U.S. patent application number 13/735638 was filed with the patent office on 2013-08-15 for live microbial microbicides.
This patent application is currently assigned to The USA, as represented by the Secretary, Department of Health and Human Services. The applicant listed for this patent is The USA, as represented by the Secretary, Department of Health and Human Services, The USA, as represented by the Secretary, Department of Health and Human Services. Invention is credited to Dean HAMER.
Application Number | 20130209407 13/735638 |
Document ID | / |
Family ID | 35949951 |
Filed Date | 2013-08-15 |
United States Patent
Application |
20130209407 |
Kind Code |
A1 |
HAMER; Dean |
August 15, 2013 |
LIVE MICROBIAL MICROBICIDES
Abstract
The present invention relates, e.g., to a commensal bacterium
which can colonize the genitourinary and/or gastrointestinal
mucosa, and which, under suitable conditions, secretes a
heterologous antimicrobial polypeptide, wherein the secreted
antimicrobial polypeptide is effective to inhibit infectivity by,
or a pathogenic activity of, a pathogen. In a most preferred
embodiment, the antimicrobial polypeptide inhibits HIV infection
(e.g., fusion) and/or pathogenesis. Also described are preventive
or therapeutic compositions comprising the commensal bacteria, and
methods to inhibit infectivity and/or pathogenesis, using the
bacteria.
Inventors: |
HAMER; Dean; (Washington,
DC) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Department of Health and Human Services; The USA, as represented by
the Secretary, |
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|
US |
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Assignee: |
The USA, as represented by the
Secretary, Department of Health and Human Services
Bethesda
MD
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Family ID: |
35949951 |
Appl. No.: |
13/735638 |
Filed: |
January 7, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11710512 |
Feb 26, 2007 |
8349586 |
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13735638 |
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PCT/US2005/030216 |
Aug 25, 2005 |
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11710512 |
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60604051 |
Aug 25, 2004 |
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60688376 |
Jun 8, 2005 |
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Current U.S.
Class: |
424/93.2 ;
435/252.3 |
Current CPC
Class: |
C07K 14/005 20130101;
C12N 2740/16122 20130101; A61K 35/74 20130101; A61K 2035/11
20130101; A61K 38/164 20130101; C12R 1/01 20130101; A61K 38/1774
20130101; C12N 2740/15022 20130101; A61K 38/195 20130101; C12R 1/46
20130101; A61K 35/741 20130101; A61K 38/162 20130101; C12R 1/385
20130101; C12R 1/07 20130101; C12N 15/72 20130101; C07K 2319/02
20130101; A61K 2039/523 20130101; C12R 1/36 20130101; C12R 1/19
20130101; A61K 38/1793 20130101; A61K 38/1709 20130101 |
Class at
Publication: |
424/93.2 ;
435/252.3 |
International
Class: |
A61K 35/74 20060101
A61K035/74 |
Claims
1. A commensal bacterium which can colonize genitourinary and/or
gastrointestinal mucosa, and which, under suitable conditions,
secretes a recombinant, antimicrobial polypeptide in an amount
effective to inhibit infectivity by, and/or a pathogenic activity
of, a pathogen, wherein the bacterium is Lactobacillus and the
recombinant, antimicrobial polypeptide is cyanovirin.
2-4. (canceled)
5. The commensal bacterium of claim 1, wherein the antimicrobial
polypeptide is fused in phase to a carboxyterminal secretion signal
of the hemolysin A gene.
6-13. (canceled)
14. The commensal bacterium of claim 1, wherein the pathogen is
HIV.
15-18. (canceled)
19. The commensal bacterium of claim 1, wherein the secretion is
mediated by a secretion signal that is a secretion-effective
C-terminal fragment of HlyA.
20. The commensal bacterium of claim 1, wherein the expression is
under the control of an expression control sequence that comprises
a constitutive promoter.
21. The commensal bacterium of claim 20, wherein the expression
control sequence comprises a promoter from the E. coli lac operon
and a translational control sequence from bacteriophage T7.
22. The commensal bacterium of claim 1, which comprises, stably
integrated into its chromosome, sequences encoding an antimicrobial
polypeptide fused in frame to a secretory signal, operably linked
to an expression control sequence.
23. A pharmaceutical composition, comprising an effective amount of
a commensal bacterium of claim 1 and a pharmaceutically acceptable
carrier.
24-25. (canceled)
26. A method for inhibiting HIV infectivity and/or pathogenicity in
a subject in need of such treatment, comprising administering to
the subject an effective amount of a commensal bacterium of claim
14, under conditions in which the anti-microbial polypeptide is
secreted in an amount effective to inhibit HIV infectivity and/or
pathogenicity.
27-35. (canceled)
Description
[0001] This application is a Continuation-In-Part of PCT
application, PCT/US2005/030216, filed Aug. 25, 2005, and claims the
benefit of the filing date of U.S. provisional applications, Ser.
No. 60/604,051, filed Aug. 25, 2004 and 60/688,376, filed Jun. 8,
2005, both of which are incorporated herein in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates, e.g., to a commensal
bacterium that can colonize the mucosa of the gastrointestinal
and/or genitourinary tract and, under suitable conditions, can
block the infectious and/or disease-causing activity of a pathogen
by secreting a heterologous antimicrobial polypeptide. Also
described are preventive and therapeutic compositions comprising
such bacteria, and methods to inhibit a pathogen, comprising
administering such a bacterium to a subject in need of such
treatment.
BACKGROUND INFORMATION
[0003] The global HIV/AIDS epidemic continues to grow at an
alarming rate. There are now more than 40 million people infected
with HIV, most of whom will die in the next decade, and last year
alone there were 5 million new infections. Most HIV transmission
worldwide is through unprotected vaginal intercourse. Unprotected
anal intercourse, another high risk activity, is also practiced
globally by both homosexual and heterosexual individuals. Ingestion
of HIV-containing breast milk by infants is a third common route of
infection.
[0004] Attempts to slow the spread of HIV by behavioral measures
have had little success, and no widely available biomedical
intervention is available. The development of a safe and effective
vaccine has proven to be extraordinarily difficult. Topical vaginal
and anal microbicides are a promising alternative to vaccines
because they can in principle be formulated from already-known HIV
inhibitors such as reverse transcriptase inhibitors or monoclonal
antibodies. However, they suffer the same fundamental problem as
condoms: they have to be used each time people have sex.
[0005] A new approach is urgently needed. The present application
describes an approach in which benign, commensal bacteria that can
colonize mucosa of human beings are genetically engineered to
secrete anti-HIV polypeptides, which then inhibit HIV infection
and/or pathogenesis. The approach can also be used to genetically
engineer commensal bacteria to inhibit a variety of pathogens other
than HIV; e.g., other viruses, pathogenic bacteria, fungi and
parasites.
DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1 diagrams the structure of some antimicrobial
polypeptides and the use of the hemolysin system to obtain their
secretion. FIG. 1A is a scheme of recombinant DNA constructs used
to express the control peptide Etag-HlyA218 and the HIV fusion
inhibitor peptides C52-HlyA218, C52-HlyA103, and C52-HlyA53. C52 is
a peptide derived from the C-terminal region of HIV gp41; Etag is a
linker sequence containing a common epitope; and HlyA is the
carboxy-terminal secretion signal sequence of E. coli hemolysin A.
FIG. 1B illustrates the roles of the HlyB, HlyD, and TolC proteins
in the secretion of the antimicrobial peptides into the culture
media.
[0007] FIG. 2 is a gel which shows secretion and biological
activity of HIV fusion inhibitor peptides from bacteria. FIG. 2A
shows E. coli Nissle 1917 that were electroporated with a
chloramphenicol resistance plasmid encoding hlyB and hlyD and with
an ampicillin resistance plasmid encoding an hlyA fusion peptide.
The doubly transformed bacteria were grown in LB for 8 hours and
the cell-free supernatants were analyzed by SDS gel electrophoresis
and Commassie Blue staining (1) no hlyA plasmid. (2)
Etag-hlyA.sub.218 (3) C52-hlyA.sub.218 (4) C52-hlyA.sub.103 (5)
C52-hlyA.sub.53. The markers on lanes at each end of the gel have
approximate molecular weight masses of 188, 98, 62, 49, 38, 28, 17,
14, 6 and 3 kDa. FIG. 2B is a graph which shows PBMC that were
incubated with various concentrations of hlyA peptides, infected
with an HIV or MuLV-pseudotyped HIV-GFP reporter virus, and assayed
for GFP reporter expression by FACS after 2 days. .largecircle.
MuLV+Etag-hlyA.sub.218; .box-solid. MuLV+C52-hlyA.sub.218; .DELTA.
HIV+Etag-hlyA.sub.218; HIV+C52-hlyA.sub.218; .quadrature.
HIV+C52-hlyA.sub.103; .tangle-solidup. HIV+C52-hlyA.sub.53.
[0008] FIG. 3 shows the colonization of the mouse gastrointestinal
and genitourinary tract by genetically modified E. coli Nissle
1917. Female mice, strain CD1, were administered 5.times.10.sup.8
E. coli Nissle 1917::C52-HlyA208::HlyB HlyD, either orally or
rectally. The mice were either treated with no antibiotic,
pretreated for one day prior to inoculation with antibiotic, or
both pre-treated and continuously post-treated with antibiotic.
FIG. 3A shows the number of the recombinant bacteria recovered in
feces at time intervals. FIG. 3B shows the number of recombinant
bacteria recovered from segments of the gastrointestinal tracts and
from the vagina after 1 week of colonization.
[0009] FIG. 4 shows the colonization and tissue distribution of
anti-HIV bacteria in Rhesus Macaque. FIG. 4A shows colonization
data; FIG. 4B shows tissue distribution.
[0010] FIG. 5 shows a Macaque Protection Experiment. Experimental
Macaques were inoculated with a mixture of E. coli Nissle 1917
expressing a mixture of antiviral peptides, as indicated in Example
II, and were challenged rectally with the challenge virus SHIV
162p3 at a high dose (100% infection of control animals). FIG. 5A
shows 4 control animals which did not receive the bacteria; all
four animals were infected (100% infected); none was protected
against viral infection. FIG. 5B shows 4 animals that were
inoculated with the bacteria. Two of the four animals were
protected against viral infection.
DESCRIPTION OF THE INVENTION
[0011] The present invention relates, e.g., to a commensal
bacterium that can colonize genitourinary and/or gastrointestinal
mucosa, and that, under suitable conditions, secretes an
antimicrobial polypeptide that inhibits the infectiousness and/or
pathogenicity of a pathogen. The commensal bacterium can be, for
example, a non-pathogenic strain of a gram negative bacterium, such
as E. coli; or the pathogen can be, for example, a virus,
bacterium, fungus or parasite.
[0012] In preferred embodiments of the invention, HIV infection
and/or pathogenesis is inhibited. Commensal microorganisms of the
invention (e.g., genetically modified E. coli) can colonize the
genitourinary and/or gastrointestinal mucosa, which are the major
sites of HIV transmission through vaginal and anal intercourse and
breast feeding, and are normally coated by a biofilm of commensal
microorganisms. By using strains that can compete with the
preexisting vaginal, intestinal and oral microflora, these
genetically engineered bacteria can persistently colonize the
mucosa, and can serve as a continuous source of secreted
antimicrobial polypeptide. The secreted polypeptide can, e.g.,
prevent HIV infection directly by binding to the virus or
indirectly by binding to cellular receptors and/or co-receptors for
the virus. The microorganism secreting the polypeptide is
self-sustaining and even transmissible from person to person. The
methods of the invention can be used to prevent new infections, or
to inhibit viral rebound in infected and antiretroviral-treated
individuals when HAART (Highly Active Anti-Retroviral Therapy) is
stopped or becomes ineffective due to drug resistance.
[0013] Among the advantages of the methods of the present invention
are that they are inexpensive and easy to administer in
resource-poor settings, and require one or infrequent rather than
daily applications. The protective microbes have the potential to
be spread by sexual and casual contact as well as by deliberate
administration. Perhaps most important, HIV has no opportunity to
evolve evasive measures--unlike the case for traditional
vaccines.
