more genetically
modified genes to auxotrophy. In a preferred embodiment, the
present invention provides Salmonella mutants comprising a
genetically modified msbB gene and a genetically modified zwf
gene.
[0123] Characteristics of CO.sub.2-Resistant Gram-Negative
Bacterial Mutant
[0124] The primary characteristic of CO.sub.2-resistant
gram-negative bacterial mutants is the enhanced percentage of their
recovery on LB agar in CO.sub.2 relative to the parental strain of
bacteria from which they were derived. In one embodiment, the
percent recovery of CO.sub.2-resistant gram-negative mutants grown
under CO.sub.2 conditions is approximately 2% to approximately 95%,
approximately 2% to approximately 75%, approximately 2% to
approximately 50%, approximately 2% to about 40%, approximately 2%
to about 30%, approximately 2% to about 25%, approximately 2% to
about 20% or about 2% to approximately 10% greater than the
recovery of the parental strain of bacteria from which the
CO.sub.2-resistant gram-negative bacterial mutants were derived
grown under the same conditions.
[0125] A secondary characteristic of CO.sub.2-resistant
gram-negative bacterial mutants with mutations in lipid
biosynthesis genes that suppress TNF-.alpha. induction is that the
derived mutant retains the same low-level induction of TNF-.alpha..
In one embodiment, the percent TNF-.alpha. induction is
approximately 2% to approximately 95%, approximately 2% to
approximately 75%, approximately 2% to approximately 50%,
approximately 2% to about 40%, approximately 2% to about 30%,
approximately 2% to about 25%, approximately 2% to about 20% or
about 2% to approximately 10% that of the wild type strain of
bacteria grown under the same conditions.
[0126] As the pH tends to drop during incubation in 5% CO.sub.2,
some CO.sub.2-resistant gram-negative mutants may have increased
growth in acidified media relative to the parental strain of
bacteria from which they were derived. Thus, CO.sub.2-resistant
clones may be tested for resistance to acidic pH (such as pH 6.7 or
lower), utilizing the methods described infra. In one embodiment,
CO.sub.2-resistant gram-negative mutants grow approximately 2% to
approximately 95%, approximately 2% to approximately 75%,
approximately 2% to approximately 50%, approximately 2% to
approximately 40%, approximately 2% to approximately 30%,
approximately 2% to approximately 25%, or approximately 2% to
approximately 10% less in acidified media than the parental strains
of bacteria from the CO.sub.2-resistant gram-negative mutants were
derived.
[0127] In addition, some CO.sub.2-resistant gram-negative mutants
may be more attenuated than the parental strains of bacteria from
which they were derived.
[0128] Characteristics of Acid pH-Resistant Gram-Negative Bacterial
Mutants
[0129] The primary characteristic of acid pH-resistant
gram-negative bacterial mutants is their ability to grow in liquid
media under acidic pH conditions (e.g., pH 6.7, pH 6.6, pH 6.5, pH
6.25, pH 6.0, pH 5.5, pH 5.0, pH 4.5, pH 4.0, pH 3.5, pH 3.0, pH
2.5, pH 2.0, pH 1.5, pH 1.0 or lower) relative to the parental
strain of bacteria from which they were derived. In one embodiment,
the growth of the acid pH-resistant gram-negative mutants in
acidified media is approximately 2% to approximately 95%,
approximately 2% to approximately 75%, approximately 2% to
approximately 50%, approximately 2% to approximately 40%,
approximately 2% to approximately 30%, approximately 2% to
approximately 20% or approximately 2% to approximately 10% or
higher than the growth of the parental strain of bacteria from
which the acid pH-resistant gram-negative bacterial mutants were
derived grown under the same conditions. In a preferred embodiment,
the growth of the acid pH-resistant gram-negative mutants in
acidified media is approximately 40% to 100% higher than the growth
of the parental strain of bacteria from which the acid pH-resistant
gram-negative bacterial mutants were derived grown under the same
conditions.
[0130] A secondary characteristic of acidic pH-resistant
gram-negative bacterial mutants with mutations in lipid
biosynthesis genes that suppress TNF-.alpha. induction is that the
derived mutant retains the same low-level induction of TNF-.alpha..
In one embodiment, the percent TNF-.alpha. induction is
approximately 2% to approximately 95%, approximately 2% to
approximately 75%, approximately 2% to approximately 50%,
approximately 2% to about 40%, approximately 2% to about 30%,
approximately 2% to about 25%, approximately 2% to about 20% or
about 2% to approximately 10% that of the wild type strain of
bacteria grown under the same conditions.
[0131] Some acid pH-resistant gram-negative mutants under CO.sub.2
conditions may have enhanced recovery relative to the parental
strain of bacteria from which they were derived under the same
conditions. Thus, acid pH-resistant clones may be tested for
enhanced recovery under CO.sub.2 conditions, utilizing the methods
described herein.
[0132] Some acid pH-resistant gram-negative mutants may have
increased sensitivity to osmolarity. In addition, some acid
pH-resistant gram-negative mutants may be more attenuated than the
parental strains of bacteria from which they were derived.
[0133] Characteristics of Osmolarity-Resistant Gram-Negative
Bacterial Mutants
[0134] The primary characteristic of osmolarity-resistant
gram-negative bacterial mutants is their ability to survive and/or
grow in media having high osmolarity relative to the parental
strain of bacteria from which they were derived. In one embodiment,
the survival and/or growth of the osmolarity-resistant
gram-negative bacterial mutants in media having high osmolarity is
approximately 2% to approximately 95%, approximately 2% to
approximately 75%, approximately 2% to approximately 50%,
approximately 2% to approximately 40%, approximately 2% to
approximately 30%, approximately 2% to approximately 25%,
approximately 2% to approximately 20% or approximately 2% to
approximately 10% better than the survival and/or growth of the
parental strain of bacteria from which the osmolarity-resistant
gram-negative bacterial mutants were derived grown under the same
conditions. In a preferred embodiment, the survival and/or growth
of the osmolarity-resistant gram-negative bacterial mutants in
media having high osmolarity is approximately 40% to 100% better
than the survival and/or growth of the parental strain of bacteria
from which the osmolarity-resistant gram-negative bacterial mutants
were derived grown under the same conditions.
[0135] A secondary characteristic of osmolarity-resistant
gram-negative bacterial mutants with mutations in lipid
biosynthesis genes that suppress TNF-.alpha. induction is that the
derived mutant retains the same low-level induction of TNF-.alpha..
In one embodiment, the percent TNF-.alpha. induction is
approximately 2% to approximately 95%, approximately 2% to
approximately 75%, approximately 2% to approximately 50%,
approximately 2% to about 40%, approximately 2% to about 30%,
approximately 2% to about 25%, approximately 2% to about 20% or
about 2% to approximately 10% that of the wild type strain of
bacteria grown under the same conditions.
[0136] Some osmolarity-resistant gram-negative bacterial mutants
may have increased sensitivity to CO.sub.2 and/or acid pH stress
conditions relative to the parental strains of bacteria from which
they were derived. Further, some osmolarity-resistant gram-negative
bacterial mutants may have increased sensitivity to CO.sub.2 and/or
acid pH stress conditions relative to the parental strains of
bacteria from which they were derived, but the sensitivity of the
osmolarity-resistant gram-negative bacterial mutants to CO.sub.2
and/or acid pH stress conditions is compensated for by other
genetic alterations (e.g., alterations which cause resistance to
CO.sub.2 and/or acid pH stress conditions).
[0137] In addition, some osmolarity-resistant gram-negative
bacterial mutants may be more attenuated than the parental strains
of bacteria from which they were derived.
[0138] Production of Stress-Resistant Gram-Negative Bacterial
Mutants
[0139] Genetic alterations that confer resistance to one or more
environmental stresses to gram-negative bacteria, preferably
attenuated gram-negative bacteria and more preferably attenuated
gram-negative bacteria for viral infection prophylaxis or
treatment, can be produced utilizing any method well-known to one
of skill in the art. For example, stress-resistant gram-negative
bacterial mutants may be obtained by growing the bacteria under
various selective pressures or by random mutagenesis (e.g., using a
transposon library, using a multicopy plasmid library or by
exposing the bacteria to various mutagens). Examples of growth
condition parameters which may be varied to obtain stress-resistant
mutants include, but are not limited to, the temperature, the type
of media used to grow the bacteria, the pH of the media, and the
CO.sub.2 concentration/levels. Examples of mutagens which may be
used to obtain stress-resistant mutants include, but are not
limited to, ultraviolet light and nitrosoguanadine.
