U.S. patent application number 12/916889 was filed with the patent office on 2011-05-12 for human optimized bacillus anthracis protective antigen.
Invention is credited to Mark Albrecht, Stanley Goldman.
Application Number | 20110110968 12/916889 |
Document ID | / |
Family ID | 43974333 |
Filed Date | 2011-05-12 |
United States Patent
Application |
20110110968 |
Kind Code |
A1 |
Goldman; Stanley ; et
al. |
May 12, 2011 |
Human optimized Bacillus anthracis protective antigen
Abstract
The invention relates to a humanized nucleic acid construct from
Bacillus anthracis protective antigen (PA) gene and method of
modifying the gene. The humanized gene, and method of producing it,
improves the structural fidelity of expressed protein product, when
produced in mammalian host cells, to native, bacterially produced
protein. The construct is useful in nucleic acid based vaccine
formulations against B. anthracis.
Inventors: |
Goldman; Stanley; (Walnut
Creek, CA) ; Albrecht; Mark; (Washington,
DC) |
Family ID: |
43974333 |
Appl. No.: |
12/916889 |
Filed: |
November 1, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61260656 |
Nov 12, 2009 |
|
|
|
Current U.S.
Class: |
424/190.1 ;
424/200.1; 536/25.3 |
Current CPC
Class: |
A61P 37/04 20180101;
A61K 2039/55511 20130101; A61K 39/07 20130101; A61P 31/04 20180101;
A61K 2039/53 20130101; C12N 15/64 20130101; C07K 14/32
20130101 |
Class at
Publication: |
424/190.1 ;
424/200.1; 536/25.3 |
International
Class: |
A61K 39/07 20060101
A61K039/07; C07H 21/04 20060101 C07H021/04; A61P 37/04 20060101
A61P037/04; A61P 31/04 20060101 A61P031/04 |
Claims
1. An immunogenic composition comprising a recombinant Bacillus
anthracis protective antigen gene, wherein said composition is
encoded by a nucleic acid sequence wherein regions of said sequence
corresponding to regions of B. anthracis containing rare bacterial
codons are replaced with rare mammalian codons and where the codons
in the first 2 to 5% of the sequence are highly utilized human
codons.
2. The immunogenic composition of claim 1, wherein the mammalian
codons are human.
3. The immunogenic composition of claim 1, wherein the first 50
codons are highly utilized human codons.
4. The immunogenic composition of claim 1, wherein said amino acid
sequence is SEQ ID No. 1 and is encoded by nucleic acid sequence of
SEQ ID No. 2.
5. The immunogenic composition of claim 1, wherein said DNA is
expressed from a DNA or viral expression system.
6. The composition of claim 1, wherein protein product encoded from
said recombinant protective antigen gene is induced via the TPA
signal.
7. A method of modifying a recombinant bacterial gene for
expression in a mammalian host comprising: a. replacing highly
utilized host codons in the first 2 to 5 percent of the total
bacterial gene sequence; b. replacing regions of the sequence where
there are stretches of three or more rare bacterial codons with
rare host codons; c. evaluation said modified bacterial gene
sequence to identify regions of complementarity that could result
in RNA folding back on itself; d. removing said regions of
complementarity by altering the codons in these regions but
retaining the same amino acid sequence of the expressed protein;
and e. evaluating for cryptic ribosomal splice sites in order to
avoid regions of said gene being deleted by the host cell; f.
altering regions containing said splice sites to remove recognition
sequences but retaining the original amino acid sequence.
8. A method of inducing an immune response against Bacillus
anthracis comprising: a. administering to the immunogenic
composition of claim 1; b. Administering a second, boosting dose of
said immunogenic composition.
9. The method of claim 8, wherein said immunogenic composition is
expressed from a DNA or viral expression vector.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/260,656, filed 12 Nov. 2009, which is
incorporated herein by reference.
BACKGROUND OF INVENTION
[0002] 1. Field of Invention
[0003] The inventive subject matter relates to a codon optimized
nucleic acid sequence of Bacillus anthracis protective antigen
(PA). The method of codon optimization of the gene is aimed at
improving expression in mammals, including humans, as well as
enhancing immunogenicity against endogenously produced PA protein.
The inventive construct, incorporated into DNA expression systems,
can be useful as a component of immunogenic compositions against B.
anthracis, such as vaccines.