[0014] One aspect of the invention is a commensal, gram negative
bacterium which can colonize genitourinary and/or gastrointestinal
mucosa and which, under suitable conditions, secretes a recombinant
antimicrobial polypeptide, wherein the secreted antimicrobial
peptide is effective to inhibit infection by, and/or a pathogenic
activity of, a pathogen. In embodiments of the invention, the gram
negative bacterium is Bacteriodes melaminogenicus, Bacteriodes
vulgatus, Bacteriodes fragilis, Pseudomonas aeruginosa, Veillonella
parvula, or a fusobacterium. In a preferred embodiment, the
commensal, gram negative bacterium is a strain of E. coli, most
preferably the strain Nissle 1917. The antimicrobial polypeptide in
these gram negative bacteria is fused in phase to a secretion
signal, preferably a carboxyterminal secretion signal of the
hemolysin A gene.
[0015] Another aspect of the invention is a commensal bacterium
which can colonize genitourinary and/or gastrointestinal mucosa,
and which, under suitable conditions, secretes a recombinant,
antimicrobial polypeptide; wherein the secreted antimicrobial
polypeptide is effective to inhibit infectivity by, and/or a
pathogenic activity of, a pathogen; provided that the commensal
bacterium is not a commensal Lactobacillus or Streptococcus (e.g. a
readily transformable strain of one of those bacteria). In
embodiments of this aspect of the invention, the bacterium is
either gram positive or gram negative. For example, the bacterium
may be a commensal strain of E. coli, Bacillus, Lactococcus,
Bitodobacterium, Bacteriodes, or Neisseria.
[0016] Any of the above types of bacteria is sometimes referred to
herein as a "commensal bacterium of the invention." It will be
evident from context which type of bacterium is being
discussed.
[0017] In embodiments of the invention, the commensal bacterium can
colonize genitourinary mucosa and/or gastrointestinal mucosa. In
embodiments of the invention, the antimicrobial polypeptide is
directed against a virus (e.g., HIV, SHIV and/or SIV, preferably
HIV), bacterium, fungus or parasite. For example, an antimicrobial
polypeptide directed against HIV may comprise a functional
inhibitory fragment from the C-terminal region of an HIV, SHIV or
SIV gp41 protein (such as a functional fragment that consists
essentially of about 20 to about 200 amino acids, preferably about
47 amino acids or about 20 amino acids, from the C-terminal region
of gp41); or a functional inhibitory fragment comprising amino
acids from the N-terminal region of gp41 from HIV-1, HIV-2 or SIV;
an allosteric site on gp120; a predicted extracellular loop of
CCR5; a single strand anti CD4 antibody; a mini-protein mimetic of
CD4; an alpha-defensin or theta-defensin; a CD38 sequence
homologous to the V3 loop of gp120; polphemusin II, which
antagonizes CXCR4; a RANTES peptide that binds to CCR5; or a HIV
surface binding peptide such as cyanovirin. In preferred
embodiments, the secretion signal is a secretion-effective
C-terminal fragment of HlyA; and/or the expression control sequence
comprises a constitutive promoter (e.g., the expression control
sequence comprises a promoter from the E. coli lac operon and a
translational control sequence from bacteriophage T7). In one
embodiment, the bacterium comprises, stably integrated into its
chromosome, sequences encoding an antimicrobial polypeptide fused
in frame to a secretory signal, wherein the coding sequences are
operably linked to an expression control sequence.
[0018] Another aspect of the invention is a pharmaceutical
composition, comprising an effective amount of a commensal
bacterium of the invention and a pharmaceutically acceptable
carrier.
[0019] Another aspect of the invention is a method for making a
gram negative, commensal bacterium which can colonize genitourinary
and/or gastrointestinal mucosa, and which, under suitable
conditions, secretes a recombinant, antimicrobial polypeptide,
wherein the secreted antimicrobial polypeptide is effective to
inhibit infection by, or a pathogenic activity of, a pathogen. The
method comprises introducing into a gram negative commensal
bacterium that can colonize genitourinary and/or gastrointestinal
mucosa a polynucleotide that encodes a heterologous antimicrobial
polypeptide fused in phase to a secretion signal (e.g. a secretion
signal from the C-terminal region of HlyA), wherein the coding
sequences for the antimicrobial polypeptide and the fused secretion
signal are operably linked to an expression control sequence. In a
preferred embodiment, the polynucleotide is stably introduced into
the bacterium.
[0020] Another aspect of the invention is a method for inhibiting
HIV infectivity and/or pathogenicity, comprising contacting the HIV
with an effective amount of a commensal bacterium of the invention,
under conditions in which the secreted anti-microbial polypeptide
is effective to inhibit HIV infectivity and/or pathogenicity.
[0021] Another aspect of the invention is a method for inhibiting
HIV infectivity and/or pathogenicity in a subject in need of such
treatment, comprising administering to the subject an effective
amount of a commensal bacterium of the invention, under conditions
in which the secreted anti-microbial polypeptide is effective to
inhibit HIV infectivity and/or pathogenicity. In preferred
embodiments of the invention, the commensal bacterium is
administered orally, intrarectally, or intravaginally.
[0022] Another aspect of the invention is a method for providing a
source of an antimicrobial polypeptide at a genitourinary and/or
gastrointestinal mucosum in a subject in need of such treatment,
comprising administering to the subject an effective amount of a
commensal bacterium of the invention, under conditions effective
for the bacterium to colonize the mucosum and to express and
secrete an effective amount of the antimicrobial polypeptide. In
general, the antimicrobial polypeptide is present in an amount
sufficient to be detected in a sample collected from the mucosal
surface.
[0023] Another aspect of the invention is a kit for delivering an
effective dose of an antimicrobial agent to a genitourinary and/or
gastrointestinal mucosum, comprising an effective amount of a
commensal bacterium of the invention, in a pharmaceutically
acceptable solution.
[0024] Another aspect of the invention is a vector comprising a
polypeptide as described in FIG. 1A.
[0025] As used herein, a "commensal" bacterium is one that is
symbiotic with its host (e.g. a human host), is non-pathogenic, and
can occupy the genitourinary and/or gastrointestinal tracts but
does not invade other areas of the body such as the blood, lungs,
or heart. Among the microorganisms which can colonize
gastrointestinal and/or genitourinary mucosa are microorganisms
which naturally inhabit those mucosa, or microorganisms which have
been manipulated (e.g., adapted) so that they can colonize the
mucosa. Exemplary methods for adapting such microorganisms are
discussed below. A microorganism that can "colonize" a mucosum is
one that can compete with the preexisting microflora and take up
residence in the mucosum. As used herein, "gastrointestinal mucosa"
include the linings of, e.g., the rectum, colon, cecum and upper
intestine (jejunum, duodenum and ileum) as well as the oral cavity
and larynx (which are normally considered part of the upper
respiratory tract, but are herein referred to as components of the
gastrointestinal tract because they are the gateway to this portion
of the anatomy). "Genitourinary mucosa" include the lining of,
e.g., the cervix, vagina, penis, and urinary tract. Many organisms
that can colonize gastrointestinal mucosa are also able to colonize
genitourinary mucosa, and vice-versa. For example, the majority of
E. coli strains isolated from vaginal samples of normal healthy
women were also found in their own stool samples (Foxman et al.
(2002) Am J Epidemiol 156, 1133-40).
[0026] A "mucosal membrane" or a "mucosal surface" refers to a
tissue layer found lining various tubular cavities of the body such
as the oropharynx, small intestine, large intestine, rectum, penis,
vagina, mouth, uterus, etc. It is composed of a layer of epithelium
containing numerous unicellular mucous glands and an underlying
layer of areolar and lymphoid tissue, separated by a basement
membrane. This membrane is typically colonized by a variety of
bacteria even when the host is healthy.
[0027] Commensal bacteria of the invention may take any of a
variety of forms. Preferably they are strains which exhibit
favorable growth and colonization properties, and which can be
efficiently (and, in some cases, stably) transformed with
recombinant DNA constructs. By "favorable growth and colonization
properties" is meant that the microorganism can efficiently
colonize a mucosal lining and can continue to grow and/or remain
attached to the mucosal lining to the extent necessary to secrete
effective amounts of the antimicrobial polypeptide. In some cases
the commensal bacterium is a genetically engineered version of a
species that naturally inhabits the mucosum which is being
colonized. In other cases it is a modified version of a strain that
has been previously administered to humans as a probiotic and
thereby known to be a good colonizer and non-pathogenic.
[0028] Humans are inhabited by over 1000 different species of
bacteria which inhabit and/or can colonize normal healthy mucosa,
and which can be used in methods of the invention (see, e.g.,
Guarner et al. (2003) Lancet 361, 512-9; Salminen et al. (1995)
Chemotherapy 41 Suppl 1, 5-15; and Galask, R. P. (1988) Am J Obstet
Gynecol 158, 993-5). Several specific examples of commensal
bacteria are described below, but a skilled worker will recognize
appropriate ways to modify any particular disclosure herein so as
to be applicable to additional species, strains and isolates of
bacteria. Bacteria can be used which naturally exhibit desired
growth and colonization behavior. Alternatively, bacteria can be
manipulated, using conventional procedures, to enhance their
ability to colonize a mucosal surface. For example, a first method
involves repetitively selecting for rapid colonizing bacteria on
animal or human mucosal layers. For example, one applies a wild
type bacterial strain to a mucosal surface and repetitively
isolates and in vitro cultures bacteria, returning at each step to
the mucosal surface. Ultimately, a bacterium with an enhanced
colonizing ability is obtained. A second method involves expression
of fusion proteins on the surface of recombinant bacteria. The
fusion protein consists of a host-binding domain linked to a
polypeptide of interest. The host-binding domain will allow the
bacteria to bind to certain determinants (protein or carbohydrate)
on a selected host mucosal surface with high affinity, thus
conferring the bacteria a survival advantage over the resident
microflora. In addition, one can use bacterial strains known to be
non-pathogenic and efficient colonizers by virtue of their use as
probiotics, which are live microorganisms which when administered
in adequate amounts confer health benefits on the host. Typically,
probiotic bacteria have demonstrated safety in human use, survival
in the intestine, adhesion to mucosa, and at least temporary
colonization of the gut. Some examples of suitable bacteria are
described below.
[0029] E. coli: E. coli is an almost universal member of the normal
human intestinal and rectal microflora. It is the first species to
colonize the gut in infants, and reaches concentrations from
10.sup.9 to 10.sup.12 CFU/gm feces in adults (Guarner et al. (2003)
Lancet 361, 512-9; Salminen et al. (1995) Chemotherapy 41 Suppl 1,
5-15). E. coli is also frequently found in the vagina. Foxman et
al. (2002), supra found E. coli in 28% of asymptomatic women, with
the highest rate in sexually active individuals. It is therefore
anticipated that expression of anti-HIV agents by E. coli will
protect strongly against rectal transmission by anal intercourse,
and very likely against vaginal transmission by vaginal intercourse
and oral transmission by breast milk as well.
[0030] One preferred strain of E. coli is Nissle 1917, which was
isolated in 1917 from the stool of a German soldier who, unlike his
comrades, survived an outbreak of enterocolitis. This strain is
widely used as a probiotic in Europe, where it is produced under
the trade name of Mutoflor, to treat intestinal disorders including
diarrhea, irritable bowel disease, ulcerative colitis and Crohn's
disease. Nissle 1917 is an excellent colonizer of the human gut,
both in adults who have undergone antibiotic therapy and in
infants, and does not produce CNF toxin, hemolysin, P-fimbrae, or
S-fimbrae (Altenhoefer et al. (2004) FEMS Immunol Med Microbiol 40,
223-9; Blum-Oehler et al. (2003) Res Microbiol 154, 59-66;
Lodinova-Zadnikova et al. (1997) Biol Neonate 71, 224-32; Rembacken
et al. (1999) Lancet 354, 635-9; Stentebjerg-Olesen et al. (1999) J
Bacteriol 181, 7470-8; Blum et al. (1995) Infection 23, 234-6). As
shown below, Nissle 1917 retains colonizing activity following
genetic manipulation to produce anti-HIV peptides.
[0031] Other preferred strains of E. coli are those capable of
growing both in the gastrointestinal and genitourinary tracts and
of colonizing new hosts. For example, Foxman et al. (2002), supra
described 57 strains of E. coli that appear to satisfy these
criteria by virtue of having been isolated from both the feces and
vaginal washes of normal health women and the feces of their male
sexual partners.
[0032] Other Gram Negative Bacteria:
[0033] A number of gram negative bacteria besides E. coli inhabit
the mucosal surfaces of interest. Such strains are especially
suitable because they have the potential to secrete antimicrobial
peptides through a gram negative type I secretion pathways such as
the hemolysin pathway discussed herein. Some specific species
suitable for this invention are Bacteroides melaminogenicus,
Bacteriodes vulgatus, Bacteriodes fragilis, Pseudomonas aeruginosa,
Veillonella parvula, and fusobacteria.