[0140] Identification and Selection of Stress-Resistant
Gram-Negative Bacterial Mutants
[0141] Gram-negative bacteria, preferably attenuated gram-negative
bacteria, and/or preferably attenuated gram-negative bacteria for
viral infection prophylaxis or treatment, with resistance to one or
more environmental stresses can be identified and selected for
utilizing any method well-known to one of skill in the art. In
general, a pool of bacteria with genetic variations is subjected to
one or more selection criteria and the resistant clones are
isolated. A pool of gram-negative bacteria with genetic variations
may be composed of spontaneous mutants, a library of transposon
mutants or mutants transformed with a library of cloned DNA in a
multicopy plasmid. The selected techniques that a pool of
gram-negative bacteria with genetic variations is subjected to
varies depending upon the particular stress-resistant mutant that
one is attempting to select. Selection techniques for particular
stress-resistant gram-negative bacteria may include, e.g., plating
the bacteria to LB agar plates under the stress condition, growing
the bacteria in LB broth under the stress condition, and then
plating the bacteria to LB agar plates. Individual colonies are
then isolated from the agar plates and tested for growth under the
particular stress condition. Colonies with greater growth ability
than the parental strain of bacteria from which they were derived
are deemed to be resistant to the particular stress condition. In a
specific embodiment, individual colonies of gram-negative bacteria
with genetic variations are deemed to be resistant to a particular
stress condition if they grow 2 fold, preferably 4 fold, 6 fold, 8
fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 45
fold, 50 fold, 55 fold, 60 fold, 65 fold, 70 fold, 75 fold or
higher levels under the stress condition than the parental strain
of bacteria from which they were derived. In another embodiment,
individual colonies of gram-negative bacteria with genetic
variations are deemed to have resistance to a particular stress
condition if their growth is 5%, preferably 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95% or greater under stress conditions than the parental strain of
bacteria from which they were derived.
[0142] In order to distinguish spontaneous stress-resistant mutants
from transposon and plasmid-based clones, the transposon or plasmid
can be transferred to another strain using selection for the
appropriate antibiotic marker found on the transposon or plasmid.
Several sibling colonies may then be isolated and tested for
resistance to the particular stress condition, thus avoiding
spontaneous mutants. In order to simultaneously transfer large
numbers of transposon or plasmid-based stress-resistant clones to
distinguish them from spontaneous stress-resistant clones,
following the first selection where the bacteria are grown under
stress conditions, the clones may be pooled together, the genetic
marker transferred, and then multiple sibling clones tested for
growth under the stress condition.
[0143] Identification and Selection of CO.sub.2-Resistant
Gram-Negative Bacterial Mutants
[0144] Gram-negative bacteria, preferably attenuated gram-negative
bacteria, with resistance to CO.sub.2 can be identified and
selected for utilizing any method well-known to one of skill in the
art. In general, a pool of bacteria with genetic variations is
subjected to one or more selection criteria and the resistant
clones are isolated. A pool of gram-negative bacteria with genetic
variations may be composed of spontaneous mutants, a library of
transposon mutants or mutants transformed with a library of cloned
DNA in a multicopy plasmid. Selection techniques for isolating
CO.sub.2-resistant gram-negative bacteria may include, but are not
limited to, growing the bacteria on LB agar plates at 37.degree. C.
under CO.sub.2 conditions, growing the bacteria in LB broth at
37.degree. C. in CO.sub.2, and then growing the bacteria on LB agar
plates at 37.degree. C. under CO.sub.2 conditions or air.
Individual colonies are then isolated from the agar plates and
tested for plating efficiency on LB agar at 37.degree. C. in air
and LB agar at 37.degree. C. in CO.sub.2. Colonies with greater
than 0.5%, preferably 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, or 50% recover on the LB agar at 37.degree. C. in
CO.sub.2 are deemed to have enhanced resistance to CO.sub.2
relative to the parental strain of bacteria from which they were
derived.
[0145] In order to distinguish spontaneous CO.sub.2-resistant
mutants from transposon and plasmid-based clones, the transposon or
plasmid can be transferred to another strain using selection for
the appropriate antibiotic marker found on the transposon or
plasmid. Several sibling colonies may then be isolated and tested
for resistance to CO.sub.2, thus avoiding spontaneous mutants. In
order to simultaneously transfer large numbers of transposon or
plasmid-based stress-resistant clones to distinguish them from
spontaneous CO.sub.2-resistant clones, following the first
selection where the bacteria are grown on LB agar plates under
CO.sub.2 conditions, the clones may be pooled together, the genetic
marker transferred, and then multiple sibling clones tested for
growth under the CO.sub.2 conditions.
[0146] Identification and Selection of Acid pH-Resistant
Gram-Negative Bacterial Mutants
[0147] Gram-negative bacteria, preferably attenuated gram-negative
bacteria, with resistance to acidic pH can be identified and
selected for utilizing any method well-known to one of skill in the
art. In general, a pool of bacteria with genetic variations is
subjected to one or more selection criteria and the resistant
clones are isolated. A pool of gram-negative bacteria with genetic
variations may be composed of spontaneous mutants, a library of
transposon mutants or mutants transformed with a library of cloned
DNA in a multicopy plasmid. Selection techniques for isolating acid
pH-resistant gram-negative bacteria may include, but are not
limited to, plating the bacteria on LB agar plates at 37.degree. C.
at an acidic pH (e.g., pH 6.7, pH 6.6, pH 6.5, pH 6.4, pH 6.0, pH
5.5, pH 5.0, pH 4.5, pH 4.0, pH 3.5, pH 2.0, pH 2.5 or pH 1.0),
growing the bacteria in LB broth at 37.degree. C. at an acidic pH
(e.g., pH 6.7, pH 6.6, pH 6.5, pH 6.4, pH 6.0, pH 5.5, pH 5.0, pH
4.5, pH 4.0, pH 3.5, pH 2.0, pH 2.5 or pH 1.0), and then plating
the bacteria on LB agar plates at 37.degree. C. Individual colonies
are then isolated from the agar plates and tested for growth in
acidified media at 37.degree. C. Colonies with greater growth
ability than the parental strain of bacteria from which they were
derived are deemed to be resistant to a particular acid pH (e.g.,
pH 6.5, pH 6, pH 5, pH 4.5, pH 4, pH 3.5, pH 2, pH 2.5 or pH 1). In
a specific embodiment, individual colonies of gram-negative
bacteria with genetic variations are deemed to be resistant to an
acidic pH (e.g., pH 6.5, pH 6, pH 5, pH 4.5, pH 4, pH 3.5, pH 2, pH
2.5 or pH 1) if they grow 2 fold, preferably 4 fold, 6 fold, 8
fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 45
fold, 50 fold, 55 fold, 60 fold, 65 fold, 70 fold, 75 fold or
higher levels in acidified media than the parental strain of
bacteria from which they were derived. In another embodiment,
individual colonies of gram-negative bacteria with genetic
variations are deemed to have resistance to an acidic pH if their
growth is 5%, preferably 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater in
acidified media than the parental strain of bacteria from which
they were derived.
[0148] In order to distinguish spontaneous acid pH-resistant
mutants from transposon and plasmid-based clones, the transposon or
plasmid can be transferred to another strain using selection for
the appropriate antibiotic marker found on the transposon or
plasmid. Several sibling colonies may then be isolated and tested
for resistance to an acidic pH, thus avoiding spontaneous mutants.
In order to simultaneously transfer large numbers of transposon or
plasmid-based stress-resistant clones to distinguish them from
spontaneous acid pH-resistant clones, following the first selection
where the bacteria are grown on LB agar plates at an acidic pH, the
clones may be pooled together, the genetic marker transferred, and
then multiple sibling clones tested for growth in acidified
media.
[0149] Identification and Selection of Osmolarity-Resistant
Gram-Negative Bacterial Mutants
[0150] Gram-negative bacteria, preferably attenuated gram-negative
bacteria, and more preferably attenuated gram-negative bacteria,
with resistance to high osmolarity can be identified and selected
for utilizing any method well-known to one of skill in the art. In
general, a pool of bacteria with genetic variations is subjected to
one or more selection criteria and the resistant clones are
isolated. A pool of gram-negative bacteria with genetic variations
may be composed of spontaneous mutants, a library of transposon
mutants or mutants transformed with a library of cloned DNA in a
multicopy plasmid. Selection techniques for isolating high
osmolarity-resistant gram-negative bacteria may include, but are
not limited to, growing the bacteria on agar plates having high
osmolarity at 37.degree. C., growing the bacteria in nutrient broth
having high osmolarity at 37.degree. C., and then growing the
bacteria on agar plates having or not having high osmolarity at
37.degree. C. Examples of agents that result in high osmolarity
include, but are not limited to, salts (e.g., NaCl or KCl) and
sugars (e.g., sucrose or glucose). Individual colonies are then
isolated from the agar plates and tested for growth in media having
high osmolarity at 37.degree. C. Colonies with greater growth
ability than the parental strain of bacteria from which they were
derived are deemed to have resistance to high osmolarity. In a
specific embodiment, individual colonies of gram-negative bacteria
with genetic variations are deemed to have resistance to high
osmolarity if they grow to 2 fold, preferably 4 fold, 6 fold, 8
fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 45
fold, 50 fold, 55 fold, 60 fold, 65 fold, 70 fold, 75 fold or
higher levels in media having high osmolarity than the parental
strain of bacteria from which they were derived. In another
embodiment, individual colonies of gram-negative bacteria with
genetic variations are deemed to have resistance to high osmolarity
if their growth is 5%, preferably 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or
greater in media having high osmolarity than the parental strain of
bacteria from which they were derived.