[0004] 2. Background Art
[0005] B. anthracis, the etiological agent of anthrax, is a
spore-forming, gram positive bacterium. Infection can occur through
a variety of routes including cutaneous and gastrointestinal,
however, inhalational anthrax is the most widely recognized and
feared (Baillie, J. Appl Microbiol., 91: 609-613 (2001)). Following
inhalation, the majority of the aerosolized spores are immediately
phagocytized by alveolar macrophages and transported through the
lymphatic channels to hilar and tracheobronchial lymph nodes. This
rapidly leads to the multiplication and systemic circulation of
vegetative bacilli. It is believed that en route to these regional
lymph nodes the spores begin to germinate and multiply within the
macrophage.
[0006] Advanced stages of infection are predicated on B. anthracis'
anti-phagocytic capsule and the secretion of a tripartite exotoxin
consisting of a cell binding component, Protective Antigen (PA),
which binds to two enzymatically active subunits: Lethal Factor
(LF) or Edema Factor (EF) to form lethal toxin (LeTx) and edema
toxin (EdTx), respectively. The currently available licensed human
vaccine for B. anthracis (BioThrax) is a filtered extract from B.
anthracis absorbed to alum and is primarily composed of PA.
SUMMARY OF THE INVENTION
[0007] An object of the invention is a humanized, i.e., codon
optimized, DNA construct of Bacillus anthracis protective antigen.
The modifications enable efficient translation of PA in mammals,
including humans.
[0008] Another object of the invention is a method of codon
optimization utilizing rare host codons in place of rare bacterial
codons, rather than those most highly utilized by the host. This
enables ribosomal stalling at appropriate places along the gene to
ensure intra-molecular associations occur within the nascent
protein similar to that which would occur naturally. Correct
folding of PA would result in a more efficacious immune response
against naturally occurring B. anthracis expressed PA.
[0009] A further object of the invention is a method of human
optimization whereby codon optimization does not consist of
replacing all bacterial codons throughout the length of the gene
with the most highly or frequently used codons in the host cell.
Instead, the inventive method utilizes consideration of a number of
factors in order to afford increased expression efficiency in a
mammalian (e.g., human) host cell, as well as an yielding an
expressed protein similar in structure to the native, B. anthracis,
PA protein.
[0010] The first factor considered is protein expression
efficiency. By incorporating codons that are highly utilized in the
mammalian host cell for the first 50 codons of the bacterial
sequence, the mammalian ribosome will effectively engage the mRNA,
decreasing the likelihood of early termination of the ribosome is
minimized. Another factor is to maximize the opportunity for
correct protein folding. This is afforded by first searching for
regions in the native PA sequence where rare codons are utilized in
the bacterial gene. Regions were rare codons are heavily utilized
may result, in normal, native PA expression, specifically proper
folding of the expressed protein. In the modified sequence, these
use of rare codons is maintained by substituting rare codons from
the human bias table. This would permit the ribosome to stall,
where it normally would when expressing native PA in the bacteria,
and permit normal folding to occur. Another factor is to ensure
against unwarranted deletions of mRNA. Therefore, the bacterial
sequence is analyzed to search for ribosomal splice sites to ensure
that post-transcriptional machinery of mammalian cells did not
delete sections of the mRNA. Finally, an analysis of the bacterial
sequence is undertaken to identify any regions of complementarity.
These regions are important since they could potentially result in
the single stranded RNA folding back on itself resulting in
unwanted host cell operations, such as ribosomal stalling or
premature termination of translation.
BRIEF DESCRIPTION OF DRAWINGS
[0011] FIG. 1(A-C). Alignment of the human optimized sequence and
the parent sequence. Humanized protective antigen (HoPA) (upper
sequence) is the human optimized gene and PA (lower sequence) is
the parent wild-type gene. Asterisks denote nucleotides that align.
FIG. 1A-C shows alignment of by 1-780; 781-1560; and 1561-2208,
respectively.
[0012] FIG. 2. Evaluation of DNA vaccines. During this 56 day study
A/J mice (n=8 per group) injected IM with pDNAVACCultura2.TM.-HoPA
encoding the human optimized PA gene with the tissue plasminogen
activator (TPA) signal sequence elicited a robust anti-PA IgG
response during the 14 days following the second boost. This
response gradually contracted over the final 14 days of the study.
FIG. 3. Anti-PA IgG titers in response to homologous
prime-boost-boost with pDNAVACCultra2.TM.-HoPA. Eight groups of
mice (n=10) were immunized IM with pDNAVACCultra2.TM.-HoPA on three
separate occasions 28 days apart. Control groups were injected with
pDNAVACCultra2 without HoPA, the lipid adjuvant dioleoyl
phosphatidylethanolamine-dimethyl dioactadecylammoniium bromide
(DDAB-DOPE) only, and 10 .mu.g of rPA injected with Alum adjuvant.