[0034] Streptococcus:
[0035] Another suitable bacterium is Streptococcus. A particularly
preferable species is Streptococcus gordonii, which is capable of
colonizing the human vagina, and which has been used to express a
number of heterologous polypeptides (see, e.g., Beninati et al.
(2000) Nat Biotechnol 18, 1060-4 and Giomarelli et al. (2002) Aids
16, 1351-6). Other suitable Streptococcus strains include
Streptococcus sps, S. mitis, S. oralis, S. salivalius, and S.
pneumoniae, all of which naturally inhibit and/or colonize at least
the nasal/pharynx mucosa of healthy individuals.
[0036] Lactobacillus:
[0037] Another suitable bacterium is Lactobacillus, which is a
major component of the human vaginal microflora. Any Lactobacillus
isolate with favorable growth and colonization properties, and
which can be transformed efficiently with heterologous DNA, is
suitable for use in the present invention. A natural vaginal
isolate of Lactobacillus jensenii-Lactobacillus jensenii strain
1153--that exhibits favorable growth and colonization properties
has been identified by Chang et al. (2003) Proc Natl Acad Sci USA
100, 11672-7. See also US Pat. Pub. 20030228297. Chang et al.
report that this isolate can be used in conjunction with a
Lactococcus-based plasmid to express and secrete foreign proteins
and that, when engineered to express biologically active two-domain
CD4, it inhibits HIV infectivity of susceptible cultured cell
lines. This construct is unlikely to be clinically useful because
of the relatively low expression levels and modest potency that
were observed. However, other constructs expressed in this or in
other suitable Lactobacillus isolates may be more suitable for
clinical use. Other suitable lactobacillus strains include, e.g.,
Lactobacillus sps, L. crispatus, L. fermentum, L. casei (e.g., L.
casei ss rhamnosus, L. casei ss alactosus), L. salivarius, L.
catenaforme, L. minutus, L. gasseri, L. acidophilus, L. plantarum,
and L. brevis.
[0038] Other suitable bacteria will be evident to the skilled
worker. These include, e.g., Lactococcus lactis, which is a
nonpathogenic gram positive bacterium frequently used to produce
fermented foods, and which has been engineered to secrete
interleukin-10 as a treatment for murine colitis (Steidler et al.
(2000) Science 289, 1352-5) and Bacillus, which has been employed
as a probiotic (Hoa et al. (2000) Appl Environ Microbiol 66,
5241-7). Other suitable bacteria include Staphylococcus sps, S.
epidermidis, S. aureus, and Neisseria sps, all of which naturally
inhabit at least the nasal/oral pharynx of healthy individuals; and
Corynebacterium sps, which naturally inhabits vaginal mucosa.
Furthermore, vectors are also available for Bifodobacteria, which
are among the most common bacteria in the human intestine (van der
Werf et al. (2001) J Agric Food Chem 49, 378-83).
[0039] A variety of antimicrobial polypeptides are encompassed by
the invention. As used herein, the terms "polypeptide," "peptide,"
and "protein" are used interchangeably to refer to a polymer of
amino acid residues. These terms as used herein encompass amino
acid chains of any length, including full-length proteins, wherein
the amino acid residues are linked by covalent bonds. A preferred
length is from about 100 to 275 amino acids (approximately 13,500
to 37,125 daltons), which the inventor has shown to be sufficiently
long to display potent antimicrobial activity, yet sufficiently
short to permit efficient secretion into the surrounding
environment (see Examples below).
[0040] An antimicrobial polypeptide of the invention can be used to
prevent any step in pathogenesis, including, e.g., initial
infection of a naive host by the pathogen; continuing reinfection
of a chronically infected host by the pathogen; detrimental
biochemical, physiological, and immunological effects caused by
infection with the pathogen; and spread of the pathogen to other
hosts.
[0041] The pathogens which can be inhibited by methods of the
invention include, e.g., viruses, bacteria, fungi and parasites.
The antimicrobial polypeptides of the invention can block these
pathogens either directly by binding to them, or indirectly, e.g.
by preventing their interaction with host cell components.
[0042] Examples of antimicrobial polypeptides that inhibit
infection by directly binding to the pathogen include antibodies,
antibody fragments, and single-chain antibodies that recognize an
external protein of the pathogen, such as a viral envelope or cell
membrane protein. Also included in this category are receptor or
receptor domains that a viral or bacterial pathogen binds to infect
a host, or a functional virus-binding fragment of the receptor.
Such direct binding proteins can inhibit infection or pathogenicity
by a variety of mechanisms. For example, they can act as decoys and
block entry of the pathogen into the cell. Alternatively, the
antimicrobial polypeptide can be an agent that binds to the
pathogen and thereby, e.g., inhibits pathogen replication,
viability, entry, etc. For example, since viruses require binding
to a receptor on the target cell surface for infection, strategies
directed at inhibiting the interaction of a virus with its host
receptor are effective at preventing infection. In some
embodiments, a polypeptide of the invention binds or inhibits
sexually transmitted pathogens and other pathogens transmitted to
or from the vagina or the rectum.
[0043] Examples of antimicrobial polypeptides that indirectly
inhibit infection by preventing interactions of the pathogen with
the host include antibodies, antibody fragments, single-chain
antibodies, and ligands that bind to a cellular receptor and/or
co-receptor for the pathogen.
[0044] In a preferred embodiment, the pathogen which is inhibited
is a virus. Among the many viruses which can be inhibited by the
methods of the invention are rotavirus, Norwalk agent,
papillomavirus, adenovirus, respiratory syncytia virus, corona
virus, cytomegalovirus, coxsackievirus, echovirus, hepatitis A
virus, hepatitis B virus, hepatitis C virus, rhinovirus, human
immunodeficiency virus, poliovirus and other picornaviruses,
Epstein-Barr virus, influenza virus, parainfluenza virus, and
herpes simplex virus.
[0045] The skilled worker will recognize a wide variety of suitable
inhibitory polypeptides that inhibit viral entry into a host cell.
For example, agents that bind to and inhibit virus surfaces or
virus receptors on a host cell are known for Human Rhinovirus
(major group ICAM-1), Influenza A (sialic acid), Adenovirus
(vitronectin), Epstein-Barr Virus (CR2 (C3 receptor)), Herpes
Simplex Virus type I (heparin sulfate/HveA/HveC), Herpes Simplex
Virus type II (heparin sulfate/HveA/HveC), Poliovirus (Poliovirus
Receptor (PVR)), and Hepatitis B (asialoglycoprotein). The
inhibitors can be, e.g., functional fragments of the receptors
which compete with the virus for entry into the cell.
Alternatively, polypeptides can be used which bind to conserved
determinants on viral capsids and thereby prevent or inhibit their
binding to a receptor.
[0046] A "functional fragment" or an "active fragment," as used
herein, refers to a fragment that retains at least one biological
activity of the full-length molecule (e.g., the ability to bind to
the pathogen or, in the case of HIV, to inhibit HIV fusion). A
skilled worker will recognize how to generate suitable functional
fragments of an antimicrobial polypeptide, e.g. based on known
properties of the polypeptides. Some such fragments have been
disclosed.
[0047] In a particularly preferred embodiment of the invention, the
virus which is inhibited is human immunodeficiency virus (HIV),
simian immunodeficiency virus (SIV), or a chimeric simian-human
immunodeficiency virus (SHIV). "HIV," as used herein, refers to
HIV-1, HIV-2, or any subset therein. Preferably, the virus
inhibited by a method of the invention is HIV-1. In one embodiment,
the antimicrobial polypeptide is a functional anti-HIV polypeptide
derived from the C-terminal region of the gp41 Env protein of HIV
(to inhibit HIV and SHIV) or SIV (to inhibit SIV). Preferably, the
polypeptide consists essentially of about 20 to about 200 amino
acids from this C-terminal region, more preferably about 47 amino
acids or about 20 amino acids from this C-terminal region.
"Consisting essentially of," when used in the context of
polypeptides, refers to a sequence which is intermediate between
the number of amino acid residues encompassed by the term
"consisting of" and the longer length encompassed by the term
"comprising." Residues in addition to the residues encompassed by
"consisting of" language do not affect the basic and novel
characteristics (e.g., in the case of a polypeptide of the
invention, the ability to inhibit infectivity or pathogenicity of
an organism) of the molecule encompassed by the "consisting of"
language.
[0048] In one embodiment, the antimicrobial polypeptide is an amino
acid fragment, that contains about 47 amino acids (e.g., about 40
to about 55 amino acids) from the C-terminal region of gp41 of HIV
or SIV, together with additional amino acids that are added to
allow cloning into an expression vector. In a preferred embodiment,
the antimicrobial polypeptide is an amino acid fragment, henceforth
termed "C52," that contains 47 amino acids from the C-terminal
region of gp41 of HIV or SIV, together with an additional 5 amino
acids that were added to allow cloning into the expression vector.
See Root et al. (2003) Proc Natl Acad Sci USA 100, 5016-5021. The
sequence of the HIV C52 peptide is
MGGHTTWMEWDREINNYTSLIHSLIEESQNQQEK NEQELLELDKWASLWNWF (SEQ ID NO:
1) and the sequence of the SIV C52 peptide is
MGGHTTWQEWERKVDFLEENITA LLEEAQIQQEKNMYELQKLNSWDVFGNWF (SEQ ID NO:
2). Without wishing to be bound by any particular mechanism, it is
suggested that the C52 polypeptides inhibit HIV and SIV infection
by binding to the N-terminal region of HIV and SIV gp41, thereby
preventing the formation of the "trimer of hairpins" structure
required for fusion of the virus to the host cell membrane (Eckert
et al. (2001) Annu Rev Biochem. 70, 777-810).
[0049] The HIV and SIV C52 polypeptides have several notable
advantages as viral inhibitors: they are potent; the sequences are
highly conserved among different HIV isolates; the polypeptides do
not require disulfide bonding or other posttranslational
modifications; similar peptides are active against both direct and
trans infection of multiple primary cell types (Ketas et al.,
(2003) J Virol 77, 2762-7; Ketas et al. (2003) AIDS Res Hum
Retroviruses 19, 177-86); and similar polypeptides, such as the
closely related C-terminal peptide T-20 (enfuvirtide), are
currently in clinical use as a salvage antiretroviral therapy
(Kilby et al. (1998) Nat Med 4, 1302-7; Kilby et al. (2002) AIDS
Res Hum Retroviruses 18, 685-93). As described below, the C52
polypeptides and all of the other antimicrobial polypeptides
discussed herein are preferably covalently attached to additional
sequences that allow detection of the heterologous polypeptides
with antibodies (e.g., the E tag epitope) and/or that allow
secretion (e.g., the hlyA C-terminal secretion signal; see
below).
[0050] In addition to C52, several other polypeptides can inhibit
the infectivity of HIV, SIV and SHIV by blocking virus-cell fusion.
These include peptides from the N-terminal region of gp41 from HIV,
SHIV or SIV, which are thought to inhibit viral fusion by targeting
the C-terminal region of the fusion apparatus, especially when
prevented from aggregation by attachment to a soluble helix-forming
domain, (Eckert et al. (2001) Proc Natl Acad Sci USA 98, 11187-92;
Louis et al. (2003) J Biol Chem 278(22), 20278-85); and peptides
from the C-terminal region of gp41 from HIV-1, HIV-2 or SIV, which
are thought to be the target of a 5-helix construct in which three
N-terminal peptides and two C-terminal peptides from HIV are
covalently linked (Root et al. (2001) Science 291, 884-8). Other
combinations of C-terminal and N-terminal peptides from HIV, SIV
and SHIV have similar effects. One such inhibitor is the potent
fusion inhibitor, T1249, which comprises a mixture of sequences
from the C-terminal region of gp41 of HIV-1, the C-terminal region
of gp41 of HIV-2, and the C-terminal region of SIV. This peptide,
which is described in U.S. Pat. No. 6,656,906 and Eron et al.
(2004) J Infect Dis 189, 1075-83, appears to function by the same
mechanism as the C52 peptides described herein. Typically, anti-HIV
polypeptides of the invention are less than about 200 amino acids
in length; longer and shorter polypeptides are included.