[0151] In order to distinguish spontaneous osmolarity-resistant
mutants from transposon and plasmid-based clones, the transposon or
plasmid can be transferred to another strain using selection for
the appropriate antibiotic marker found on the transposon or
plasmid. Several sibling colonies may then be isolated and tested
for resistance to high osmolarity, thus avoid spontaneous mutants.
In order to simultaneously transfer large numbers of transposon or
plasmid-based stress-resistant clones to distinguish them from
spontaneous osmolarity-resistant clones, following the first
selection where the bacteria are grown on LB agar plates having
high osmolarity, the clones may be pooled together, the genetic
marker transferred, and then multiple sibling clones tested for
growth in media having high osmolarity.
[0152] Genetic Modifications to Stress-Resistant Mutants with
Transposon Insertions
[0153] Stress-resistant gram-negative mutants with transposon
insertions can be re-engineered to have a deletion and/or insertion
in the same site in order to eliminate the antibiotic resistance
and transposon element. First, the site of the transposon insertion
is determined using standard techniques well-known to those skilled
in the art. Such techniques include, e.g., cloning from chromosomal
DNA based on selection for antibiotic resistance and sequencing of
the adjacent region, using GenomeWalker.TM. (Clontech, Palo Alto,
Calif.) or direct chromosomal sequencing (Qiagen, Valencia,
Calif.). A deletion and/or insertion is then constructed using PCR
to generate the two segments necessary for the use of the sucrase
vector (Donnenberg and Kaper, 1991, Infection and Immunity 59:
4310-4317). A multiple cloning site can be engineered at the
junction of the two segments used to create an insertion. The
insertion can be non-coding DNA or coding DNA (e.g., a nucleotide
sequence encoding a therapeutic molecule such as prodrug-converting
enzyme).
[0154] The genetic modification of a spontaneous mutant may be
identified using standard techniques well-known to one of skill in
the art. One technique to identify the genetic modification(s) of a
spontaneous mutant uses linkage to transposons, as described by
Murray et al., 2001, J. Bacteriology 183: 5554-5561. Another
technique to identify the genetic modification(s) of a spontaneous
mutant is to generate a DNA library derived from the strain of
interest in a low-copy or transposon vector and to select for
resistance to a particular stress condition. The plasmid or
transposon DNA is then sequenced as described above. Another
technique to identify the genetic modification(s) of a spontaneous
mutant is to use a Genechip approach. In the Genechip approach
differences between the spontaneous mutant and the parental strain
are identified. The spontaneous deletion, rearrangement,
duplication or other form of mutation identified in the spontaneous
mutant may then be re-engineered into a multicopy plasmid such as
asd vector or a sucrase chromosomal vector as described above.
[0155] Kits
[0156] Similarly, the invention may also provide a kit comprising
(a) a first container comprising a bacterial expression codon
optimized antigen from a pathogenic avian influenza virus strain
containing unique genetically engineered restriction sites
contained within either a bacterial protein expression plasmid or a
bacterial chromosomal protein expression vector and (b) a second
container comprising bacterial vector(s) with one or more (e.g.,
fH1, fH2 or fH0) flagellar antigen(s) and/or various
non-overlapping O-antigens. Component (a) will be modifiable to
genetically match an emerging avian influenza virus using standard
in vitro molecular techniques and can be combined with component
(b) to generate one or more bacterial strains with defined
flagellar antigens which constitute a live vaccine. The
variation(s) in flagellar antigens provided by the kit provide for
more than one live vaccine strain in which a first immunization
(prime) using one strain may be followed at an appropriate time
such as 2 to 4 weeks by a second immunization (boost) using a
second strain with a different fH antigen or no fH antigen. The
live vaccine compositions are suitable for oral administration to
an individual to provide protection from avian influenza.
[0157] The invention also provides a pharmaceutical pack or kit
comprising one or more containers with one or more of the
components of the pharmaceutical compositions of the invention.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration.
[0158] In a specific embodiment of the invention, the kit comprises
one or more stress-resistant gram-negative bacterial mutants and
optionally means of administering the pharmaceutical compositions
of the invention. The different stress-resistant gram-negative
bacterial mutants may comprise nucleotide sequences encoding one or
more therapeutic molecules. The kit may further comprise
instructions for use of said stress-resistant gram-negative
bacterial mutants. In certain embodiments of the invention, the kit
comprises a document providing instruction for the use of the
composition of the invention in, e.g., written and/or electronic
form. Said instructions provide information relating to, e.g.,
dosage, method of administration, and duration of treatment.
[0159] In one embodiment, a kit of the invention comprises a
stress-resistant gram-negative bacterial mutant in a vial and
instructions for administering the stress-resistant gram-negative
bacterial mutants for viral prophylaxis or treatment, wherein the
stress-resistant gram-negative bacterial mutant is a facultative
anaerobe or facultative aerobe. In accordance with this embodiment,
the stress-resistant gram-negative bacterial mutant may be
engineered to express one or more nucleic acid molecules encoding
one or more therapeutic molecules. In another embodiment, a kit of
the invention comprises an anti-viral agent contained in a first
vial, a stress-resistant gram-negative bacterial mutant in a second
vial, and instructions for administering the anti-viral agent and
stress-resistant gram-negative bacterial mutant to a subject for
viral infection prophylaxis or treatment. In accordance with this
embodiment, stress-resistant gram-negative bacterial mutant may be
engineered to express one or more nucleic acid molecules encoding
one or more therapeutic molecules. Preferably, the stress-resistant
gram-negative bacterial mutants included in the kits of the
invention are stress-resistant gram-negative Salmonella
mutants.
[0160] In another embodiment, a kit of the invention comprises an
attenuated stress-resistant gram-negative bacterial mutant in a
vial and instructions for administering the attenuated
stress-resistant gram-negative bacterial mutant to a subject for
viral infection prophylaxis or treatment, wherein the attenuated
stress-resistant gram-negative bacterial mutant is a facultative
anaerobe or facultative aerobe. In accordance with this embodiment,
the attenuated stress-resistant gram-negative bacterial mutant may
be engineered to express one or more nucleic acid molecules
encoding one or more therapeutic molecules. In another embodiment,
a kit of the invention comprises an anti-viral agent contained in a
first vial, an attenuated stress-resistant gram-negative bacterial
mutant contained in a second vial, and instructions for
administering the anti-viral agent and attenuated stress-resistant
gram-negative bacterial mutant to a subject for viral infection
prophylaxis or treatment. In accordance with this embodiment, the
attenuated stress-resistant gram-negative bacterial mutant may be
engineered to express one or more nucleic acid molecules encoding
one or more therapeutic molecules. Preferably, the attenuated
stress-resistant gram-negative bacterial mutants included in the
kits of the invention are attenuated stress-resistant gram-negative
Salmonella mutants.
[0161] In another embodiment, a kit of the invention comprises a
stress-resistant gram-negative bacterial mutant for viral infection
prophylaxis or treatment in a vial and instructions for
administering the stress-resistant gram-negative bacterial mutant
to a subject, where the stress-resistant gram-negative bacterial
mutant is a facultative anaerobe or facultative aerobe. In
accordance with this embodiment, the stress-resistant gram-negative
bacterial mutant may be engineered to express one or more nucleic
acid molecules encoding one or more therapeutic molecules. In
another embodiment, a kit of the invention comprises an anti-viral
agent contained in a first vial, a stress-resistant gram-negative
bacterial mutant contained in a second vial, and instructions for
administering the anti-viral agent and stress-resistant
gram-negative bacterial mutant to a subject with a for viral
infection prophylaxis or treatment. In accordance with this
embodiment, the stress-resistant gram-negative bacterial mutant may
be engineered to express one or more nucleic acid molecules
encoding one or more therapeutic molecules.
[0162] The present invention incorporates a combination of
bacterial vector and protein expression technology which results in
a unique vaccine which is rapidly constructed in response to
emerging avian influenza and their highly pathogenic derivatives.
The present invention is directed to the construction bacterially
codon optimized avian and human influenza genes and their
incorporation into a Salmonella strain for therapeutic use in the
prevention of avian influenza and highly pathogenic derivatives. An
antigen-expressing plasmid or chromosomal construct in the
bacterial strains described herein may also contain one or more
transcriptional terminators adjacent to the 3' end of a particular
nucleotide sequence on the plasmid to prevent undesired
transcription into another region of the plasmid or chromosome.
Such transcription terminators thus serve to prevent transcription
from extending into and potentially interfering with other critical
plasmid functions, e.g., replication or gene expression. Examples
of transcriptional terminators that may be used in the
antigen-expressing plasmids described herein include, but are not
limited to, the TI and T2 transcription terminators from 5S
ribosomal RNA bacterial genes (see, e.g., FIGS. 1-5; Brosius and
Holy, Proc. Natl. Acad. Sci. USA, 81: 6929-6933 (1984); Brosius,
Gene, 27(2): 161-172 (1984); Orosz et al., Eur. J Biochem., 20 (3):
653-659 (1991)).