Titers were low in comparison to the control rPA treated group.
FIG. 4. Anti-PA IgG titers in response to homologous prime-boost
with pDNAVACCultra2.TM.-HoPA. Eight groups of mice (n=10) were
immunized IM with pDNAVACCultra2.TM.-HoPA on two separate occasions
28 days apart. Control groups were injected with pDNAVACCultra2
without HoPA, the lipid adjuvant (DDAB-DOPE) only, and 10 .mu.g of
rPA injected with Alum.
[0013] FIG. 5. Efficacy of a homologous prime-boost-boost and a
prime-boost with pDNAVACCultra2.TM.-HoPA. Eight groups of mice
(n=10) were immunized IM with pDNAVACCultra2.TM.-HoPA two separate
occasions 28 days apart. Fourteen days after the last immunization
all mice were challenged with LD.sub.50s of B. anthracis Sterne
strain spores. Survival was significantly improved at 90% with
three 100 .mu.g doses of pDNAVACCultra2-HoPA, designated in the
figure legend as 7162-HoPA, relative to the lower less frequent
doses. Recombinant PA protected 100% of the mice.
[0014] FIG. 6. Anti-PA IgG titers and survival in response to
homologous prime-boost-boost with humanized protective antigen.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0015] The following terms are defined:
[0016] An immunogenic composition is a composition, containing one
or more antigens, including proteins or peptides or nucleic acid
expression systems that express immunogenic proteins or peptides in
vivo for the induction of a humoral or cell mediated immune
response; a vaccine is an immunogenic composition used to induce
protective immunity; a DNA expression system is a molecular system
containing plasmid or closed loop DNA containing elements for
expressing an inserted DNA sequence as polypeptide; a viral
expression system is any viral based system, including viral like
particles or viral replicons, containing elements for expressing an
inserted DNA sequence as a polypeptide.
[0017] Immunization of susceptible individuals through the process
of vaccination has long been the most desirable approach to disease
prevention. This is particularly important in anthrax since
manifestation of the disease results in high mortality. Therefore,
it is preferred to prophylactically protect against infection
rather than attempt to administer antibiotic post-infection.
[0018] The current licensed vaccine in the U.S. for anthrax is a
cellular filtrate of B. anthracis that mostly contains PA (Baillie,
L., J Appl Microbiol, 91: 609-613 (2001)). Unfortunately, due to
the nature of the vaccine, batch to batch variability occurs,
resulting in inconsistency in efficacy.
[0019] In order to alleviate these problems, recombinant technology
has been employed. Nucleic acid based, or DNA vaccines represent a
relatively recent and attractive vaccination modality. This
interest has been stemmed by their inexpensive and easy production,
high stability, and flexibility regarding cloning and delivery
method. The basic structure of a DNA vaccine is a plasmid or closed
loop of DNA (the plasmid "backbone") that contains a selectable
marker, a mammalian promoter such as the CMV promoter for tissue
specific expression, and the gene encoding the antigen of interest,
in this case protective antigen (PA).
[0020] In principal, the DNA is delivered to either immunologically
relevant cells of the skin or to cells that have highly active
transcription and translation machinery such as muscle cells where
the PA gene is expressed via the CMV promoter, released and
displayed from the cell, and hopefully picked up by a scavenging
dendritic cell or macrophage.
[0021] Unfortunately, DNA vaccines in primates and humans often do
not elicit humoral or antibody based responses (Calarota, et al.,
Lancet, 351: 1320-1325; Coban, et al., Infect Immun., 72: 584-588
(2004); Epstein, et al., Hum Gene Ther, 13: 1551-1560 (2002);
Klinman, et al., Curr Top Microbiol Immunol, 247: 131-142 (2000);
Wang, et al., Science, 282: 476-480 (1998)) which are critical to
surviving an anthrax infection. Several approaches have been
developed to enhance the immunogenicity of DNA vaccines including
the use of adjuvants, altering the delivery system, modifying the
plasmid backbone by including CpG motifs, or altering the codon
bias (Leitner, et al., Vaccine, 18: 765-777 (1999); O'Hagan, et
al., Nat Rev Drug Discov, 2: 727-735 (2003)).