[0051] Various other peptides and short proteins that directly
recognize HIV, SIV and SHIV can inhibit infection at different
points in the viral life cycle. Short linear peptides that inhibit
HIV infection by binding to an allosteric site on gp120 have been
identified by phage display (Biorn et al. (2004) Biochemistry, 43,
1928-38). One such peptide is the peptide 12P1, which is discussed
in Ferrer et al. (1999) J Virol 73, 5795-5812. The 2D-CD4
polypeptide, which contains the first approximately 183 amino acids
of 2-domain CD4, binds to gp120 with the same affinity as the
intact protein (Salzwedel et al. (2000) J. Virol. 74, 326-333).
High affinity miniprotein mimetics of CD4 can also be isolated and
used to inhibit HIV entry (see, e.g., Dowd et al. (2002)
Biochemistry 41, 7038-46 and Li et al. (2001) J Pept Res 57,
507-18).
[0052] Other peptides can inhibit HIV, SIV and SHIV infection
indirectly, by blocking cellular receptors and co-receptors. For
example, derivatives of RANTES, which is a natural ligand for the
CCR5 chemokine receptor, block infection by R5-tropic strains of
HIV that utilize CCR5 as co-receptor (Simmons et al. (1997) Science
276, 276-9). Peptides corresponding to the predicted extracellular
loops of CCR5 also inhibit infection by R5 strains of HIV, which
are the type most frequently transmitted (Agrawal et al. (2004)
Blood 103, 1211-7). CXCR4, the other major co-receptor for HIV, can
be inhibited by peptides such as T22 (Masuda et al. (1992) Biochem
Biophys Res Commun. 189, 845-50; D. Schols (2004) Curr Top Med Chem
4, 883-93.
[0053] Other suitable, well-known anti-HIV polypeptides will be
evident to the skilled worker. These include, e.g., a mini-protein
mimetic of CD4; an alpha-defensin or theta-defensin; a CD38
sequence homologous to the V3 loop of gp120; polphemusin II, which
antagonizes CXCR4; and an HIV surface binding peptide, such as
cyanovirin (Giomarelli et al. (2002) Aids 16), 1351-6,
[0054] In another embodiment of the invention, the pathogen which
is inhibited is a bacterium. Anti-bacterial polypeptides include
those that bind to or inhibit growth or colonization by
uropathogenic E. coli. Among the bacteria that can be inhibited by
methods of the invention are bacteria which cause sexually
transmitted diseases, including, e.g., Neisseria gonorrhoeae
(gonorrhea), Treponema palladium (syphilis) and Chlamydia
trachomatis (chlamydia). Exemplary anti-bacterial polypeptides
include, e.g., permeability-increasing protein against
gram-negative bacteria (Levy (2002) Expert Opin. Investig. Drugs
11, 159-167), mammalian anti-microbial peptides, .beta.-defensins
(Ganz et al, (1995) Pharmacol. Ther. 66, 191-205), bacteriocins
(e.g., Loeffler et al. (2001) Science 294, 2170-2172) and
antibodies that specifically bind to the bacteria. Agents that
target bacterial cell walls are also included. In general, target
specificity is determined by the C-terminal domain of these
molecules (known as cell wall targeting sequences). The best
studied of these are molecules which bind specifically to choline,
which is a constituent of the cell wall of Strep. pneumoniae and a
few other bacterial species (e.g., S. oralis). These molecules
include LytA and PspA. Other bacteria-binding molecules include
RIB, which targets Listeria monocytogenes and Bacillus subtilis,
and Lysostaphin, which targets Staph. aureus. The C-terminal
(targeting) domains of such molecules to can be used. Suitable
inhibitory molecules include, e.g., LytA, a C-terminal binding
domain of PspA, a C-terminal domain of lysostaphin (SPA.sub.CWT), a
C-terminal domain of InIB, an anti-5-layer protein antibody, and an
anti-peptidoglycan antibody.
[0055] In another embodiment of the invention, the pathogen which
is inhibited is a fungus. Anti-fungal polypeptides include those
that bind to or inhibit growth or colonization by fungi such as
Candida.
[0056] In another embodiment of the invention, the pathogen which
is inhibited is a parasite. Suitable anti-parasite polypeptides
will be evident to the skilled worker.
[0057] Another class of antimicrobial polypeptides, which are
effective against viruses or other pathogens, comprises antibodies
that are specific for a surface component of a pathogen, such as a
virus, or for a cellular receptor or co-receptor involved in
pathogen binding or entry into a host. The antibody can be, e.g. a
single chain antibody, or an antibody fragment, such as an F(ab) or
a F(ab').sub.2. For example, the anti-CD4 antibody 5A8, which
allows HIV-CD4 interactions but blocks subsequent steps required
for fusion, or active fragments thereof, can be used. This antibody
is attractive for microbial expression because it potently
neutralizes virtually all strains of HIV-1, and a humanized version
has been shown to decrease viral loads and increase CD4 T cell
counts in HIV-infected subjects in a phase I clinical trial
(Kuritzkes et al. (2004) JInfect Dis 189, 286-91). Antibodies such
as b12, and improved derivatives thereof, recognize the CD4 binding
site of HIV Env protein with high potency (Kessler et al. (1997)
AIDS Res Hum Retroviruses 13, 575-82; McHugh et al. (2002) J Biol
Chem 277, 34383-90). An antibody "specific for" a polypeptide
includes an antibody that recognizes a defined sequence of amino
acids, or epitope, either present in the full length polypeptide or
in a peptide fragment thereof. In one embodiment, the antibody is a
neutralizing antibody. By "neutralizing" is meant herein that
binding of an antibody to a pathogen or its receptor inhibits or
prevents infection of the host by the pathogen.
[0058] Therapeutic polypeptides that are not antimicrobial
polypeptides are also included in the invention. Such polypeptides,
which are secreted by commensal microorganisms of the invention,
include, among many others, anti-inflammatory molecules, growth
factors, molecules that bind to, or antagonize, growth factors,
therapeutic enzymes, antibodies (including, e.g., antibody
fragments or single-chain antibodies) and molecules that inhibit or
treat cancer including cervical cancer. Anti-inflammatory molecules
include, e.g., antibodies or other molecules that specifically bind
to tumor necrosis factor (TNF) or interleukin-8 (IL-8). Other
exemplary anti-inflammatory molecules include IL-10 and IL-11.
Growth factors useful in the invention include, e.g., those
involved in local tissue repair such as keratinocyte growth factor
(KGF), heparin-binding epidermal growth factor (HB-EGF), fibroblast
growth factor (FGF) and transforming growth factor-beta
(TGF-.beta.), or antagonists of these molecules. Therapeutic
enzymes include, e.g., nitric oxide (NO) synthase. Anti-cancer
molecules include those that induce apoptosis, that regulate cell
cycle such as p53, or that act as a vaccine to target
cancer-specific epitopes.
[0059] Active fragments or variants of any of the antimicrobial
polypeptides discussed herein are included in the invention,
provided that the altered polypeptide retains at least one
biological activity of the unaltered (e.g., wild type) polypeptide
(e.g., the ability to inhibit infection by, or pathogenic activity
of, a pathogen). The fragment or variant can have the sequence of a
naturally occurring polypeptide (e.g., it can be a peptide fragment
of a longer antimicrobial polypeptide), or it can have a variant of
the sequence of a naturally occurring polypeptide. Suitable
variants may comprise small deletions, insertions or substitutions
compared to the wild type protein; preferably, the variant contains
one or more conservative amino acid substitutions. The variants may
be naturally occurring (e.g., allelic variants or strain
differences), or they may be introduced artificially, using
conventional methods. A skilled worker can readily determine if a
given polypeptide, either wild type or variant, exhibits a desired
antimicrobial activity.
[0060] Under suitable conditions, a commensal microorganism of the
invention secretes an antimicrobial polypeptide, which is effective
to inhibit infection by, or a pathogenic activity of, a pathogen.
"Suitable conditions," as used herein, include the presence of
regulatory elements (including secretion signals) that allow
effective amounts of the polypeptide to be produced and secreted.
"Suitable conditions" also include a physiological environment
which is conducive to the expression and secretion of the
polypeptide. Such conditions are found in the various mucosa to
which the microorganisms are administered. For example, suitable
conditions for expressing an inducible promoter include a
physiological environment in which an agent is present that induces
the promoter. Suitable conditions also include the presence of an
amount of the microorganism that is sufficient to compete
effectively with resident microorganisms and to colonize the
mucosal surfaces of an infected individual, thereby allowing
secretion of an effective amount of an antimicrobial
polypeptide.
[0061] A commensal microorganism of the invention that secretes an
antimicrobial polypeptide which is effective to inhibit infection
by, or a pathogenic activity of, a pathogen, can generally produce
and secrete a sufficient amount of the inhibitory peptide into the
surrounding milieu (either into a cell culture medium in vitro or
into a suitable locale in vivo) to inhibit a microbe (e.g. to
inhibit growth of a virus) without the need to purify or
concentrate the antimicrobial peptide further. For example, Example
I demonstrates that an E. coli Nissle 1918 strain of bacteria
engineered to secrete a C.sub.52-hHly.sub.218 peptide in vitro
secretes the peptide into the broth to a concentration of 40 mg/l,
which is 1.25 .mu.M. The amount of this peptide known to be
required to inactivate HIV is about 5 nM. Thus, this strain can
secrete greater than 100-fold more peptide than is required to
inactivate HIV. In embodiments of the invention, the effective
amount of an antimicrobial polypeptide that is secreted is, e.g.,
greater than about 10-fold, 20-fold, 50-fold, 75-fold etc. of the
amount known to be needed to inhibit infectivity by, and/or a
pathogenic activity of, a pathogen.
[0062] The commensal organisms of the invention preferably secrete
recombinant, antimicrobial polypeptides. These organisms comprise a
polynucleotide which encodes a heterologous antimicrobial
polypeptide fused in phase to a secretion signal, wherein the
coding sequences are operably linked to an expression control
sequence. The polynucleotide can have been introduced into the
microorganism (or an ancestor thereof) by transfection,
transformation, or the like.
[0063] Methods of making recombinant constructs, in which a
sequence encoding a polypeptide of interest is operably linked to
an expression control sequence, are conventional. In general, a
sequence of interest is operably linked to an expression control
sequence in an expression vector. A construct (a recombinant
construct) generated in this manner can express the polypeptide
when introduced into a cell.
[0064] Methods of making recombinant constructs, as well as many of
the other molecular biological methods used in conjunction with the
present invention, are discussed, e.g., in Sambrook, et al. (1989),
Molecular Cloning, a Laboratory Manual, Cold Harbor Laboratory
Press, Cold Spring Harbor, N.Y.; Ausubel et al. (1995). Current
Protocols in Molecular Biology, N.Y., John Wiley & Sons; Davis
et al. (1986), Basic Methods in Molecular Biology, Elseveir
Sciences Publishing, Inc., New York; Hames et al. (1985), Nucleic
Acid Hybridization, IL Press; Dracopoli et al. (current edition)
Current Protocols in Human Genetics, John Wiley & Sons, Inc.;
and Coligan et al. (current edition) Current Protocols in Protein
Science, John Wiley & Sons, Inc.
[0065] As used herein, the term "expression control sequence" means
a polynucleotide sequence that regulates expression of a
polypeptide coded for by a polynucleotide to which it is
functionally ("operably") linked. Expression can be regulated at
the level of the mRNA or polypeptide. Thus, the term expression
control sequence includes mRNA-related elements and protein-related
elements. Such elements include promoters, domains within
promoters, upstream elements, enhancers, ribosome binding
sequences, transcriptional terminators, etc. An expression control
sequence is operably linked to a nucleotide sequence (e.g., a
coding sequence) when the expression control sequence is positioned
in such a manner to effect or achieve expression of the coding
sequence. For example, when a promoter is operably linked 5' to a
coding sequence, expression of the coding sequence is driven by the
promoter. Examples of promoters that can be used to drive
expression in E. coli host bacteria include the trp, lac, tac, and
T7 phage promoters (Melton et al. (1984) Polynucleotide Res.
12(18), 7035-7056; Dunn et al. (1984) J. Mol. Biol. 166, 477-435;
U.S. Pat. No. 5,891,636; Studier et al., (1987) Gene Expression
Technology, in Methods in Enzymology, 85, 60-89) and synthetically
improved versions of these promoters such as those described in Liu
et al. (2004) Proc Natl Acad Sci 101, 6911-6. In the case of
translational signals, such as a ribosome binding sites, the
control element is typically inserted in between the promoter and
the start point of translation. Many potent E. coli translational
control sequences are available from highly expressed chromosomal
loci or from bacteriophage, such as bacteriophage T7.