[0163] The mutations in an attenuated bacterial host strain may be
generated by integrating a homologous recombination construct into
the chromosome or the endogenous Salmonella virulence plasmid
(Donnenberg and Kaper, 1991; Low et al. (Methods in Molecular
Medicine, 2003)). In this system, a suicide plasmid is selected for
integration into the chromosome by a first homologous recombination
event, followed by a second homologous recombination event which
results in stable integration into the chromosome. The
antigen-expressing chromosomal integration constructs described
herein comprise one or more nucleotide sequences that encode one or
more polypeptides that, in turn, comprise one or more avian
influenza antigens, such as the hemagglutinin and neuraminidase
polypeptide antigens, or immunogenic portions thereof, from avian
influenza virus and highly pathogenic derivatives. Such coding
sequences are operably linked to a promoter of transcription that
functions in a Salmonella bacterial strain even when such a
bacterial strain is ingested, i.e., when a live vaccine composition
described herein is administered orally to an individual. A variety
of naturally occurring, recombinant, and semi-synthetic promoters
are known to function in enteric bacteria, such as Escherichia coli
and serovars of S. enterica (see, e.g., Dunstan et al., Infect.
Immun., 67(10): 5133-5141 (1999)). Promoters (P) that are useful in
the invention include, but are not limited to, well known and
widely used promoters for gene expression such as the naturally
occurring Plac of the lac operon and the semi-synthetic Ptrc (see,
e.g., Amman et al., Gene, 25 (2-3): 167-178 (1983)) and Ptac (see,
e.g., Aniann et al., Gene, 69(2): 301-315 (1988)), as well as PpagC
(see, e.g., Hohmann et al., Proc. Natl. Acad. Sci. USA, 92.
2904-2908 (1995)), PpmrH (see, e.g., Gunn et al., Infect. Immun.,
68: 6139-6146 (2000)), PpmrD (see, e.g., Roland et al., J
Bacteriol., 176: 3589-3597 (1994)), PompC (see, e.g., Bullifent et
al., Vacccine, 18: 2668-2676 (2000)), PnirB (see, e.g., Chatfield
et al., Biotech. (NY), 10: 888-892 (1992)), PssrA (see, e.g., Lee
et al., J Bacteriol. 182. 771-781 (2000)), PproU (see, e.g.,
Rajkumari and Gowrishankar, J Bacteriol., 183. 6543-6550 (2001)),
Pdps (see, e.g., Marshall et al., Vaccine, 18: 1298-1306 (2000)),
and PssaG (see, e.g., McKelvie et al., Vaccine, 22: 3243-3255
(2004)), Some promoters are known to be regulated promoters that
require the presence of some kind of activator or inducer molecule
in order to transcribe a coding sequence to which they are operably
linked. However, some promoters may be regulated or inducible
promoters in E. coli, but function as unregulated promoters in
Salmonella. An example of such a promoter is the well-known trc
promoter ("Ptrc", see, e.g., Amman et al., Gene, 25(2-3): 167-178
(1983); Pharmacia-Upjohn). As with Plac and Ptac, Ptrc functions as
an inducible promoter in Escherichia coli (e.g., using the inducer
molecule isopropyl-p-D-1 8 thio-galactopyranoside, "IPTG"),
however, in Salmonella bacteria having no Lad repressor, Ptrc is an
efficient constitutive promoter that readily transcribes avian
influenza antigen-containing polypeptide coding sequences present
on antigen-expressing plasmids described herein. Accordingly, such
a constitutive promoter does not depend on the presence of an
activator or inducer molecule to express an antigen-containing
polypeptide in a strain of Salmonella.
[0164] The avian influenza antigen-expressing chromosomal
integration constructs which integrate into the live vaccine
strains also contain an origin of replication (ori) that enables
the precursor plasmids to be maintained as multiple copies in
certain the bacterial cells which carry the lambda pir element. For
the process of cloning DNA, a number of multi-copy plasmids that
replicate in Salmonella bacteria are known in the art, as are
various origins of replications for maintaining multiple copies of
plasmids. Preferred origins of replications for use in the
multi-copy antigen-expressing plasmids described herein include the
origin of replication from the multi-copy plasmid pBR322 ("pBR
ori"; see, e.g., Maniatis et al., In Molecular Cloning: A
Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring
Harbor, 1982), pp. 479-487; Watson, Gene, 70: 399-403, 1988), the
low copy origin of replication from pACYC177, and the origin of
replication of pUC plasmids ("pUC ori"), such as found on plasmid
pUC 1 8 (see, e.g., Yanish-Perron et al., Gene, 33: 103-119
(1985)). Owing to the high degree of genetic identity and homology,
any serovar of S. enterica may be used as the bacterial host for a
live vaccine composition for avian influenza, provided the
necessary attenuating mutations and antigen-expressing plasmids as
described herein are also employed. Accordingly, serovars of S.
enterica that may be used in the invention include those selected
from the group consisting of Salmonella enterica serovar
Typhimurium ("S. typhimurium"), Salmonella montevideo, Salmonella
enterica serovar Typhi ("S. typhi"), Salmonella enterica serovar
Paratyphi B ("S. paratyphi B"), Salmonella enterica serovar
Paratyphi C ("S. paratyphi C"), Salmonella enterica serovar Hadar
("S. Hadar"), Salmonella enterica serovar Enteriditis ("S.
enteriditis"), Salmonella enterica serovar Kentucky ("S.
kentucky"), Salmonella enterica serovar Infantis ("S. infantis"),
Salmonella enterica serovar Pullorum ("S. pullorum"), Salmonella
enterica serovar Gallinarum ("S. gallinarum"), Salmonella enterica
serovar Muenchen ("S. muenchen"), Salmonella enterica serovar
Anaturn ("S. anatum"), Salmonella enterica serovar Dublin ("S.
dublin"), Salmonella enterica serovar Derby ("S. derby"),
Salmonella enterica serovar Choleraesuis var. kunzendorf ("S.
cholerae kunzendorf"), and Salmonella enterica serovar minnesota
("S. minnesota").
[0165] The vaccine compositions described herein may be
administered orally to an individual in any form that permits the
Salmonella bacterial strain of the composition to remain alive and
to persist in the gut for a time sufficient to elicit an immune
response to one or more avian influenza antigens of avian influenza
virus and highly pathogenic derivatives expressed in the Salmonella
strain. For example, the live bacterial strains described herein
may be administered in relatively simple buffer or saline solutions
at physiologically acceptable pH and ion content. By
"physiologically acceptable" is meant whatever is compatible with
the normal functioning physiology of an individual who is to
receive a live vaccine composition described herein. Preferably,
bacterial strains described herein are suspended in otherwise
sterile solutions of bicarbonate buffers, phosphate buffered saline
(PBS), or physiological saline, that can be easily swallowed by
most individuals. However, "oral" routes of administration may
include not only swallowing from the mouth a liquid suspension or
solid form comprising a live bacterial strain described herein, but
also administration of a suspension of a bacterial strain through a
nasal spray or pulmonary inhaler, a nasojejunal or gastrostomy
tube, and rectal administration, e.g., by using a suppository
comprising a live bacterial strain described herein to establish an
infection by such bacterial strain in the lower intestinal tract of
the alimentary canal. Accordingly, any of a variety of alternative
modes and means may be employed to administer a vaccine composition
described herein to the alimentary canal of an individual if the
individual cannot swallow from the mouth.
[0166] FIG. 1 shows a selection scheme for isolation of transposon
insertions which confer CO.sub.2 resistance. Beginning with the
YS1646 strain which is CO.sub.2 sensitive, a library of mutants is
created using transposon insertional mutagenesis (e.g., EZ::Tn,
Epicentre, Madison, Wis.). The library is then plated to LB plates
and incubated in a 5% CO.sub.2 containing environment at 37.degree.
C. This results in numerous colonies on the plates which are
CO.sub.2 resistant, which could be either due to the transposon, or
due to spontaneous mutations. In order to isolate the
transposon-related CO.sub.2-resistant colonies, the colonies are
scraped off the plate using media and a bent glass rod in order to
pool the colonies. A phage lysate is prepared from the pooled
colonies and used to re-transduce YS1646 which is plated to
kanamycin. This results in numerous kanamycin-resistant colonies.
These colonies are then individually patched to a master plate and
replica plated to LB and incubated in a CO.sub.2 environment in
order to confirm transpon-derived CO.sub.2 resistance phenotype.
The retransduction and replica plating is then performed on an
individual colony basis. Colonies confirmed to have CO.sub.2
resistance associated with the transposon are subjected to genome
walking techniques which results in identifying the chromosomal
insertion site.
[0167] FIG. 2 shows sensitivity and resistance to CO.sub.2 shown by
comparing colony forming units (CFUs). In each of the two panels,
the number of colonies on the right is compared with the number of
colonies on the left to indicate sensitivity or resistance. Wild
type Salmonella on LB media in either air (left) or 5% CO.sub.2
showed no sensitivity to the CO.sub.2 conditions (not shown in
FIGS. 2A and 2B). FIG. 2A shows growth of VNP20009 (YS1646; 41.2.9)
on LB media in either air (left) or CO.sub.2 (right) showing strong
sensitivity to CO.sub.2. FIG. 2B shows VNP20009 .DELTA.zwf on LB
media in either air (left) or CO.sub.2 (right) showing that
.DELTA.zwf confers resistance to CO.sub.2 of an msbB.sup.-
strain.