[0022] The current invention relates to a DNA construct, useful in
vaccine formulations, utilizing a novel human codon-optimized PA
gene sequence. The current invention, unlike previously described
methods of DNA optimization, not only permits efficient expression
of recombinant protein, but also enables expression of protein
tertiary structure, with fidelity to the native bacterially
expressed protein. PA expression from B. anthracis is optimized
through consideration of a number of factors that enable efficient
expression in a mammalian host, e.g., human. The factors considered
in the process also improve the likelihood of greater tertiary
structural similarity between the expressed recombinant protein and
native, bacterially expressed PA. The result is a greater
likelihood of a more efficacious induction of adaptive
immunity.
Method for Humanizing DNA Sequence
[0023] DNA vaccines rely heavily on the natural processes of
transcription and translation by the eukaryotic host cell.
Bacterial gene structures are very different from the human host
and require specialized transcriptional and translational
apparatuses. These differences include a lack of introns (noncoding
regions that eukaryotes splice out of message RNA), the presence of
operons (multiple genes in one message), and a variety of secondary
structures within the mRNA that are foreign in eukaryotes
(Strugnell, et al., Immunol Cell Bio, 75: 364-369 (1997)).
Additionally, bacterial proteins are not glycosylated by the
bacterial system but contain amino acid motifs which are
efficiently and inappropriately glycosylated by eukaryotic cells.
Bacterial mRNA also lacks appropriate structures and sequences to
insure an effective half-life in eukaryotic cells.
[0024] A important additional difference between eukaryotic and
prokaryotic transcriptional/translational systems is the
significant differences in codon usage and the arrangement of
nucleotides in bacterial mRNA that give rise to codons that are
rare in eukaryotic mRNA (Manoj, et al., Crit Rev Clin Lab Sci, 41:
1-39 (2004)). These differences may be explained by the composition
of the tRNA pool that is available to the host for translation or
the guanine/cytosine (GC) and adenine/thymidine (AT) percentages of
the bacterial gene and their similarity to the eukaryotic host
(Saler, Nat Rev Drug Discov, 2: 727-735 2003)). Coincident with
these differences is the operation of the ribosome and the complex
combinations of RNAs and proteins that comprise the translational
machinery.
[0025] During translation, the ribosome attaches to the mRNA by a
specific recognition operation. As the ribosome proceeds down the
mRNA it specific codons are recognized leading to a defined
assembly of amino acids to ultimately build the nascent protein.
Ribosomes have been shown to complete this protein synthesis in a
complex manner moving down the mRNA at a varying rate of
progression resulting in a multitude of different structural
results. If a ribosome slows in its progression, it disconnects
from the RNA resulting in premature termination of translation.
[0026] Variations in the rate of ribosomal processivity can result
in the creation of important structural features. For example,
pausing is thought to allow proteins to create protein folds,
allowing complex intra-molecular associations to occur. These
associations can give rise to proper protein function but also
create important immunogenic motifs, that are not present from the
linear sequence.
[0027] However, when bacterial sequences are expressed from
eukaryotic host systems, variations, away from that seen in the
bacteria, can result in significant differences in ribosomal
procession, glycosylation and even premature termination of
translation. The result, therefore, in developing immunogenic
compositions, are proteins that may not mimic native protein immune
induction.
[0028] In developing a more antigenically efficient PA protein,
genetic incompatibilities between bacterial and eukaryotic genomes
were mitigated by modifying the bacterial sequence in order to
conform to optimal codon usage in eukaryotic hosts.
[0029] There are many approaches that can be taken in the effort to
produce bacterial gene sequences that are translated in human cells
more efficiently. The most common is to synthesize the new gene
sequence using only the most highly used codon in the host
organism. However, this method does not take into account
differences between prokaryotic and eukaryotic transcription and
translational machinery or the guanine/cytosine (GC) and
adenine/thymidine (AT) content of the bacterial gene.
[0030] The approach utilized in the current invention is to modify
the bacterial gene in order to permit expression resulting in a
greater likelihood of maintaining fidelity to the bacterially
expressed native protein. This is termed here as "human
optimization." The aim of this approach is to produce a recombinant
protein with a greater likelihood of inducing a more efficacious
adaptive immune response.
[0031] In the inventive method to modify proteins for efficient
expression of antigens in a eukaryotic host a number of factors are
taken into account. These are summarized as: [0032] a. efficiency
of translation; [0033] b. fidelity of protein folding; [0034] c.
minimize excision of mRNA regions by recognition by the
post-transcriptional machinery of mammalian host cell; [0035] d.
avoidance of single-stranded RNA folding due to resultant mRNA
sequence complementarity.