[0066] In a preferred embodiment of the invention, the fusion
polypeptide is expressed under the control of a constitutive
promoter, e.g. a promoter from the E. coli lac operon, and a
translational control sequence, e.g. from bacteriophage T7. When
these elements are present in a high copy number plasmid, they lead
to constitutive high level expression of the antimicrobial fusion
polypeptide. Alternatively, promoters that are induced in the
conditions in which the host bacteria colonizes the mucosa can be
employed. For example, promoters that are active in the vagina,
e.g. after the introduction of semen, have been described (see,
e.g., U.S. Pat. No. 6,242,194). Promoters regulated by iron are
activated in the gastrointestinal tract.
[0067] To promote secretion of an antimicrobial polypeptide of the
invention, the polypeptide is preferably fused in phase to a
suitable secretion signal. An especially preferred method of this
invention uses the secretory apparatus of hemolysin in a gram
negative bacterial host. A polypeptide of interest is fused to the
carboxy-terminal secretion signal of the hemolysin A gene (hlyA).
The Hly system specifically secretes target proteins from the
bacterial cytoplasm into the extracellular medium without a
periplasmic intermediate. This is one of very few gram negative
bacterial systems that allow protein secretion into the culture
medium, and has been extensively used in live vaccines (Andersen,
C. (2003) Rev Physiol Biochem Pharmacol 147, 122-65; Gentschev et
al. (2002) Trends Microbiol 10, 39-45). The protein machinery of
the Hly type I secretory apparatus consists of two operon-specific
inner membrane components, HlyB and HlyD, and the chromosomally
encoded outer membrane protein, TolC, which form a protein channel
between the inner and outer membranes. The HlyB-HlyD complex
recognizes the carboxy-terminal portion of HlyA, thereby allowing
the secretion of polypeptides fused to this signal sequence.
[0068] In a preferred method of this invention, a construct is
generated which encodes a fusion polypeptide comprising an
antimicrobial polypeptide of interest fused to the carboxyterminal
about 50 to 250 amino acids of the HlyA gene from E. coli strain
LE2001 (G ray et al. (1989) J Cell Sci Suppl 11, 45-57; Mackman et
al. (1984) Mol Gen Genet 193, 312-5; Mackman et al. (1984) Mol Gen
Genet 196, 129-34). This carboxyterminal region comprises a
secretion signal. For example, as shown in the Examples herein, the
construct may encode one of the following 218, 103 or 53 amino acid
sequences from the carboxyterminal portion of the HlyA gene:
TABLE-US-00001 HlyA218 (SEQ ID NO: 3)
NSLAKNVLSGGKGNDKLYGSEGADLLDGGEGNDLLKGGYGNDIYRYLSGYGHHIIDD
EGGKDDKLSLADIDFRDVAFKREGNDLIMYKAEGNVLSIGHKNGITFKNWFEKESDDL
SNHQIEQIFDKDGRVITPDSLKKAFEYQQSNNKVSYVYGHDASTYGSQDNLNPLINEISK
IISAAGNFDVKEERSAASLLQLSGNASDFSYGRNSITLTASA; HlyA103 (SEQ ID NO: 4)
LSNHQIEQIFDKDGRVITPDSLKKAFEYQQSNNKVSYVYGHDASTYGSQDNLNPLINEIS
KIISAAGNFDVKEERSAASLLQLSGNASDFSYGRNSITLTASA; HlyA53 (SEQ ID NO: 5)
LNPLINEISKIISAAGNFDVKEERSAASLLQLSGNASDFSYGRNSITLTASA
[0069] Preferably, the constructs are introduced into a bacterial
cell on a high copy plasmid, or on a low copy number plasmid or
chromosomal integration site in conjunction with a strong promoter.
The HlyB and HlyD genes from E. coli LE2001 are preferably
introduced into the bacterial cell as a single operon carried on a
low copy number plasmid or integrated into a chromosomal site.
Alternatively, the HylB and HlyD genes can be introduced into the
cell on two separate low copy number plasmids, or can be integrated
independently into different chromosomal sites. The precise ratio
of hlyA fusion polypeptide to hlyB and hlyD gene products is not
critical. TolC protein is an endogenous gram negative bacterial
protein and does not need to be artificially introduced into the
bacteria. Example I illustrates the production of an anti-HIV
polypeptide using the hlyA secretory system.
[0070] Additional suitable secretion signals will be evident to the
skilled worker. For example, secretory signals that can be used for
gram positive bacteria include secretion signals derived from the
Lactococcus lactis S-protein, Lactobacillus amylovorus
alpha-amylase, or Streptococcus pyogenes M6 protein genes. Some
signal sequences that are suitable for use in lactobacilli are
described in US Pat. Pub. 20030228297.
[0071] Methods to introduce constructs of the invention into
bacterial cells will be evident to the skilled worker. The most
common are chemical transformation, electroporation, and infection
or transduction with a phage vector.
[0072] Recombinant polypeptides of the invention can be expressed,
e.g., from a vector, such as a plasmid or phage, or from a stably
integrated sequence. Plasmids are particularly useful for
experimental procedures, such as those described in the Examples
herein. In general, such a plasmid contains a selectable marker,
such as resistance to an antibiotic, which is used to select for
the plasmid and to maintain it in the cell. A large number of
suitable selectable markers are known in the art, as are methods
employing them. If desired, a plasmid bearing an antibiotic
resistance gene can be introduced into a subject along with the
antibiotic, in order to facilitate the establishment of the
bacterium in the mucosum.
[0073] However, because antibiotic resistance markers might be
transferred to opportunistic human pathogens such as Staphylococci
and Enterococci, commensal microorganisms of the invention may be
modified to lack such antibiotic resistance markers when designated
for use in the clinic. In this case, constructs of the invention
are stably integrated into the chromosome of a commensal
microorganism, so that a resistance marker is not required. A
resistance marker that is used to select a stable transformant can
be removed after the stable transformant is obtained by a variety
of conventional genetic methods. Alternatively, a construct lacking
a resistance marker can be introduced into the chromosome by
homologous recombination.
[0074] Methods for inserting a sequence of interest into a
bacterial genome in a stable fashion are conventional. For example,
a number of bacteriophage vectors have been developed for use in
different bacteria. A bacteriophage vector based on the temperate
bacteriophage phi adh can be used (see, e.g., Raya et al. (1992) J.
Bacteriol. 174, 5584-5592 and Fremaux et al. (1993) Gene 125,
61-66). This vector undergoes site-specific integration into the
host chromosome at defined phage (attP) and bacterial (attB)
attachment sites. Similarly, Lactobacillus-specific bacteriophage
can be used to transduce vectors or other polynucleotides into the
Lactobacillus chromosome. Lactobacillus-specific phage include mv4
(Auvray et al. (1997) J. Bacteriol., 179, 1837-1845), phi adh
(Fremaux et al. (1993) Gene 126, 61-66), phi gle (Kakikawa et al.
(1996) Gene 175, 157-165, and those belonging to Bradley's groups A
or B in vaginal lactobacillus isolates (Kilic et al. (2001) Clin.
Diagn. Lab. Immunol. 8, 31-39).
[0075] A commensal bacterium of the invention can be used to treat
and/or prevent conditions (e.g., diseases) mediated by a pathogen.
It can be used to inhibit infectivity by, and/or a pathogenic
activity of, a pathogen. As used herein, the singular forms "a,"
"an," and "the" include plural referents unless the context clearly
dictates otherwise. For example, "a" pathogenic activity, as used
above, means one or more pathogenic activities. Among the
pathogenic activities that can be inhibited are, e.g., any feature
in the life cycle of the pathogen following its initial infection
of the host, which allows the pathogen to elicit a detrimental
(pathogenic) effect, or the resulting pathologic effects of that
infection. Initial infection, or infectivity, by a pathogen
includes binding of the pathogen to the host and/or its entry into
the host. For enveloped viruses such as HIV, fusion of the virus to
the host is included. A commensal bacterium of the invention can,
e.g., prevent, inhibit, stabilize, and/or reverse infectivity
and/or a pathogenic activity of a pathogen, and/or can regulate or
modulate the susceptibility of a cell or tissue to infection by the
pathogen.
[0076] One aspect of the invention is a method for inhibiting
infection by, or a pathogenic activity of, a pathogen (e.g.,
inhibiting HIV infection) in vitro or in vivo, comprising
contacting the pathogen with an effective amount of a commensal
bacterium of the invention which secretes a recombinant
antimicrobial polypeptide. Such "contacting," as used herein,
either in the context of in vitro or in vivo methods, need not
involve direct contact of a pathogen with a bacterium of the
invention. For example, the bacterium may be at a distance from the
pathogen, and an antimicrobial polypeptide secreted by the
bacterium may act on the pathogen. Exemplary methods for performing
this method are illustrated in the Examples.
[0077] Another aspect of the invention is a method for inhibiting
infection by, or a pathogenic activity of, a pathogen (e.g.,
inhibiting HIV infectivity) in a subject (e.g., a patient) in need
of such treatment, comprising administering to the subject an
effective amount of a commensal bacterium of the invention which
secretes an effective amount of a recombinant antimicrobial
polypeptide. Another aspect of the invention is a method for
treating a patient infected by, or subject to infection by, a
pathogen, or for preventing the spread of a pathogen (such as a
viral pathogen, particularly HIV) from an infected patient to
others, comprising administering to the patient an effective amount
of a commensal bacterium of the invention, under conditions
effective for the antimicrobial agent secreted by the commensal
microorganism to inhibit infectivity by, or a pathogenic activity
of, a pathogen of interest.
[0078] The hosts (or targets) for administration and colonization
by the genetically altered bacteria include: uninfected individuals
who are at risk for infection by the pathogen of interest;
individuals already infected with the pathogen of interest; various
animals infected by or subject to infection by a pathogen, e.g., a
mammal, such as an experimental animal, a farm animal, pet, or the
like. In preferred embodiments, the animal is a primate, most
preferably a human.
[0079] The microorganism can be administered to a subject using any
of a variety of routes of administration, which will be evident to
the skilled worker. Preferably, the microorganism is administered
through the oral cavity, or is applied directly to the rectum or
the vagina, using conventional methods. Optionally, antibiotic
pretreatment of the subject can be used to pre-clear the mucosal
surface of resident bacteria prior to introduction of the bacteria
of the invention into the rectum, vagina or gastrointestinal tract.
See, e.g., Freter et al. (1983) Infect. Immun. 39, 686-703 and
Example I herein. Antibiotics can be provided orally or can be
applied directly, e.g. to the vagina or rectum.
[0080] Certain agents that do not irritate mucosal epithelial cells
may also be added to a unit dose of the bacteria in capsules or
tablets to aid in colonization. Many bacteria on mucosal surfaces
secrete capsular materials that coalesce to form a biofilm that
covers the entire mucosal surface. It may be beneficial to add an
enzyme that digests this biofilm material to promote penetration of
the engineered bacteria into the biofilm for more successful
colonization. The enzymes include DNAses, peptidases, collagenases,
hyaluronidases, and other carbohydrate degrading enzymes.
Antibiotics to which the engineered bacteria itself is not
susceptible may also be added to decrease the number of resident
bacteria on the mucosal surface in order to make room for the
engineered bacteria.
[0081] Delivery of engineered bacteria to a desired mucosal surface
depends on the accessibility of the area and the local conditions.
For example, engineered bacteria may be placed in a
pharmaceutically acceptable solution, such as a saline solution, or
in a foam for delivery onto the vaginal or rectal mucosa. Foams can
include, e.g., one or more hydrophobically modified polysaccharides
such as cellulosics and chitosans. Cellulosics include, for
example, hydroxyethyl cellulose, hydroxypropyl cellulose, methyl
cellulose, hydroxypropylmethyl cellulose, hydroxyethyl methyl
cellulose, and the like. Chitosans include, for example, the
following chitosan salts; chitosan lactate, chitosan salicylate,
chitosan pyrrolidone carboxylate, chitosan itaconate, chitosan
niacinate, chitosan formate, chitosan acetate, chitosan gallate,
chitosan glutamate, chitosan maleate, chitosan aspartate, chitosan
glycolate and quaternary amine substituted chitosan and salts
thereof, and the like. Foam can also include other components such
as water, ethyl alcohol, isopropyl alcohol, glycerin, glycerol,
propylene glycol, and sorbitol. Spermicides are optionally included
in the bacterial composition. Further examples of foams and foam
delivery vehicles are described in, e.g., U.S. Pat. Nos. 5,595,980
and 4,922,928.