[0168] FIGS. 3A-3D show that msbB.sup.- confers growth sensitivity
in liquid media under CO.sub.2 conditions containing physiological
amounts of salt and is suppressed by zwf.sup.-. Two sets of
Salmonella strains, YS873 and YS873 zwf.sup.-, and ATCC 14028 and
ATCC 14028 zwf.sup.- were grown on either LB or LB-0 in either air
or CO.sub.2. FIG. 3A: In LB media under ambient air conditions,
YS873 and YS873 zwf.sup.- show a normal growth curve. However,
under CO.sub.2 conditions, the YS873 strain is highly inhibited and
shows as reduction in the number of CFUs whereas the YS873
zwf.sup.- strain grows at a much greater rate. FIG. 3B: In LB-0,
the CO.sub.2 sensitivity is much less, and is not suppressed by the
zwf mutation. FIGS. 3C and 3D: Wild type Salmonella strain ATCC
14028 and 14028 zwf.sup.- show similar growth properties in either
LB or LB-0 with or without CO.sub.2.
[0169] FIG. 4 shows results of .beta.-galactosidase release assays
which confirm cell lysis in LB in the presence of 5% CO.sub.2 and
that zwf confers resistance. Release of .beta.-galactosidase from
the cytosol of the bacteria was used to test if the decrease in CFU
observed in YS873 in LB in the presence of 5% CO.sub.2 resulted
from cell lysis. The strains used were Salmonella YS873 and YS873
zwf.sup.- grown under either ambient air or 5% CO.sub.2 conditions.
After 2 hours growth, there is little difference between the
strains under either of the growth conditions. After 6 hours of
growth, significant cell lysis, as measured by the release of the
cytoplasmic enzyme .beta.-galactosidase, is observed in YS873 grown
in the presence of 5% CO.sub.2. Furthermore, a loss-of-function
mutation in zwf significantly reduces cell lysis in YS873. No
significant cell lysis is observed in the absence of CO.sub.2.
[0170] FIGS. 5A-5D show that zwf suppresses sensitivity to acidic
pH in LB broth. Two sets of Salmonella strains, YS873 and YS873
zwf.sup.-, and ATCC 14028 and ATCC 14028 zwf.sup.- were grown on LB
at either low pH (pH 6.6) or physiological pH (pH 7.6) in either
air or 5% CO.sub.2. FIG. 5A: Under ambient air conditions, YS873 is
strongly growth inhibited at pH 6.6, compared to the YS873
zwf.sup.- which suppresses the inhibition and restores normal
growth, while at pH 7.6, both strains grow normally. FIG. 5B: Under
5% CO.sub.2, the zwf mutation suppressed the sensitivity to acid pH
compared to the YS873 strain, which lost viability during the
6-hour time period. Moreover, the zwf mutation changed the pH
optimum of the strain, which now grew better at pH 6.6 than at pH
7.6. FIGS. 5C and 5D: Wild type Salmonella strain ATCC 14028 and
14028 zwf.sup.- show similar growth properties an either pH 6.6 or
pH 7.6 with or without CO.sub.2.
[0171] FIGS. 6A and 6B show results of .beta.-galactosidase assays
which confirm cell lysis in LB broth, pH 6.6 and that zwf confers
resistance. Release of .beta.-galactosidase from the cytosol of the
bacteria was used to test if the decrease in CFU observed in YS873
in LB at pH 6.6+/- the presence of 5% CO.sub.2 resulted from cell
lysis. The strains used were Salmonella YS873 and YS873 zwf.sup.-
grown in LB broth at either pH 6.5 or pH 7.5 under either ambient
air or 5% CO.sub.2 conditions. FIG. 6A: Under ambient air
conditions after 8 hours, significant cell lysis occurs after
growth of YS873 in LB broth, pH 6.5 but not pH 7.5. Furthermore, a
loss-of-function mutation in zwf significantly reduces cell lysis
of YS873 grown in LB broth pH 6.6. FIG. 6B: Under 5% CO.sub.2
conditions after 8 hours, cell lysis is suppressed only in the
YS873 zwf.sup.- strain at pH 6.5, again showing a shift in pH
optimum for this strain.
[0172] FIG. 7 shows that the zwf mutation suppresses both
msbB-induced CO.sub.2 sensitivity and osmotic defects. Different
media and growth conditions were used to indicate the ability of
small patches of bacteria (3 each) to grow using the replica
plating technique. The strains used are listed on the left: wt,
wild type Salmonella typhimurium ATCC 14028; YS1, Salmonella
typhimurium ATCC 14028 containing the msbB mutation; YS1 zwf::kan,
the YS1 strain with a kanamycin containing transposing insertion
into the zwf gene; YS873, the YS1 strain with a deletion in the
somA gene; YS873 zwf::kan, the YS873 strain with a kanamycin
containing Tn5 transposon disrupting the zwf gene. Growth
conditions maintained at 37.degree. C. used included: A, LB media
in air; B, LB media in 5% CO.sub.2; C, msbB media; D, msbB media in
5% CO.sub.2; E, LB-0 media in air; F, LB-O media in 5% CO.sub.2; G,
LB-0 media containing sucrose (total 455 milliosmoles); H, LB-0
media containing sucrose and 5% CO.sub.2; I, LB-0+gluconate
(glucon.) in air; J, LB-0+gluconate in 5% CO.sub.2.
[0173] FIG. 8 shows a selection scheme for isolation of transposon
insertions which confer acidic pH resistance. Beginning with the
YS1646 strain which is acidic pH sensitive, a library of mutants is
created using transposon insertional mutagenesis (e.g., EZ::Tn,
Epicentre, Madison, Wis.). The library is then plated to LB plates
at pH.ltoreq.6.6. This results in numerous colonies on the plates
which are acidic pH resistant, which could be either due to the
transposon, or due to spontaneous mutations. In order to isolate
the transposon-related acidic-resistant colonies, the colonies are
scraped off the plate using media and a bent glass rod in order to
pool the colonies. A phage lysate is prepared from the pooled
colonies and used to re-transduce YS1646 which is plated to
kanamycin. This results in numerous kanamycin-resistant colonies.
These colonies are then individually patched to a master plate and
replica plated to LB at pH.ltoreq.6.6 and incubated in order to
confirm transposon-derived acidic pH resistance phenotype. The
retransduction and replica plating is then performed on an
individual colony basis. Colonies confirmed to have an acidic pH
resistant phenotype associated with the transposon are subjected to
genome walking techniques which results in identifying the
chromosomal insertion site.
[0174] FIG. 9 shows a selection scheme for isolation of transposon
insertions which confer osmolarity resistance. Beginning with the
YS1646 strain which is osmolarity sensitive, a library of mutants
is created using transposon insertional mutagenesis (e.g., EZ::Tn,
Epicentre, Madison, Wis.). The library is then plated to LB plates
(containing salt). This results in numerous colonies on the plates
which are osmolarity resistant, which could be either due to the
transposon, or due to spontaneous mutations. In order to isolate
the transposon-related osmolarity-resistant colonies, the colonies
are scraped off the plate using media and a bent glass rod in order
to pool the colonies. A phage lysate is prepared from the pooled
colonies and used to re-transduce YS1646 which is plated to
kanamycin. This results in numerous kanamycin-resistant colonies.
These colonies are then individually patched to a master plate and
replica plated to LB and incubated in order to confirm
transpon-derived osmolarity resistance phenotype. The
retransduction and replica plating is then performed on an
individual colony basis. Colonies confirmed to have an osmolarity
resistant phenotype associated with the transposon are subjected to
genome walking techniques which results in identifying the
chromosomal insertion site.
[0175] In order to more fully illustrate the invention, the
following non-limiting examples are provided.
Example 1: Isolation and Identification of a Gene Involved in
Resistance to CO.sub.2, Acidic pH and/or Osmolarity
[0176] Isolation of CO.sub.2 Resistant Strains Using Transposon
Libraries.
[0177] Throughout the procedures, msbB.sup.+ strains were grown in
Luria-Bertani (LB) broth containing 10 g tryptone, 5 g yeast
extract, 10 g NaCl, pH adjusted as indicated using either 1N NaOH
or 1N HCl, or LB plates containing 1.5% agar at 37.+-.2.degree. C.
msbB.sup.- strains were grown in modified LB referred to as MSB
media (msbB media), containing 10 g tryptone, 5 g yeast extract 2
mL 1N CaCl.sub.2 and 2 mL 1N MgSO.sub.4 per liter, adjusted to pH
7.0 to 7.6 using 1N NaOH, or in LB broth or LB plates lacking NaCl,
referred to as LB-0. For transductions, LB lacking EGTA was used.