[0036] In the inventive method, the early (i.e., first region) of
the gene utilizes codons most highly utilized by the mammalian host
cell. This consideration, therefore, improves the efficiency of
gene expression by minimizing the likelihood of early termination
of the ribosome. Although the extent of the sequence that is left
unaltered varies from gene to gene, the region is typically up to
100 bp.
[0037] An important consideration is the fidelity of the tertiary
structure and folding of the protein produced in eukaryotic cells
to the native, bacterially expressed protein. It is recognized that
important immunogenic epitopes are likely to exist beyond the
linear or even secondary peptide structure. Rather, proper protein
folding can bring amino acids or even peptide sequences, that are
normally considerably downstream of each other, into juxtaposition,
creating important immunogenic conformational epitopes.
[0038] To improve the likelihood of producing these epitopes in the
recombinant protein, a search of the native PA sequence is
undertake in order to ascertain and identify regions containing
relatively heavy concentrations of rare codons. These regions may
represent domains with specific folding motifs within the normal,
native PA protein. Therefore, retention of these regions in the
modified sequence is incumbent upon substituting the rare bacterial
codons with complimentary rare codons from the human bias table.
This would permit ribosome progression to slow, where it normally
would in the bacteria, and permit normal folding to occur.
[0039] In order to ensure against unwarranted deletions of mRNA the
bacterial sequence is analyzed with the aim of identifying
ribosomal splice sites. Alteration of these regions, therefore,
will ensure that post-transcriptional machinery of mammalian cells
does not inadvertently delete sections of the mRNA
[0040] Finally, an analysis of the bacterial sequence is undertaken
to identify any regions of complementarity. These regions are of
importance since these regions could potentially result in single
stranded RNA folding back on itself, resulting in unwanted host
cell operations, such as ribosomal stalling or premature
termination of translation.
[0041] Collectively, the inventive method avoids "over
optimization" of the bacterial gene sequence. Instead, the method
provides a more deliberate procedure leading to an expressed
protein with greater antigenic similarity to native, bacterially
expressed protein.
Example
Design of Humanized Bacillus anthracis Protective Antigen (PA)
[0042] In order to illustrate the inventive method, the human
optimization of PA (HoPA) was undertaken. As discussed above, the
factors that were considered in the development of the humanized
gene sequence. The features of HoPA include: [0043] a. highly used
codons for the first 50 codons of the sequence, thereby effectively
engaging the ribosome and reducing premature termination; [0044] b.
using rare codons from the human codon bias table in the same
positions where the wildtype PA gene sequence used rare codons from
the Bacillus anthracis codon usage table thus insuring that where
the bacterial ribosome paused during protein synthesis in the
bacteria, the mammalian ribosome did as well; [0045] c. ensuring
that regions, where there are many rare codons close together, are
maintained but the actual number of rare codons reduced in order to
minimize the likelihood of ribosomal progression slowing or
stalling; [0046] d. a search for cryptic ribosomal splice sites was
undertaken to ensure that the post-transcriptional machinery of the
mammalian cells did not delete sections of the mRNA; [0047] e.
secondary structure determinations to ensure that the resulting
mRNA did not have long regions of complementarity that would result
in a single stranded RNA that was folded back onto itself, into a
secondary structure that could not be resolved by the ribosome also
leading to premature termination of the translation process.
[0048] Human optimization was performed on the non-proprietary
wild-type PA gene (GenBank Accession no. AAA22637.1). In designing
the new sequence, the factors, above, were considered and
incorporated into the new human optimized sequence (HoPA). The new
HoPA gene sequence is illustrated in FIG. 1 (A-C) adjacent to the
native sequence. The optimized sequence is also listed in SEQ ID
No. 1.
[0049] Unlike in the native sequence, the new nucleotide sequence
lacks many of the rare codons and motifs that hinder expression in
eukaryotes while using human rare codons to emulate the overall
spacing of rare codons. An important consideration is to avoid over
optimization of the gene sequence. Over optimization may result in
the most common eukaryotic codons depleting the available reservoir
of normally abundant tRNAs. This process may artificially
accelerate the processivity of the ribosome, increasing the chance
that the nascent protein will not fold into the proper secondary
structure.
[0050] The newly synthesized PA gene also included Sap 1
restriction sites at the N- and C-terminal ends to allow effective
cloning into the multi-cloning site of the pDNAVACCultra2.TM.