[0082] Alternatively, the bacteria can be delivered as a
suppository or pessary. See, e.g., U.S. Pat. No. 4,322,399. In some
embodiments, the bacteria of the invention are prepared in a
preservation matrix such as described in U.S. Pat. No. 6,468,526
and are delivered in a dissolvable element made of dissolvable
polymer material and/or complex carbohydrate material selected for
dissolving properties, such that it remains in substantially solid
form before use, and dissolves due to human body temperatures and
moisture during use to release the agent material in a desired
timed release and dosage. See, e.g., U.S. Pat. No. 5,529,782. The
bacteria can also be delivered in vaginal foam or a sponge delivery
vehicle such as described in U.S. Pat. No. 4,693,705.
[0083] In one embodiment, the bacteria are administered orally,
e.g. using a solution of about 0.16M sodium bicarbonate (pH about
8.5) to pre-gavage the subjects and to deliver the bacteria to
neutralize the acidic environment of the stomach.
[0084] In one embodiment, a commensal bacterium of the invention is
administered to a subject by coating, at least in part, a
biologically compatible prosthetic device or dildo-like device with
the bacterium, and then inserting the coated device into the
subject. The biologically compatible device may comprise polymers
such as fluorinated ethylene propylene, sulfonated polystyrene,
polystyrene, or polyethylene terephthalate, or glass. The device
may be, e.g., a catheter such as a urinary or peritoneal catheter,
an IUD, or another intravaginal, intrauterine, or intraurethral
device. In another embodiment, the device is a condom. In another
embodiment, the device is a dildo-like device (e.g., a dildo
comprising a small camera at one end, which allows one to follow
the administration of the substance). Alternatively, if desired,
the device (e.g., a relatively long term device, such as an IUD)
can be coated in vivo by administering the commensal bacterium
prior to insertion of the device, and allowing an indigenous
protective flora to be formed on the device.
[0085] The particular mode of administration and the dosage regimen
will be selected by the attending clinician, taking into account
the particulars of the case (e.g., the subject, the degree of the
infection, etc.). Treatment may involve yearly, monthly, daily or
multi-daily doses, over a period of a few days to months, or even
years. Even less frequent treatments can be used if the commensal
organism remains stably associated with the mucosa and continues to
secrete the antimicrobial polypeptide for an extended period of
time.
[0086] The dosage form of a pharmaceutical composition will be
determined by the mode of administration chosen. For example,
topical and oral formulations can be employed. Topical preparations
can include creams, ointments, sprays and the like. Oral
formulations may be liquid (e.g., syrups, solutions or
suspensions), or solid (e.g., powders, pills, tablets, or
capsules). Sprays or drops, such as oral sprays or drops, are also
included. For solid compositions (e.g., lyophilized bacteria),
conventional non-toxic solid carriers can include pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. In as
preferred application, live bacteria are lyophilized and placed in
an enteric-coated capsule, which allows the bacteria not to be
released until they have passed through the acidic stomach and
reached the more alkaline colon. Actual methods of preparing
suitable dosage forms are known, or will be apparent, to those
skilled in the art.
[0087] Effective dosages of the inhibitory commensal organisms of
the invention will be evident to the skilled worker. The exact
amount (effective dose) of the agent will vary from subject to
subject, depending on, i.a., the species, age, weight and general
or clinical condition of the subject, the severity or mechanism of
any disorder being treated, the particular agent or vehicle used,
the method and scheduling of administration, and the like. A
therapeutically effective dose can be determined empirically, by
conventional procedures known to those of skill in the art. See,
e.g., The Pharmacological Basis of Therapeutics, Goodman and
Gilman, eds., Macmillan Publishing Co., New York. For example, an
effective dose can be estimated initially either in cell culture
assays or in suitable animal models. The animal model may also be
used to determine the appropriate concentration ranges and routes
of administration. Such information can then be used to determine
useful doses and routes for administration in humans. A therapeutic
dose can also be selected by analogy to dosages for comparable
therapeutic agents. In general, normal dosage amounts may vary from
about 10.sup.8 bacteria to 10.sup.11 bacteria per person per 1 to
30 days. For example, a daily dose of about 10.sup.9 to 10.sup.10
E. coli Nissle 1917 is typically used for therapy of
gastrointestinal complaints. A dose of 10.sup.8 lactobacilli can be
used to restore the normal urogenital flora. See, e.g., Reid et al.
(2001) FEMS Immuno. Med. Microbiol. 32, 37-41.
[0088] In some embodiments, applications of engineered bacteria to
a mucosal surface will need to be repeated on a regular basis;
optimal dosing intervals are routine to determine, but will vary
with different mucosal environments and bacterial strains. The
dosing intervals can vary, e.g., from once daily to once every 2-4
weeks. In a most preferred embodiment, the bacteria need be
delivered very infrequently (e.g., only once year).
[0089] A commensal microorganism of the invention can be formulated
as pharmaceutical composition, which comprises the commensal
microorganism (e.g., a therapeutically effective amount of the
commensal microorganism) and a pharmaceutically acceptable carrier,
using conventional components and methodologies. "Therapeutic"
compositions and compositions in a "therapeutically effective
amount" are compositions that can elicit at least a detectable
amount of inhibition or amelioration of infection by, or a
pathogenic activity of, a pathogen.
[0090] Such pharmaceutical compositions are normally formulated
with a solid or liquid carrier, depending upon the particular mode
of administration chosen. The pharmaceutically acceptable carriers
useful in this disclosure are conventional. Pharmaceutically and
physiologically acceptable fluid vehicles, such as water,
physiological saline, other balanced salt solutions, aqueous
dextrose, glycerol or the like, may be employed. Excipients that
can be included are, for instance, other proteins, such as human
serum albumin or plasma preparations. If desired, the
pharmaceutical composition to be administered may also contain
minor amounts of non-toxic auxiliary substances, such as wetting or
emulsifying agents, preservatives, and pH buffering agents and the
like, for example sodium acetate or sorbitan monolaurate.
[0091] The present disclosure also includes combinations of agents
of the commensal microorganisms of the invention with one another,
and/or with one or more other agents useful in the treatment of a
pathogenic infection. For example, commensal microorganisms of the
invention may be administered in combination with effective doses
of other anti-pathogenic agents, such as an antiretroviral drug.
The term "administration in combination" refers to both concurrent
and sequential administration of the active agents. The combination
therapies are of course not limited to the agents provided herein,
but include any composition for the treatment of pathological
infections.
[0092] Another aspect of the invention is a kit for carrying out
any of the methods of the invention. For example, one embodiment is
a kit for inhibiting an infection by a pathogen, comprising an
effective amount of amount of an inhibitory commensal bacterium of
the invention and, optionally, (e.g., if the infection is in a
subject in vivo) means for storing or packaging the inhibitory
commensal bacterium, or for administering it to a subject.
[0093] The components of the kit will vary according to which
method is being performed. Optionally, the kits comprise
instructions for performing the method. Kits of the invention may
further comprise a support on which a cell can be propagated (e.g.,
a culture vessel). Other optional elements of a kit of the
invention include suitable buffers, media components, or the like;
containers; or packaging materials. The reagents of the kit may be
in containers in which the reagents are stable, e.g., in
lyophilized form or stabilized liquids. The reagents may also be in
single use form, e.g., in single dosage form for use as
therapeutics.
[0094] In the foregoing and in the following examples, all
temperatures are set forth in uncorrected degrees Celsius; and,
unless otherwise indicated, all parts and percentages are by
weight.
EXAMPLES
Example I
Expression of an Anti-HIV Peptide by a Commensal Strain of E.
coli
A. Experimental Design: Choice of Inhibitory Peptide, Secretion
System and Bacterial Strain
[0095] The inventors engineered E. coli commensal strain Nissle
1917 to express an HIV fusion inhibitor peptide fused to the
carboxy-terminal secretion signal of the hemolysin A gene (hlyA)
and showed that the hybrid polypeptide secreted into the culture
medium can block HIV infection. Moreover the genetically modified
bacteria were capable of colonizing the gastrointestinal tract and
the vagina of an experimental animal, the mouse.
[0096] As an HIV blocker, a 52 amino acid peptide comprising 47
amino acids from the C-terminal region of gp41 and 5 amino acids
added to allow cloning into the expression vector-052--was chosen.
For a further description of C52, see Root et al. (2003) Proc Natl
Acad Sci USA 100, 5016-5021 and discussions elsewhere herein. The
C52 peptide coding sequences were cloned into a high copy number,
ampicillin-resistant expression vector containing the E-tag epitope
and the final 218 amino acids from the C-terminal secretion signal
sequence of hlyA (Fernandez et al. (2000) Appl Environ Microbiol
66, 5024-9). Subsequently, the hlyA secretion signal sequence was
further truncated to either 103 or 53 amino acids. The structures
of the recombinant genes are shown in FIG. 1. The sequences of the
recombinant antimicrobial polypeptides encoded by these constructs
are:
TABLE-US-00002 C52-HlyA218 (SEQ ID NO: 6)
MGGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF
PGGAPVPYPDPLEPAGENNSLAKNVLSGGKGNDKLYGSEGADLLDGGEGNDL
LKGGYGNDIYRYLSGYGHHIIDDEGGKDDKLSLADIDFRDVAFKREGNDLIMYKAEGN
VLSIGHKNGITFKNWFEKESDDLSNHQIEQIFDKDGRVITPDSLKKAFEYQQSNNKVSY
VYGHDASTYGSQDNLNPLINEISKIISAAGNFDVKEERSAASLLQLSGNASDFSYGRNSI
TLTASA; C52-HlyA103 (SEQ ID NO: 7)
MGGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFPGGAP
VPYPDPLEPAGENLSNHQIEQIFDKDGRVITPDSLKKAFEYQQSNNKVSYVYGHDASTY
GSQDNLNPLINEISKIISAAGNFDVKEERSAASLLQLSGNASDFSYGRNSITLTASA;
C52-HlyA53 (SEQ ID NO: 8)
MGGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFPGGAP
VPYPDPLEPAGENLNPLINEISKIISAAGNFDVKEERSAASLLQLSGNASDFSYGRNSIT
LTASA.
[0097] The HIV C52 fragment was prepared by PCR of DNA from HIV
strain NL4-3 with the following two primers:
TABLE-US-00003 C52NcoTop: (SEQ ID NO: 9)
5'gatggccatgggcggtcacacgacctggatggag3' C52XmaBot: (SEQ ID NO: 10)
5'attccccgggaaaccaattccacaaacttgc3'
[0098] The PCR product was purified, treated with NcoI and XmaI,
and cloned into pEHLYA2-SD cleaved with NcoI and XmaI. Truncations
of the HlyA secretion sequence were introduced by
oligonucleotide-mediated mutagenesis.
B. Secretion of Anti-HIV Peptides by Nissle 1917
[0099] The recombinant plasmids were electroporated into E. coli
Nissle 1917 (obtained from a commercial preparation of the
probiotic Mutaflor from Arceypharm, Herdecke, Germany) together
with a low copy number chloramphenicol-resistance plasmid that
encodes hlyB and hlyD (Fernandez et al. (2000) Appl Environ
Microbiol 66, 5024-9). Colonies carrying both plasmids were
selected on agar plates containing ampicillin and chloramphenicol.
These strains were grown in rich medium (Luria broth) to mid log
phase, treated with IPTG, and allowed to reach late log phase. The
cell-free culture supernatants were then examined by SDS gel
electrophoresis. In each case, the C52-hlyA fusion protein was
observed as the predominant species secreted into the culture
medium (See FIG. 2A). Similar results were obtained in early log
phase and in stationary overnight cultures. Moreover, a similar
level of expression was observed even in the absence of IPTG
induction. The identity of the hybrid proteins was confirmed by
Western blotting with an anti-HIV gp41 monoclonal antibody and by
complex formation with 5-Helix protein. Similar experiments using
C-peptide sequences from SIV.sub.mac239 led to even higher levels
of secreted peptides.
[0100] The amount of C.sub.52-HlyA.sub.218 peptide secreted by
Nissle 1917 was estimated to be 40 mg/liter, representing a peptide
concentration of 1.25 .mu.M. Similar expression levels were found
in cultures grown to late log phase in the presence or absence of
IPTG and in saturated overnight cultures without inducer; this lack
of dependence on IPTG was expected due to titration of the
available Lac repressor by the high copy number Lac operator.
Several other commensal strains of E. coli isolated from feces or
vagina of normal healthy volunteers (C594.72; C641.72; and C105.72)
also expressed the C.sub.52-HlyA.sub.218 peptide, but the levels of
secreted protein were no greater than or lower than for Nissle
1917.