For sucrose resolutions, LB lacking NaCl and containing 5% sucrose
at 30.+-.2.degree. C. was used. Auxotrophic mutants are determined
on minimal media 56 (M56): 0.037 M KH.sub.2PO.sub.4, 0.06 M
Na.sub.2HPO.sub.4, 0.02% MgSO.sub.47H.sub.2O, 0.2%
(NH.sub.4).sub.2SO.sub.4, 0.001% Ca (NO.sub.3).sub.2, 0.00005%
FeSO.sub.47H.sub.2O, with a carbon source (e.g., glucose 0.1 to
0.3%) as sterile-filtered additive, and further supplemented with
the appropriate nutrients, 0.1 mg/ml thiamine and 50 mg/ml each of
adenine. Solid M56 media is made by preparing separate autoclaved
2.times. concentrates of the mineral salts and the agar, which are
combined after sterilization. Media are also supplemented with
antibiotics used as needed to select for resistance markers,
including tetracycline (Sigma) at 4 mg/ml from a stock: 10 mg/ml in
70% ethanol stored in darkness at -20.degree. C. or ampicillin at
100 mg/ml from a stock: 100 mg/ml in H.sub.2O, sterile filtered and
stored at -20.degree. C. The bacteria used are listed in Table
1.
TABLE-US-00001 TABLE 1 Parental Strain strain Genotype Derivation
or source S. enterica Wild type Wild type ATCC 14028 serovar
Manassas, VA Typhimurium 14028 14028 zwf 14028 .DELTA.zwf
Replacement of zwf YS1646 14028 .DELTA.msbB gene with .DELTA.zwf by
(VNP20009) .DELTA.purI homologous YS1 14028 msbB1::.OMEGA.tet
recombination msbB1::.OMEGA.tet Low et al., pp 47-59, In: Suicide
Gene Therapy: Methods and Protocols, C. Springer (ed), Humana
Press, 2003. Murray et al. 2001, J. Bacteriol. 183: 5554-5561. YS1
zwf YS1 zwf: Tn5 (Kan.sup.R) P22 zwf: Tn5 (Kan.sup.R)X
msbB1::.OMEGA.tet YS1 .fwdarw. Kan.sub.20.sup.r Murray somA YS873
14028 zbj10: Tn10 et al. 2001, J. msbB1::.OMEGA.tet Bacteriol. 183:
somA zbj10: Tn10 zwf: 5554-5561. Tn5 YS873 zwf YS873 (Kan.sup.R)
P22 zwf: Tn5 (Kan.sup.R)X YS873 .fwdarw. Kan.sub.20.sup.r
[0178] CO.sub.2 resistant mutants were obtained as outlined in FIG.
1. A Tn5 transposome (EZ::TN, Epicentre, Madison, Wis.) was used to
directly generate a library in YS1646, plated to MSB agar plates
with the appropriate antibiotic (kanamycin for Tn5) and grown
overnight at 37.degree. C. in ambient air. The plates were then
flooded with MSB broth and the colonies scraped from the plates,
pooled and frozen in aliquots at -80.degree. in 15% glycerol.
[0179] The library was screened by plating dilutions of the library
onto MSB agar and incubating them in 5% CO.sub.2 at 37.degree. C.
overnight. In particular, colonies were tested for CO.sub.2
resistance by plating serial dilutions of the library of bacteria
onto MSB plates and incubating the plates at 37.degree. C. in
either ambient air or in air with 5% CO.sub.2. Colonies that were
recovered from MSB plates incubated at 37.degree. C. in air with 5%
CO.sub.2 were deemed resistant to CO.sub.2. The resistance of these
colonies to CO.sub.2 could be due to either the presence of the
transposon insertion or to a spontaneous mutant. In order to
eliminate any background of spontaneous mutants, the
CO.sub.2-resistant colonies were pooled, P22 lysates prepared (P22
phage transduction by the method of Davis et al., 1980, Advanced
Bacterial Genetics, Cold Spring Harbor Press, Cold Spring Harbor,
N.Y.), and the Tn5 insertions transferred to YS1646 and plates for
individual colonies in MSB-kanamycin at 37.degree. C. in ambient
air. Individual colonies were then gridded and replica plated to
MSB plates and incubated at 37.degree. C. in either ambient air or
in air with 5% CO.sub.2. Those colonies which tested positive by
the replica plating were chosen for further study of retransduction
to confirm the phenotype. These clones were also tested to ensure
that there was no significant increase in TNF-.alpha. induction
using standard techniques such as those described by Low et al.,
1999. The CO.sub.2 resistant clones chosen to undergo further
testing include the clones designated 14.2, 32.2 and 37.2.
[0180] An example of the CO.sub.2 sensitivity and resistance
observed using the plating efficiency method is shown in FIG. 2.
The percent growth of the bacteria under a stress condition such as
CO.sub.2 is determined by plating to MSB agar plates and incubated
in either air or CO.sub.2, and dividing the number of clones on the
stress-subjected plate to the number of clones in the
non-stress-subjected plate. General observation of the plate is
often sufficient to determine sensitivity and resistance. The wild
type bacteria do not show any obvious reduction in the number of
CFUs observed, whereas the strain YS1646 shows a dramatic reduction
in the number of bacteria observed. The CO.sub.2 resistant mutant
32.2 shows approximately the same number of colonies when grown
either under ambient air conditions or 5% CO.sub.2, indicating
CO.sub.2 resistance. The GenomeWalker (Clonetech, Palo Alto,
Calif.) kit was used to determine the chromosomal insertion site in
clones designated 14.2, 32.2 and 37.2. The Tn5 was determined to be
located in the zwf gene in all three clones, two of them (clones
14.2 and 32.2) were located after base pair 1019 and the third
(clone 37.2) was located after base pair 1349. Therefore, zwf.sup.-
confers CO.sub.2 resistance in the msbB.sup.- strain YS1646.
[0181] Suppression of CO.sub.2-Mediated Growth Inhibition by
zwf.sup.- Mutants.
[0182] In order to further analyze the effect of the zwf mutation
on growth of the msbB.sup.- Salmonella, growth rates under
different conditions were studied. To generate growth curves, 3 ml
broth tubes were inoculated with single colonies and grown on a
shaker overnight at 37.degree. C. An adequate amount of LB or LB-0
broth was then inoculated 1:1000 with cells. Cells were held on ice
until all inoculations were completed. Triplicate 3 ml aliquots
were then placed in a 37.degree. C. shaker with 250 rpm in air or
5% CO.sub.2. O.D..sub.600 was measured every 60 minutes and
dilutions of bacteria were plated onto MSB or LB agar plates to
calculate the number of colony forming units (CFU) per ml.
[0183] FIG. 3 shows the growth of wild type ATCC 14028, 14028
zwf.sup.-, YS873, and YS873 zwf.sup.- in LB and LB-0 broth, grown
in the presence or absence of 5% CO.sub.2. The growth of YS873
(FIG. 3A), but not ATCC 14028 (FIG. 3C) is greatly impaired LB
broth in the presence of 5% CO.sub.2. A significant decrease in CFU
is observed (FIG. 3A), indicating that YS873 cells lose viability
in the presence of 5% CO.sub.2 in LB broth. When a loss-of-function
mutation in zwf is incorporated into YS873, no loss in viability is
observed under identical conditions, although there is a longer lag
phase of growth and the CFU does not increase at the same rate as
in LB broth in the absence of 5% CO.sub.2 (FIG. 3A). In LB-0 broth,
there are no growth defects in 14028 or 14028 zwf.sup.- (FIG. 3D).
For YS873 and YS873 zwf.sup.-, the growth defects in LB-0 in the
presence of 5% CO.sub.2 are attenuated in comparison to those
observed in LB broth. There is no decrease in viability in YS873 in
LB-0 in 5% CO.sub.2, although there is a decreased growth rate in
both YS873 and YS873 zwf.sup.- in LB-0 in the presence of CO.sub.2
compared to growth in the absence of CO.sub.2 (FIG. 3B).
[0184] Suppression of CO.sub.2 Mediated Cell Lysis by zwf.sup.-
Mutants.
[0185] To test if the decrease in CFU observed in YS873 in LB in
the presence of 5% CO.sub.2 resulted from cell lysis, release of
.beta.-galactosidase (a cytoplasmic enzyme not normally present in
the culture supernatant) was determined. For .beta.-galactosidase
expression, lacZ was cloned into the high copy vector pSP72
(Promega) and screened for bright blue colonies on LB agar
containing 40 .mu.g/ml X-gal. .beta.-gal assays were performed
according to the instructions for the Galacto-Star.TM.
chemiluminescent reporter gene assay system (Applied Biosystems,
Bedford, Mass.). Briefly, 1 ml of bacterial culture expressing
.beta.-Gal from pSP72 was pelleted at 13,000.times.g for 5 min.
Supernatants were filtered through a 0.2 mm syringe filter and then
assayed immediately or frozen at -80.degree. C. until assayed with
no further processing. Cell pellets were quickly freeze-thawed and
suspended in 50 .mu.l or 200 ml B-Per.TM. bacterial cell lysis
reagent (Pierce Chemical) containing 10 mg/ml lysozyme (Sigma).