(Nature Technology, Lincoln, Nebr.) construct. At the same time,
the amino terminal Bacillus leader peptide was eliminated since
cloning into pDNAVACCultra2.TM. places the human TPA leader peptide
upstream and in-frame of the PA sequence. This modification
effectively increases extracellular trafficking of the recombinant
PA (rPA) protein by eukaryotic cells. Ultimately, due to codon
redundancy, when both genes are translated they result in the same
wild-type amino acid sequence, as illustrated in SEQ ID No. 1.
[0051] In order to evaluate the expression of HoPA in a eukaryotic
cell line Chinese hamster ovary (CHO) cells strain K1 was
transfected with pDNAVACCultra2.TM.-HoPA and
pDNAVACCultra2.TM.-HoPA carrying the green fluorescent protein
(GFP). Efficient transfection and the transcription/translation of
HoPA were verified by the expression of GFP from the modified
HoPA-GFP vector. Western blot analysis of supernatants from the
transfected CH0-K1 cells after 20 hr demonstrated the presence of
PA. This study confirmed that HoPA with it's codon optimizations,
could be expressed from a eukaryotic cell line using the host cell
machinery.
[0052] Referring to FIG. 2, analysis of the mouse antibody response
following immunizations with HoPA cloned into NTC's
pDNAVACCultra2.TM. DNA vaccine expression vector demonstrated that
the animals had mounted PA specific IgG responses. The efficacy of
the human TPA sequence at the beginning of HoPA, to direct the
synthesized PA protein out of the cell for detection and processing
by circulating immune cells, was evaluated.
[0053] In the first study 50 .mu.g pDNAVACCultra2.TM.-HoPA or
pDNAVACCultra2.TM.-null (no insert) were mixed with a lipid
adjuvant prior to being intramuscularly injected into mice (n=8) on
three separate occasions 28 days apart. Serum anti-PA IgG titers
were tracked for 56 days and demonstrated that following the second
homologous boost on day 28 the rPA specific IgG titer increased
from baseline to 33.8 .mu.g/ml (FIG. 2).
[0054] In another animal study the efficacy following multiple
administrations was evaluated (FIG. 3). In this study efficacy was
evaluated after injecting various doses (100, 75, 50, 25, and 12.5
.mu.g) of pDNAVACCultra2.TM.-HoPA once, twice, and three times.
Serum IgG titers were tracked as before. Dose and frequency related
responses were observed, with the highest antigen specific titers
(FIG. 3) achieved with the highest dose of DNA injected three
times. The same doses of vaccine given twice (days 28 and 42) (FIG.
4) or once did not generate as robust an IgG response as the triple
vaccination schedule or rPA.
[0055] Two weeks after the last vaccination the mice were
challenged intraperitoneally with 40 LD.sub.50s of B. anthracis
Sterne strain spores (4.03.times.10.sup.5 CFU/mouse). Survival was
tracked over the course of 14 days (FIG. 5). Survival was
significantly improved (p<0.05) with 100 .mu.m of
pDNAVACCultra2.TM.-HoPA (designated as 7162-HoPA in the figure
below) when administered three times. Doses less than 100 .mu.g or
immunization schedules that lacked a second booster are not
efficacious. In comparison, 10 .mu.g of rPA protected 100% of the
challenged mice. These results speak to the efficacy of HoPA when
cloned into the pDNAVACCultra construct. In FIG. 5, the negative
control is the use of the adjuvant Dioleoyl
phosphatidylethanolamine-dimethyl dioctadecylammonium bromide
(DDAB-DOPE).
[0056] FIG. 6 illustrates the immunoglobulin concentration
following immunization with humanized PA. As seen in panel A, a
significant humoral response is evident following either
recombinant PA protein or HoPA. However, significantly less
antibody response is seen following immunization with the DNA
expressed HoPA than following immunization with rPA protein. The
clear dichotomy of immunoglobulin induction seen in FIG. 5 between
HoPA (expressed from an administered DNA vector) and rPA protein
administration induced between is likely dependent on the
expression efficiency of the DNA expression system.
[0057] Additionally, antibody induced by HoPA was capable of
efficiently neutralizing lethal toxin, as evidenced by toxin
neutralization activity (TNA) assay. These results are summarized
in Table 1. The assay was conducted as described by Quinn, C. P.,
et al., J. Infect. Dis. 190: 1228-1236 (2004). Prior to testing,
recombinant PA (rPA) and recombinant LF (rLF) were titrated for
toxin potency with J774A.1 cells. The concentrations of rPA and rLF
that resulted in more than 99% cell lysis at a fixed cell density
of 2.times.10.sup.4 cells/well were 45.1 and 36.1 ng/ml,
respectively. Serum samples were serially diluted starting at 1:50
out to 1:102400 and were assayed in quadruplicate. The resulting
serum neutralization curve (antibody dilution factor versus optical
density was analyzed with a four-parameter logistic log fit curve.