[0101] As shown, fusion peptides having 103 or 53 amino acids from
the C-terminus of HlyA were secreted efficiently into the culture
medium. By contrast, a fusion peptide having only 43 amino acids
from the C-terminus did not show any peptide secretion. These data
define the N-terminal boundary of the HlyA secretion signal
sequence at 43-53 amino acids upstream of the C terminus of the
protein.
[0102] One advantage of working with E. coli is the extensive
knowledge of the cis and trans-acting signals that can be
manipulated to increase foreign gene expression. We have previously
found that gene aadA1 originating from Tn21 transposon, which
confers resistance to aminoglycoside antibiotics, increases the
expression of a broad spectrum of cellular proteins in bacteria.
Introduction of aadA1 into Nissle 1917 upregulated
C.sub.52-HlyA.sub.218 peptide expression by 3-fold without any
adverse effect on the growth of the bacteria.
C. Inhibition of HIV Infection by Secreted Peptides
[0103] The ability of the secreted peptides to block HIV-GFP
reporter virus infection was tested in PBMC (peripheral blood
mononuclear cells). Various concentrations of the antimicrobial
recombinant proteins were incubated with the reporter virus
HIV.sub.NL4-3GFP, in which a GFP (green fluorescent protein) gene
allows monitoring of infection by FACS (fluorescence-activated cell
sorter). Efficient inhibition was observed for each of the C52-hlyA
peptides, but not for the control Etag-hlyA peptide. Inhibition was
specific for the HIV envelope glycoprotein as shown by the lack of
any effect on pseudovirions encapsidated with MuLV rather then HIV
Env (See FIG. 1B). The potency of C52-hlyA.sub.218 was close to
that of 5-Helix, having an IC.sub.50 value of approximately 1 nM.
Similar results were obtained using a HIV.sub.JR-CSFGFP reporter
virus. Thus the anti-HIV peptides can block infection by both X4
and R5 tropic viruses.
D. Colonization of Mouse Gastrointestinal and Genitourinary
Tracts
[0104] The ability of the genetically modified E. coli Nissle 1917
to colonize the gastrointestinal and genitourinary tracts was
investigated using the mouse as a small animal model system. Female
mice, strain CD-1 (Charles River laboratories) were administered
5.times.10.sup.8 E coli Nissle 1917::C52-HlyA208, either orally (by
gavage using 0.2 ml of a 0.16 M sodium bicarbonate solution, pH
8.5) or rectally (by injection using 0.2 ml of a PBS solution). The
mice were either treated with no antibiotic, pretreated for one day
prior to inoculation with antibiotic (streptomycin sulfate, 2.5
mg/l in drinking water), post-treated for 12 days with antibiotic
(ampicillin, 2.4 mg/l in drinking water), or both pre-treated and
post-treated with antibiotic. At intervals, feces and vaginal wash
samples were collected and assayed for the presence of the
recombinant bacteria by plating on agar containing chloramphenicol
and ampicillin. In addition, mice were sacrificed at 2 and 7 days
post-inoculation, and segments of the gastrointestinal tract and
genitourinary tract were dissected and assayed for recombinant
bacteria.
[0105] Substantial colonization of the entire gastrointestinal
tract and of the vagina were observed (FIG. 3). The concentrations
of bacteria in the feces reached approximately 10.sup.10 per gram,
which is as high or higher than the concentration of endogenous E.
coli in the absence of antibiotic treatment. In animals that
received no antibiotic, the number of recombinant bacteria peaked
at 1 to 3 days and then declined. However, in mice pre-treated or
post-treated with antibiotics, the concentration of bacteria found
in feces remained high throughout the 12 day observation period.
The maintenance of such a high concentration of bacteria clearly
demonstrates that the altered microorganisms are undergoing growth
and cell division in the mice.
[0106] To investigate the cis and trans-acting signals involved in
the maintenance of gastrointestinal tract colonization, mice were
given a single treatment with ampicillin, orally administered
various bacterial strains, and then maintained without antibiotic
for 25 days. Colonization levels of Nissle 1917 expressing
C.sub.52-HlyA.sub.218 fell more than 10.000-fold over this period
(Table 1).
TABLE-US-00004 TABLE 1 Effects of cis and trans-acting signals on
mouse colonization Log cfu/g feces Nissle 1917 Plasmid Day 1 Day 25
Log diff. wt pC.sub.52-HlyA.sub.218 8.35 (0.27) 3.61 (0.16) 4.74
aadA1 pC.sub.52-HlyA.sub.218 8.42 (0.52) 4.62 (0.79) 3.80 wt
Etag-HlyA218 7.84 (0.50) 3.02 (0.27) 4.81 wt C52-HlyA103 7.98
(0.36) 5.99 (0.91) 1.99 wt C52-HlyA53 8.91 (0.13) 7.00 (0.91)
1.91
Mice were orally administered 5.times.10.sup.8 cfu of the indicated
strain and maintained without antibiotic. Feces were assayed for
Ap.sup.r Cm.sup.r cfu at intervals and the results at day 1 and day
25 are shown as the mean (standard error) of the log.sub.10 cfu/g
feces #, with errors in parentheses.
[0107] This drop in bacterial levels was not simply due to the
amount of peptide secreted as shown by the somewhat better
retention of bacteria that overproduced the peptide due to the
introduction of aadA1 gene. The drop was also not exclusively due
to the presence of HIV sequences in the secreted peptide as shown
by the poor maintenance of a strain expressing Etag-HlyA.sub.218.
By contrast, the extent of HlyA C-terminal sequences did appear to
play an important role as demonstrated by the ability of bacteria
expressing the C-terminal deletion mutants C.sub.52-HlyA.sub.103
and C.sub.52-HlyA.sub.53 to undergo more persistent colonization
than C.sub.52-HlyA.sub.218, with levels at day 25 more than
100-fold higher than for the full length construct. This conclusion
was confirmed by a competitive colonization experiment in which
mice were fed an equal mixture of bacteria expressing
C.sub.52-HlyA.sub.218 and C.sub.52-HlyA.sub.53 and subsequently
analyzed for the ratio of colonies expressing the different length
peptides. At day 1, a slight excess of bacteria expressing the
HlyA.sub.218 construct was excreted, but by day 8 and thereafter
only bacteria expressing the short HlyA.sub.53 construct could be
recovered.
[0108] To examine the potential of the genetically engineered
Nissle for long-term, stable colonization in the absence of
antibiotics, mice were administered bacteria expressing a C.sub.52
peptide both orally and rectally, then maintained on ampicillin for
50 days prior to removal of the antibiotic. The rational for this
prolonged initial antibiotic treatment was that it would eliminate
much of the competing indigenous microflora while allowing the
genetically engineered bacteria to adapt to the nutritional
environment of the intestine. In the absence of antibiotics,
bacteria expressing C.sub.52-HlyA.sub.218 were again eliminated
from the mice reaching undetectable levels by 77 days. However,
bacteria secreting C.sub.52-HlyA.sub.103 and C.sub.52-HlyA.sub.103
were maintained in the mice at levels of approximately 10.sup.6
cfu/g feces for up to 50 days after the removal of drug selection.
Bacteria recovered from the mouse feces after prolonged
colonization were still capable of secreting high levels of the
C.sub.52 peptides, indicating there was no strong selection against
peptide secretion in vivo.
E. In Vivo Growth Patterns and Peptide Secretion.
[0109] The distribution of the anti-HIV bacteria in different
tissues was examined in mice that has been pre-treated with
ampicillin to reduce the endogenous microflora, then orally or
rectally administered Nissle 1917 expressing C.sub.52-HlyA.sub.53.
The highest concentrations of bacteria (10.sup.8 to 10.sup.9 cfu/g)
were present in the colon and cecum following both oral and rectal
administration (FIG. 3B). Lower levels of bacteria (10.sup.5 to
10.sup.6 cfu/g) were also recovered along the upper intestine
including duodenum, jejunum and ileum in mice that were orally
inoculated, whereas bacteria were recovered from rectum primarily
in mice that were rectally inoculated. Nissle 1917 was also
recovered from vagina in approximately one third of rectally
inoculated animals. In summary, the commensal bacteria secreting an
anti-HIV peptide were capable of colonizing the gastrointestinal
and genitourinary tract including the mucosal surfaces at which HIV
infection most often occurs.
[0110] Tissues from colonized animals were also processed for
histopathology and immunohistochemistry. None of the target organs
demonstrated any inflammation, necrosis, or other pathology.
Detailed examination of the colon revealed the presence of numerous
monomorphic, hematoxylin-eosin stained bacterial colonies in
animals inoculated with Nissle 1917 expressing C.sub.52-HlyA.sub.53
but not in control animals inoculated with PBS. As expected for E.
coli, these bacteria were gram negative. The bacterial colonies
were present through the lumen, often in close association with the
epithelial surface. In some samples, the colon and cecum of Nissle
1917-inocculated animals demonstrated goblet cell hyperplasia and
copious mucus secretion into the lumen that was not evident in
control PBS-inoculated animals.
[0111] To examine peptide secretion, colon samples were subjected
to immunohistochemistry using the human monoclonal antibody 2F5,
which recognizes an epitope present in the C.sub.52 peptide. Clear
staining was observed through the lumen in samples from animals
inoculated with Nissle 1917 expressing pC.sub.52-HlyA.sub.53, but
not in control samples processed without antibody or from animals
expressing control Etag peptide.
Example II
Efficacy Tests, Using Rhesus Macaques
A. Experimental Design: Choice of Bacteria, Test Animals, and
Challenge Virus.
[0112] The inventor used macaques to demonstrate that a commensal
strain of E. coli expressing an HIV fusion inhibitor peptide can
colonize the gastrointestinal tract of a primate and that the
genetically modified bacteria inhibit infection by an HIV-SIV
hybrid virus. The bacteria used for this experiment were E. coli
Nissle 1917 expressing C52-HlyA hybrid peptides because the
previous data showed that these organisms can secrete high levels
of biologically active peptide and can efficiently colonize mucosal
surfaces of the gastrointestinal tract (see, 1 e.g., Example I).
The test animals were rhesus macaque of Chinese origin, which were
selected because they are a well-studied nonhuman primate model for
immunodeficiency virus infection and pathognesis. The challenge
virus was SHIV 162p3, an HIV-SIV hybrid virus. This strain was
chosen because (i) it uses the same envelope protein as HIV and is
therefore a suitable test target for HIV-specific peptides; (ii) it
uses the same replication machinery as SIV and therefore can grow
and cause disease in macaques; (iii) it utilizes the CCR5
coreceptor, which is the same employed in mucosal transmission of
HIV in humans; and (iv) it is a well studied model for HIV
microbicide research and carefully characterized and tittered viral
stocks are available.
B. Colonization of Macaque Gastrointestinal Tract
[0113] Sexually mature male and female Rhesus macaques of Chinese
origin were used for the colonization and challenge experiments. In
the experiment shown in FIG. 4A, the animals were pretreated
starting five days prior to inoculation with oral Cefixime and
Cephaclor. At two days prior to inoculation the animals were put on
a clear liquid diet, and at one day prior to inoculation PEG and
Metronidazole were administered to further reduce endogenous
microflora. The animals were then inoculated with a mixture of E.
coli Nissle 1917 expressing C52-Hly218, C52-Hly103 and C52-HlyA53
both orally and rectally with 10.sup.10 bacteria in 5 ml of PBS
every other day for a total of one week. At intervals, feces
samples were collected and assayed for the presence of the
recombinant bacteria by plating on agar containing chloramphenicol
and ampicillin and by PCR of boiled extracts using plasmid and
C52-specific primers. Positive colonies were further verified by
growth in rich LB broth and analysis of the secreted peptides by
SDS-PAGE.
[0114] FIG. 4A demonstrates that the recombinant anti-HIV bacteria
were capable of colonizing the macaque gastrointestinal tract at
levels between 10.sup.6 and 10.sup.10 colony forming units/gm
feces. Furthermore these levels could be maintained as long at 10
days. In additional experiments, colonization was also observed in
animals treated with ampicillin rather than Cefixime, Cephaclor and
Metronidale, and to a lesser but still significant extent in
animals without any antibiotic treatment. The administration of the
recombinant bacteria to the macaques had no pathogenic effects as
determined by clinical observations, body weight, hematology, and
serum clinical chemistry.