Bacteria were allowed to lyse for 10-20 min. at room temperature
and then placed on ice. All reagents and samples were allowed to
come to room temperature before use. Filtered supernatants and
bacterial lysates were diluted as needed in Galacto-Star.TM. Lysis
Solution or assayed directly. 3-gal standard curves were made by
preparing recombinant .beta.-gal (Sigma, 600 units/mg) to 4.3 mg/ml
stock concentration in 1.times.PBS. The stock was diluted in Lysis
Solution to prepare a standard curve of 100 ng/ml-0.05 ng/ml in
doubling dilutions. 20 ml of standard or sample was added to each
well of a 96-well tissue culture plate. 100 ml of Galacto-Star.TM.
Substrate diluted 1:50 in Reaction Buffer Diluent was added to each
well and the plate rotated gently to mix. The plate was incubated
for 90 minutes at 25.degree. C. in the dark and then read for 1
second/well in an L-Max.TM. plate luminometer (Molecular Devices).
Sample light units/ml were compared to the standard curve and
values converted to units .beta.-gal/ml. Percent release of
.beta.-gal was determined by dividing units/ml supernatant by total
units/ml (units/ml supernatant+units/ml pellet). All samples were
assayed in triplicate. As shown in FIG. 4, after 6 hours of growth,
significant cell lysis, as measured by the release of the
cytoplasmic enzyme .beta.-galactosidase, is observed in YS873 grown
in the presence of 5% CO.sub.2. No significant cell lysis is
observed in the absence of CO.sub.2. Furthermore, a
loss-of-function mutation in zwf significantly reduces
CO.sub.2-mediated cell lysis in YS873.
[0186] Suppression of Acidic pH Mediated Growth Inhibition by zwf
Mutants.
[0187] To test if increased or reduced pH would reduce sensitivity
to CO.sub.2, LB media was buffered to pH 6.6 or 7.6 and cultures
were grown in the presence or absence of 5% CO.sub.2. As shown in
FIG. 5, wild type ATCC 14028 and ATCC 14028 zwf.sup.- grow normally
under all conditions (FIGS. 5C and 5D). In contrast, the growth of
YS873 is significantly impaired when the pH of LB is 6.6 under
ambient air conditions, with no significant increase in CFU after 6
hours (FIG. 5A). In contrast, when the pH of LB is 7.6, YS873 grows
well (FIG. 5A). A loss-of-function mutation in zwf allows for YS873
to grow well in LB broth at a pH of 6.6 (FIG. 5A). Under 5%
CO.sub.2, the zwf mutation suppressed the sensitivity to acid pH
compared to the YS873 strain (FIG. 5B), which lost viability during
the 6-hour time period. Moreover, the zwf mutation changed the pH
optimum of the strain, which now grew better at pH 6.6 than at pH
7.6.
[0188] Suppression of Acidic pH Mediated Cell Lysis by zwf
Mutants.
[0189] Release of .beta.-galactosidase from the cytosol of the
bacteria was used to test if the decrease in CFU observed in YS873
in LB at pH 6.6+/- the presence of 5% CO.sub.2 resulted from cell
lysis (FIGS. 6A and 6B). The strains used were Salmonella YS873 and
YS873 zwf.sup.- grown in LB broth at either pH 6.5 or pH 7.5 under
either ambient air or 5% CO.sub.2 conditions. Under ambient air
conditions after 8 hours, significant cell lysis occurs after
growth of YS873 in LB broth, pH 6.5 but not pH 7.5 (FIG. 6A).
Furthermore, a loss-of-function mutation in zwf significantly
reduced cell lysis of YS873 grown in LB broth pH 6.6. Under 5%
CO.sub.2 conditions after 8 hours, cell lysis is suppressed only in
the YS873 zwf.sup.- strain at pH 6.5, again showing a shift in pH
optimum for this strain (FIG. 6B).
[0190] Determination of Osmolarity and Gluconate Sensitivity
Properties of zwf Mutants.
[0191] Phenotypes of strains were determined by replica plating
(FIG. 7). Master plates were made on either MSB or LB-0 agar.
Plates were supplemented with sucrose (455 mOsmol) instead of NaCl,
or 0.33% gluconate, a downstream product in the same pathway as
zwf, thus restoring or enhancing the pathway using a metabolic
supplement. Replica plating was performed using a double velvet
technique. Plates were incubated for 16 hours at 37.degree. C. in
either ambient air or 5% CO.sub.2. For comparative purposes, the
wild type Salmonella ATCC 14028, YS1, YS1 zwf::kan, YS873 and YS873
zwf::kan were used.
[0192] As shown in the replica series of FIG. 7, growth of
unsuppressed YS1 is inhibited on LB (FIG. 7A) but YS1 grew well on
MSB and LB-0 agar (FIGS. 3C and 3D. In contrast, growth of YS1 on
MSB and LB-0 agar is completely inhibited when the plates are
incubated in the presence of 5% CO.sub.2. The introduction of the
zwf mutation completely compensates for the phenotype and allows
the bacteria to grow under 5% CO.sub.2 on all three media (FIG. 7
B, D, F). When NaCl in LB plates is substituted with sucrose at
iso-osmotic concentrations (FIG. 7 G), growth of YS1 is also
inhibited, indicating osmosensitivity of YS1. Introduction of the
zwf mutation improves growth of YS1 on LB and on LB-0 5% sucrose
agar, indicating that the zwf mutation can partially compensate for
the msbB-induced osmotic growth defect. YS873, which contains the
EGTA and salt resistance suppressor mutation somA (Murray et al.,
2001), grows well on LB, MSB, LB-0 and LB-0 sucrose agar plates in
air, but not when the plates are incubated in 5% CO.sub.2. In
contrast, the strain YS873 zwf.sup.- is able to grow on all plates
in CO.sub.2, indicating that the zwf mutation can compensate for
the growth defect of msbB strains in CO.sub.2. YS873 zwf.sup.- was
not able to grow on LB-0 gluconate in 5% CO.sub.2 (FIG. 7, I+J),
confirming the role of the zwf pathway in CO.sub.2 sensitivity.
[0193] Isolation and Identification of Genes Involved in Resistance
to Acid pH.
[0194] The Tn5 insertion library described above is screened to
identify mutants with resistance to acidity FIG. 8. The library is
screened by dilution of the library onto MSB agar or broth buffered
to pH 6.6 with 100 mM sodium phosphate buffer and incubating them
at 37.degree. C. overnight. Colonies that were recovered from
acidified MSB plates incubated at 37.degree. C. in air are deemed
acidic pH resistant. The resistance of these colonies could be due
to either the presence of the transposon insertion or to
spontaneous mutants. In order to eliminate any background of
spontaneous mutants, the acidic pH-resistant colonies are pooled,
P22 lysates prepared (P22 phage transduction by the method of Davis
et al., 1980, Advanced Bacterial Genetics, Cold Spring Harbor
Press, Cold Spring Harbor, N.Y.), and the Tn5 insertions
transferred to YS1646 and plated for individual colonies in pH 7.4
MSB-kanamycin at 37.degree. C. in ambient air. Individuals colonies
were then gridded and replica plated to pH 6.6 and pH 7.4 MSB
plates and incubated at 37.degree. C. Those colonies which tested
positive by the replica plating are chosen for further study of
retransduction to confirm the phenotype. These clones are also
tested to ensure that there was no significant increase in
TNF-.alpha. induction using standard techniques such as those
described by Low et al., 1999. The GenomeWalker (Clonetech, Palo
Alto, Calif.) kit is used to determine the chromosomal insertion
site in the acidic pH-resistant clones.
[0195] Isolation and Identification of Genes Involved in Resistance
to Osmolarity.
[0196] The Tn5 insertion library described above is screened to
identify mutants with resistance to osmolarity FIG. 9. The library
is screened by dilution of the library onto MSB agar or broth
containing sucrose such that it results in greater than 100
mOsmoles, at physiological osmolarity, (approx 300 mOsmole) or
greater (e.g., 450 mOsmole) and incubating them at 37.degree. C.
overnight. Colonies that were recovered from physiological
osmolarity or greater on MSB plates incubated at 37.degree. C. in
air are deemed osmolarity resistant. The resistance of these
colonies could be due to either the presence of the transposon
insertion or to a spontaneous mutant. In order to eliminate any
background of spontaneous mutants, the osmolarity-resistant
colonies are pooled, P22 lysates prepared (P22 phage transduction
by the method of Davis et al., 1980, Advanced Bacterial Genetics,
Cold Spring Harbor Press, Cold Spring Harbor, N.Y.), and the Tn5
insertions transferred to YS1646 and plated for individual colonies
in MSB-kanamycin at 37.degree. C. in ambient air. Individuals
colonies were then gridded and replica plated to MSB and
MSB-sucrose plates and incubated at 37.degree. C. Those colonies
which tested positive by the replica plating are chosen for further
study of retransduction to confirm the phenotype. These clones are
also tested to ensure that there was no significant increase in
TNF-.alpha. induction using standard techniques such as those
described by Low et al., 1999. The GenomeWalker (Clonetech, Palo
Alto, Calif.) kit is used to determine the chromosomal insertion
site in the osmolarity-resistant clones.
Example 2. Incorporation of Stress Resistance Genes for Use in
Genetically Stabilized and Isolated Strains with Defined Flagellar
Antigens and their Use in Protection Against Avian Influenza and
Highly Pathogenic Derivatives
[0197] Construction of an Antibiotic-Sensitive Non-Polar Deletion
in zwf.