The primary endpoint calculated from this 4-PL curve is the 50%
effective antibody dilution (ED.sub.50) that protects 50% of the
eukaryotic cells in the assay. This value is reported as the
reciprocal of the antibody dilution corresponding to the inflection
point ("c" parameter) of the four-parameter logistic log fit of the
serum neutralization curve. An ED.sub.50 greater than 200 is
correlated with survival (Pitt, M. L., et al., Vaccine 19:
4768-4773 (2001). Vaccination with pDNAVACCulture2-HoPA three times
elicited antibody responses by day 69 (FIG. 6A) with sufficiently
high toxin neutralization capacities greater than 200
ED.sub.50.
TABLE-US-00001 TABLE 1 Mouse ED.sub.50 1.3 587.8 1.4 409.4 1.7
1144.7 1.8 272.2
[0058] A further demonstration of the effectiveness of the humoral
response induced by HoPA is illustrated in FIG. 6B. In FIG. 6B,
mice were immunized with either rPA protein or plasmid-HoPA
expression plasmid (pDNAVACCuItra.TM.). In FIG. 6B, the survival of
mice following anthrax challenge, that had been immunized with
HoPA, was equivalent to that observed for the recombinant protein,
despite the much higher levels of immunoglobulin induced. The
improved efficacy of the immunoglobulin over that induced from rPA
protein likely represents the greater similarity of HoPA to native
PA structure. Additionally, HoPA may induce higher affinity anti-PA
antibody or higher concentrations of antibody specific to regions
on the native PA molecule that are more relevant to immune
protection. One explanation is that HoPA may induce helper T-cell
response to induce IL4. This may suppress IgG2a and IgG2b responses
and increase IgG1.
[0059] Although the example shown expressed the novel PA in the
pDNAVACCultra.TM. construct, expression of the optimized PA
construct can be inserted and expressed by other suitable
expression systems. This could include other DNA and viral
expression systems.
Sequence CWU 1
1
212208DNABacillus anthracis 1gaagtgaagc aggagaatcg gctgctgaat
gaatccgaga gcagctctca gggcttgctg 60gggtattact tctctgacct gaatttccag
gcacccatgg tcgtcacatc ctctaccacc 120ggagacctat cgattccctc
aagcgagctg gagaatatcc cttcagagaa ccagtatttt 180caatccgcaa
tctggtccgg cttcattaaa gtgaagaagt ctgatgaata caccttcgcc
240acttccgcag acaatcacgt taccatgtgg gtggacgacc aagaagtcat
taataaagcc 300tctaattcca ataaaatcag actcgaaaag ggcagacttt
atcagatcaa gattcagtac 360cagagggaga atcccacaga aaaaggtttg
gatttcaaat tgtattggac agatagtcag 420aataagaaag aggtgatatc
ctctgacaac ctccagctgc ccgaattgaa acaaaaatca 480tcaaattcac
ggaagaagcg ctctacctca gccggcccaa ccgtccctga cagagataac
540gacgggattc cagattctct ggaggttgag ggctacacag tggacgtaaa
gaacaagaga 600acctttttga gtccttggat ttcaaacatt catgagaaga
agggacttac taagtacaag 660tctagcccag agaaatggag cacagcctcc
gatccatact cggacttcga gaaggtcacc 720ggacgcatcg ataagaatgt
aagcccagag gctcggcatc cactggtggc tgcctatccc 780attgtgcacg
tggatatgga aaatattatt ctcagcaaga acgaggatca gtccactcaa
840aacacggact ccgagacaag aaccatcagc aagaacacat ctacaagtag
aactcataca 900tcagaggtgc acggcaacgc cgaggttcac gccagtttct
tcgatatcgg gggcagtgtt 960agcgccggat tttctaactc caacagctct
acagtggcta tcgaccactc tctgagtctc 1020gcaggggagc ggacgtgggc
ggaaaccatg ggcctgaaca ccgccgatac agccaggttg 1080aatgcaaaca
ttcgctatgt gaatacaggt accgctccca tctataatgt ccttcctact
1140acatctctcg tgttggggaa aaatcagacc ctggcaacaa tcaaggccaa
ggagaatcag 1200ctgagtcaga tactcgcacc caataactac tacccatcaa
agaatcttgc acctatagct 1260ctgaacgccc aggatgattt tagcagcacc
ccaattacta tgaattataa ccagttcctg 1320gagttggaga agactaagca
attgaggctg gatacagacc aagtgtacgg caatatagct 1380acttataact
tcgagaatgg gagggttagg gtggacacag ggagcaattg gtcggaagtg
1440cttccacaaa ttcaggagac cacagccaga atcatcttta atggcaagga
cctcaacctt 1500gtggaaagga gaattgccgc agttaaccca agtgacccct
tggaaacaac caagccagac 1560atgactctga aggaagccct gaagatcgct
ttcgggttta atgaaccaaa cggcaacctg 1620cagtaccagg gaaaggacat
taccgagttt gactttaatt tcgatcagca gacctcccaa 1680aacatcaaga
atcaactggc cgagctgaac gctacaaata tttataccgt gctcgacaag
1740attaaattga acgcgaagat gaatatcttg atacgcgata agcgctttca