[0115] To determine the distribution of the bacteria along the
intestinal tract, a macaque was orally and rectally administered
10.sup.10 recombinant Nissle then sacrificed one day later for
microbiological and histological analysis of tissue. FIG. 4B shows
that the highest concentrations of bacteria were present in the
colon, cecum and rectum. Somewhat lower but still significant
levels of bacteria were also recovered along the upper intestine
including duodenum, jejunum, ileum, and the ileal junction. Nissle
1917 was also recovered from anus whereas all tested lymphatic
tissues were negative. Microscopic examination revealed heavy
concentrations of bacteria through the gastrointestinal tract with
no indication of experimentally caused inflammation, necrosis or
other pathology.
C. Efficacy Testing.
[0116] Four macaques were colonized with the anti-HIV Nissle 1917
as above, then on day 3 challenged with SHIV 162p3, a pathogenic
HIV-SIV hybrid that resembles most naturally transmitted HIV-1
strains by using CCR5 as coreceptor. The dose used in this study (1
ml of a 1:10 dilution of a tittered stock virus) gave 100%
infection in control animals (FIG. 5A). By contrast, among the
animals colonized with anti-HIV Nissle, two were completely
protected against viral challenge as determined both by plasma
viral RNA measurements (FIG. 5B) and antibody measurements (not
shown). Two of the experimental animals were infected, but the
viral load levels were on average approximately 10-fold lower than
in the control animals. This result suggests that the E. coli-based
microbicide is capable of protecting roughly 50% of animals against
rectal HIV challenge.
[0117] In further studies, rhesus macaques of Indian or Chinese
origin are used for efficacy tests of bacteria expressing
HIVC52-HlyA, SIVC52-HlyA, T1249, and/or other effective
HIV-inhibiting peptides, or combinations thereof. Initially,
animals are administered a range of doses from about 10.sup.8 to
10.sup.11 of genetically engineered bacteria by oral gavage, rectal
gavage, or intra-vaginal inoculation, with or without pretreatment
with ampicillin or other agents as noted above. The extent of
colonization and peptide expression are determined by fecal plating
assays and/or Western blots. Once animals that stably express the
HIV- or SIV-inhibiting and control peptides are obtained, they are
challenged with SHIV or SIV by the rectal, vaginal, or oral route.
The ability of the bacteria to protect against viral infection is
determined by measurements of viral load and CD4 T cells at
periodic intervals. The virus stocks used in these studies are
first titered and standardized by established procedures used for
vaccine candidate testing. It is expected that macaques colonized
with bacteria expressing the anti-HIV and anti-SIV peptides will be
infected poorly or not at all, whereas macaques colonized with
control bacteria will be infected at the normal rate.
REFERENCES
[0118] U.S. Pat. Nos. 5,705,160; 5,804,179; 5,821,081; 5,733,540;
6,277,370; 6,365,156; 6,605,286; 6,180,100; U.S. Patent application
20020086020; U.S. Patent application 20030228297; U.S. Patent
application 20050003510; Lee et al. (2000) J Biol Chem 275,
15809-819.
[0119] From the foregoing description, one skilled in the art can
easily ascertain the essential characteristics of this invention,
and without departing from the spirit and scope thereof, can make
changes and modifications of the invention to adapt it to various
usage and conditions.
[0120] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, utilize the
present invention to its fullest extent. The preceding preferred
specific embodiments are, therefore, to be construed as merely
illustrative, and not limitative of the remainder of the disclosure
in any way whatsoever.
[0121] The entire disclosure of all applications, patents and
publications, cited above and below and in the figures are hereby
incorporated by reference.
Sequence CWU 1
1
10152PRTHuman immunodeficiency virus 1Met Gly Gly His Thr Thr Trp
Met Glu Trp Asp Arg Glu Ile Asn Asn 1 5 10 15 Tyr Thr Ser Leu Ile
His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln 20 25 30 Glu Lys Asn
Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu 35 40 45 Trp
Asn Trp Phe 50 252PRTSimian immunodeficiency virus 2Met Gly Gly His
Thr Thr Trp Gln Glu Trp Glu Arg Lys Val Asp Phe 1 5 10 15 Leu Glu
Glu Asn Ile Thr Ala Leu Leu Glu Glu Ala Gln Ile Gln Gln 20 25 30
Glu Lys Asn Met Tyr Glu Leu Gln Lys Leu Asn Ser Trp Asp Val Phe 35
40 45 Gly Asn Trp Phe 50 3217PRTEscherichia coli 3Asn Ser Leu Ala
Lys Asn Val Leu Ser Gly Gly Lys Gly Asn Asp Lys 1 5 10 15 Leu Tyr
Gly Ser Glu Gly Ala Asp Leu Leu Asp Gly Gly Glu Gly Asn 20 25 30
Asp Leu Leu Lys Gly Gly Tyr Gly Asn Asp Ile Tyr Arg Tyr Leu Ser 35
40 45 Gly Tyr Gly His His Ile Ile Asp Asp Glu Gly Gly Lys Asp Asp
Lys 50 55 60 Leu Ser Leu Ala Asp Ile Asp Phe Arg Asp Val Ala Phe
Lys Arg Glu 65 70 75 80 Gly Asn Asp Leu Ile Met Tyr Lys Ala Glu Gly
Asn Val Leu Ser Ile 85 90 95 Gly His Lys Asn Gly Ile Thr Phe Lys
Asn Trp Phe Glu Lys Glu Ser 100 105 110 Asp Asp Leu Ser Asn His Gln
Ile Glu Gln Ile Phe Asp Lys Asp Gly 115 120 125 Arg Val Ile Thr Pro
Asp Ser Leu Lys Lys Ala Phe Glu Tyr Gln Gln 130 135 140 Ser Asn Asn
Lys Val Ser Tyr Val Tyr Gly His Asp Ala Ser Thr Tyr 145 150 155 160
Gly Ser Gln Asp Asn Leu Asn Pro Leu Ile Asn Glu Ile Ser Lys Ile 165
170 175 Ile Ser Ala Ala Gly Asn Phe Asp Val Lys Glu Glu Arg Ser Ala
Ala 180 185 190 Ser Leu Leu Gln Leu Ser Gly Asn Ala Ser Asp Phe Ser
Tyr Gly Arg 195 200 205 Asn Ser Ile Thr Leu Thr Ala Ser Ala 210 215
4103PRTEscherichia coli 4Leu Ser Asn His Gln Ile Glu Gln Ile Phe
Asp Lys Asp Gly Arg Val 1 5 10 15 Ile Thr Pro Asp Ser Leu Lys Lys
Ala Phe Glu Tyr Gln Gln Ser Asn 20 25 30 Asn Lys Val Ser Tyr Val
Tyr Gly His Asp Ala Ser Thr Tyr Gly Ser 35 40 45 Gln Asp Asn Leu
Asn Pro Leu Ile Asn Glu Ile Ser Lys Ile Ile Ser 50 55 60 Ala Ala
Gly Asn Phe Asp Val Lys Glu Glu Arg Ser Ala Ala Ser Leu 65 70 75 80
Leu Gln Leu Ser Gly Asn Ala Ser Asp Phe Ser Tyr Gly Arg Asn Ser 85
90 95 Ile Thr Leu Thr Ala Ser Ala 100 552PRTEscherichia coli 5Leu
Asn Pro Leu Ile Asn Glu Ile Ser Lys Ile Ile Ser Ala Ala Gly 1 5 10
15 Asn Phe Asp Val Lys Glu Glu Arg Ser Ala Ala Ser Leu Leu Gln Leu
20 25 30 Ser Gly Asn Ala Ser Asp Phe Ser Tyr Gly Arg Asn Ser Ile
Thr Leu 35 40 45 Thr Ala Ser Ala 50 6287PRTArtificial
sequenceSynthetic construct 6Met Gly Gly His Thr Thr Trp Met Glu
Trp Asp Arg Glu Ile Asn Asn 1 5 10 15 Tyr Thr Ser Leu Ile His Ser
Leu Ile Glu Glu Ser Gln Asn Gln Gln 20 25 30 Glu Lys Asn Glu Gln
Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu 35 40 45 Trp Asn Trp
Phe Pro Gly Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu 50 55 60 Glu
Pro Ala Gly Glu Asn Asn Ser Leu Ala Lys Asn Val Leu Ser Gly 65 70
75 80 Gly Lys Gly Asn Asp Lys Leu Tyr Gly Ser Glu Gly Ala Asp Leu
Leu 85 90 95 Asp Gly Gly Glu Gly Asn Asp Leu Leu Lys Gly Gly Tyr
Gly Asn Asp 100 105 110 Ile Tyr Arg Tyr Leu Ser Gly Tyr Gly His His
Ile Ile Asp Asp Glu 115 120 125 Gly Gly Lys Asp Asp Lys Leu Ser Leu
Ala Asp Ile Asp Phe Arg Asp 130 135 140 Val Ala Phe Lys Arg Glu Gly
Asn Asp Leu Ile Met Tyr Lys Ala Glu 145 150 155 160 Gly Asn Val Leu
Ser Ile Gly His Lys Asn Gly Ile Thr Phe Lys Asn 165 170 175 Trp Phe
Glu Lys Glu Ser Asp Asp Leu Ser Asn His Gln Ile Glu Gln 180 185 190
Ile Phe Asp Lys Asp Gly Arg Val Ile Thr Pro Asp Ser Leu Lys Lys 195
200 205 Ala Phe Glu Tyr Gln Gln Ser Asn Asn Lys Val Ser Tyr Val Tyr
Gly 210 215 220 His Asp Ala Ser Thr Tyr Gly Ser Gln Asp Asn Leu Asn
Pro Leu Ile 225 230 235 240 Asn Glu Ile Ser Lys Ile Ile Ser Ala Ala
Gly Asn Phe Asp Val Lys 245 250 255 Glu Glu Arg Ser Ala Ala Ser Leu
Leu Gln Leu Ser Gly Asn Ala Ser 260 265 270 Asp Phe Ser Tyr Gly Arg
Asn Ser Ile Thr Leu Thr Ala Ser Ala 275 280 285 7173PRTArtificial
sequenceSynthetic construct 7Met Gly Gly His Thr Thr Trp Met Glu
Trp Asp Arg Glu Ile Asn Asn 1 5 10 15 Tyr Thr Ser Leu Ile His Ser
Leu Ile Glu Glu Ser Gln Asn Gln Gln 20 25 30 Glu Lys Asn Glu Gln
Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu 35 40 45 Trp Asn Trp
Phe Pro Gly Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu 50 55 60 Glu
Pro Ala Gly Glu Asn Leu Ser Asn His Gln Ile Glu Gln Ile Phe 65 70
75 80 Asp Lys Asp Gly Arg Val Ile Thr Pro Asp Ser Leu Lys Lys Ala
Phe 85 90 95 Glu Tyr Gln Gln Ser Asn Asn Lys Val Ser Tyr Val Tyr
Gly His Asp 100 105 110 Ala Ser Thr Tyr Gly Ser Gln Asp Asn Leu Asn
Pro Leu Ile Asn Glu 115 120 125 Ile Ser Lys Ile Ile Ser Ala Ala Gly
Asn Phe Asp Val Lys Glu Glu 130 135 140 Arg Ser Ala Ala Ser Leu Leu
Gln Leu Ser Gly Asn Ala Ser Asp Phe 145 150 155 160 Ser Tyr Gly Arg
Asn Ser Ile Thr Leu Thr Ala Ser Ala 165 170 8122PRTArtificial
sequenceSynthetic construct 8Met Gly Gly His Thr Thr Trp Met Glu
Trp Asp Arg Glu Ile Asn Asn 1 5 10 15 Tyr Thr Ser Leu Ile His Ser
Leu Ile Glu Glu Ser Gln Asn Gln Gln 20 25 30 Glu Lys Asn Glu Gln
Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu 35 40 45 Trp Asn Trp
Phe Pro Gly Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu 50 55 60 Glu
Pro Ala Gly Glu Asn Leu Asn Pro Leu Ile Asn Glu Ile Ser Lys 65 70
75 80 Ile Ile Ser Ala Ala Gly Asn Phe Asp Val Lys Glu Glu Arg Ser
Ala 85 90 95 Ala Ser Leu Leu Gln Leu Ser Gly Asn Ala Ser Asp Phe
Ser Tyr Gly 100 105 110 Arg Asn Ser Ile Thr Leu Thr Ala Ser Ala 115
120 934DNAArtificial sequenceSynthetic primer 9gatggccatg
ggcggtcaca cgacctggat ggag 341031DNAArtificial sequenceSynthetic
primer 10attccccggg aaaccaattc cacaaacttg c 31
* * * * *