[0198] A non-polar deletion in zwf was generated by constructing a
pCVD442 vector capable of deleting the entire zwf coding region by
homologous recombination with the Salmonella chromosome (Donnenberg
and Kaper, 1991 Infection & Immunity 59:4310-4317; Low et al.,
2003, Methods in Molecular Medicine; Suicide Gene Therapy, C.
Springer (ed), Humana Press, pp 47-59, expressly incorporated in
their entirety herein). Primers for PCR were designed that would
generate one product immediately upstream of the 5' ATG start codon
and a separate product immediately downstream of the 3' stop codon
of the zwf coding region. The two separate products could then be
ligated sequentially into the pCVD442 vector. The primers were:
TABLE-US-00002 zwf-5'-reverse: (SEQ ID No.: 1) 5'-GTG TGA GCT CGT
GGC TTC GCG CGC CAG CGG CGT TCC AGC-3' (with added SacI) and zwf-5'
forward: (SEQ ID No.: 2) 5'-GTG TGC ATG CGG GGG GCC ATA TAG GCC GGG
GAT TTA AAT GTC ATT CTC CTT AGT TAA TCT CCT GG-3' (with added
SphI); and zwf-3' reverse: (SEQ ID No.: 3) 5'-GTG TGC ATG CGG GGT
TAA TTA AGG GGG CGG CCG CAT TTG CCA CTC ACT CTT AGG TGG-3' and
3-forward: (SEQ ID No.: 4) 5'-GTG TGT CGA CCC TCG CGC AGC GGC GCA
TCC GGA TGC-3'.
[0199] The primers also generate internal NotI, PacI, SphI, SfiI,
and SwaI in order to facilitate cloning of DNA fragments, such as
the influenza H5 and N1 antigens into the .DELTA.zwf for stable
chromosomal integration without antibiotic resistance. This vector
is referred to as pCVD442-.DELTA.zwf. Presence of the deletion, in
Amp.sup.S Suc.sup.R colonies, was detected with PCR using the
following primers:
TABLE-US-00003 zwf-FL-forward: (SEQ ID No.: 5)
5'-ATATTACTCCTGGCGACTGC-3' and zwf-FL-reverse: (SEQ ID No.: 6)
5'-CGACAATACGCTGTGTTACG-3'.
[0200] Determination of Improved Penetration and Persistence in Gut
Tissues.
[0201] Standard methods are utilized to determine increased
penetration and persistence in gut tissues. In all cases,
comparison of different dose levels is performed, comparing the
parental, CO.sub.2, acidic pH, and/or osmolarity sensitive strain
with the CO.sub.2, acidic pH and/or osmolarity resistant strain(s).
1) Total recovery from gut material. Mice are orally administered
the parental strain and the resistant strains at different dose
levels. At fixed times between days 1 and 21 (e.g., d. 1, 7, 14
& 21) the mice are euthanized (avoiding CO.sub.2 asphyxiation)
and their gut collected by dissection. The gut is then homogenized
and serial dilutions plated for Salmonella on Salmonella selective
media such as SS agar, bismuth sulfite agar, or Hecktoen enteric
agar. The number of Salmonella present for the parental and
resistant strains at different times and dosing levels can then be
compared to demonstrate improved penetration and persistence in the
gut. 2) Determination of gut lining-associated Salmonella. Mice are
orally administered the parental strain and the resistant strains
at different dose levels. At fixed times between days 1 and 21
(e.g., d. 1, 7, 14 & 21) the mice are euthanized (avoiding
CO.sub.2 asphyxiation) and their gut collected by dissection. The
gut is then repeated flushed with a saline solution containing 100
ug/ml of gentamicin, an antibiotic that does not enter cells and
will therefore not kill any bacteria that have penetrated the gut
mucosal cells. The gut is then washed with saline to remove traces
of gentamicin, homogenized and serial dilutions plated for
Salmonella on Salmonella selective media such as SS agar, bismuth
sulfite agar, or Hecktoen enteric agar. The number of Salmonella
present for the parental and resistant strains at different times
and dosing levels can then be compared in order to demonstrate
improved gut penetration and persistence in the gut at lower
doses.
[0202] Determining Immune Response to H5N1-Expressing Bacteria.
[0203] Live bacterial vaccines for H5N1 influenza prophylaxis or
treatment described by Bermudes (WO/2008/03908) are engineered as
described above to have an additional mutation in a
stress-resistance gene such as zwf. Experimental determination of
vaccine activity is known to those skilled in the arts. By way of
non-limiting example, determination of an antibody response is
demonstrated.
[0204] 1) Vertebrate animals including mice, birds, dogs, cats,
horses, pigs or humans are selected for not having any known
current or recent (within 1 year) influenza infection or
vaccination. Said animals are pre-bled to determine background
binding to, for example, H5 and N1 antigens.
[0205] 2) The Salmonella expressing H5 and N1 are cultured on LB
agar overnight at 37.degree.. Bacteria expressing other H and or N
antigens may also be used.
[0206] 3) The following day the bacteria are transferred to LB
broth, adjusted in concentration to OD.sub.600=0.1
(.about.2.times.10.sup.8 cfu/ml), and subjected to further growth
at 37.degree. on a rotator to OD.sub.600=2.0, and placed on ice,
where the concentration corresponds to approx. 4.times.10.sup.9
cfu/ml.
[0207] 4) Following growth, centrifuged and resuspended in 1/10 the
original volume in a pharmacologically suitable buffer such as PBS
and they are diluted to a concentration of 10.sup.4 to 10.sup.9
c.f.u./ml in a pharmacologically suitable buffer on ice, warmed to
room temperature and administered orally or intranasally in a
volume appropriate for the size of the animal in question, for
example 50 .mu.l for a mouse or 10 to 100 ml for a human. The
actual dose measured in total cfu is determined by the safe dose as
described elsewhere in this application.
[0208] 5) After 2 weeks, a blood sample is taken for comparison to
the pretreatment sample. A booster dose may be given. The booster
may be the same as the initial administration, a different species,
a different serotype, or a different flagellar antigen (H1 or H2)
or no flagellar antigen.
[0209] 6) After an additional 2 to 4 weeks, an additional blood
sample may be taken for further comparison with the pretreatment
and 2-week post treatment.
[0210] 7) A comparison of preimmune and post immune antibody
response is performed by immunoblot or ELISA. A positive response
is indicated by a relative numerical value 2.times. greater then
background/preimmune assay.
[0211] Immunization with H5N1 Bacterial Vaccine Strains.
[0212] Live bacterial vaccines for H5N1 influenza prophylaxis or
treatment described by Bermudes (WO/2008/03908) are engineered as
described above to have an additional mutation in a
stress-resistance gene such as zwf. An experiment to determine if
H5N1 strains of Salmonella are capable of providing protection from
challenge with the wildtype strain. Ducks are immunized orally with
5.times.10.sup.9 cfu of bacteria when 4 weeks old, then challenged
with the standard challenge model of avian influenza at 6-weeks
age.
[0213] Birds in Group A are immunized with empty vector. Group B
receive Salmonella H5N1. Group C is immunized with Salmonella
expressing the Tamiflu resistant neuraminidase mutations. Birds in
Group D are not immunized. Each group is further divided into +/-
Tamiflu treatment. Results of these experiments can be used to
demonstrate the effectiveness of the vaccine on Tamiflu resistant
strain, with and without Tamiflu treatment.
[0214] Immunization with a Trimeric Hemagglutinin Antigen.
[0215] The bacteria described above in "Immunization with H5N1
Bacterial Vaccine Strains", are further engineered to contain a
trimeric immunogen described by Wei et al. 2008 (J Virology 82:
6200-6208, expressly incorporated by reference in its entirety
herein). The antigen is further modified to contain the HlyA
C-terminal 60 amino acids in-frame, in order to guide secretion
together with HlyBD (and a functional tolC). Immunization and
efficacy evaluations are performed as described above.
[0216] Control of Bacterial Infection with Gluconate.
[0217] As described herein, in an msbB.sup.- zwf.sup.- strains are
sensitive to physiological concentrations of CO.sub.2 in the
presence of gluconate. The ability of gluconate to control
excessive bacterial infections, such as might occur in a patient
who becomes immunocompromised or otherwise has their health
complicated such that the proliferation of the bacteria requires
control, can be modeled using immunocompromised mice, such as nude
(nu/nu) or severe combined immunodeficient (SCID) mice.
[0218] 1) The msbB.sup.- zwf.sup.- Salmonella cultured on LB agar
overnight at 37.degree.. 2) The following day the bacteria are
transferred to LB broth, adjusted in concentration to
OD.sub.600=0.1 (.about.2.times.10.sup.8 cfu/ml), and subjected to
further growth at 37.degree. on a rotator to OD.sub.600=2.0, and
placed on ice, where the concentration corresponds to approx.
4.times.10.sup.9 cfu/ml.
[0219] 3) Following growth, centrifuged and resuspended in 1/10 the
original volume in a pharmacologically suitable buffer such as PBS
and they are diluted t