ttacgaccgc 1800aacaatatag ccgttggcgc cgacgaatct gtcgtaaaag
aggcccacag agaagtgatt 1860aactccagca ccgaagggct cctgctgaac
attgataaag acatccgcaa gatcctgtcc 1920gggtacatag tggagatcga
ggacaccgag ggactcaaag aagtaatcaa cgaccggtac 1980gatatgctga
atatctcaag cctccggcaa gacgggaaga ctttcataga tttcaagaaa
2040tacaacgaca agctgccact ctatatctca aaccccaatt acaaggtgaa
cgtttacgca 2100gtcaccaaag agaacaccat cattaatcca tcagaaaatg
gtgacacaag tactaatggt 2160attaagaaga ttctcatttt cagcaagaag
gggtatgaga tcggctaa 22082735PRTBacillus anthracis 2Glu Val Lys Gln
Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu
Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro 20 25 30Met Val
Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser 35 40 45Glu
Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile 50 55
60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65
70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu
Val 85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys
Gly Arg 100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn
Pro Thr Glu Lys 115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp
Ser Gln Asn Lys Lys Glu 130 135 140Val Ile Ser Ser Asp Asn Leu Gln
Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Arg Lys
Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro 165 170 175Asp Arg Asp
Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr 180 185 190Thr
Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser 195 200
205Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu
210 215 220Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys
Val Thr225 230 235 240Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala
Arg His Pro Leu Val 245 250 255Ala Ala Tyr Pro Ile Val His Val Asp
Met Glu Asn Ile Ile Leu Ser 260 265 270Lys Asn Glu Asp Gln Ser Thr
Gln Asn Thr Asp Ser Gln Thr Arg Thr 275 280 285Ile Ser Lys Asn Thr
Ser Thr Ser Arg Thr His Thr Ser Glu Val His 290 295 300Gly Asn Ala
Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val305 310 315
320Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His
325 330 335Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met
Gly Leu 340 345 350Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile
Arg Tyr Val Asn 355 360 365Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu
Pro Thr Thr Ser Leu Val 370 375 380Leu Gly Lys Asn Gln Thr Leu Ala
Thr Ile Lys Ala Lys Glu Asn Gln385 390 395 400Leu Ser Gln Ile Leu
Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu 405 410 415Ala Pro Ile
Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile 420 425 430Thr
Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu 435 440
445Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe
450 455 460Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser
Glu Val465 470 475 480Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile
Ile Phe Asn Gly Lys 485 490 495Asp Leu Asn Leu Val Glu Arg Arg Ile
Ala Ala Val Asn Pro Ser Asp 500 505 510Pro Leu Glu Thr Thr Lys Pro
Asp Met Thr Leu Lys Glu Ala Leu Lys 515 520 525Ile Ala Phe Gly Phe
Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly 530 535 540Lys Asp Ile
Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln545 550 555
560Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr
565 570 575Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu
Ile Arg 580 585 590Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala
Val Gly Ala Asp 595 600 605Glu Ser Val Val Lys Glu Ala His Arg Glu
Val Ile Asn Ser Ser Thr 610 615 620Glu Gly Leu Leu Leu Asn Ile Asp
Lys Asp Ile Arg Lys Ile Leu Ser625 630 635 640Gly Tyr Ile Val Glu
Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile 645 650 655Asn Asp Arg
Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly 660 665 670Lys
Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr 675 680
685Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu
690 695 700Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr
Asn Gly705 710 715 720Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly
Tyr Glu Ile Gly 725 730 735
* * * * *