SAFE POTENT SINGLE PLATFORM VACCINE AGAINST TIER 1 SELECT AGENTS AND OTHER PATHOGENS
20190240310 ยท 2019-08-08
Assignee
Inventors
Cpc classification
C07K14/20
CHEMISTRY; METALLURGY
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A61K9/0019
HUMAN NECESSITIES
A61K9/0053
HUMAN NECESSITIES
International classification
A61K9/00
HUMAN NECESSITIES
Abstract
A method of immunizing a susceptible host against a pathogen comprising administering to the host a vaccine that comprises an attenuated recombinant live vaccine strain lacking a polynucleotide encoding CapB (LVS capB), wherein the LVS capB expresses an antigen of at least one pathogen from Table 1; or administering to the host a vaccine that comprises an attenuated Listeria monocytogenes expressing the antigen of the pathogen from Table 1; or administering to the host a prime vaccine and a heterologous booster vaccine where the prime vaccine comprises an attenuated recombinant live vaccine strain lacking a polynucleotide encoding CapB (LVS capB), wherein the LVS capB expresses an antigen of at least one pathogen from Table 1 and the heterologous booster vaccine comprises an attenuated Listeria monocytogenes expressing the antigen of the pathogen from Table 1.
Claims
1. An immunogenic composition comprising: a recombinant attenuated Francisella tularensis subspecies bolarctica live vaccine Strain (LVS) having a deleted CapB gene (SEQ ID NO: 1) which comprises a F. tularensis promoter that expresses a fusion protein comprising an antigenic polypeptide epitope present in a Francisella tularensis, Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei or Burkholderia mallei polypeptide; wherein: the fusion protein expressed by the F. tularensis promoter is less than 80 kilodaltons in size; and the antigenic polypeptide epitope elicits an immune response in a mammalian host when the immunogenic composition is administered orally, intradermally (i.d.), subcutaneously (s.q.), intramuscularly (i.m.), or intranasally (i.n.) to the mammalian host.
2. The immunogenic composition of claim 1, wherein: (a) the F. tularensis promoter expresses: a fusion protein comprising at least 8 contiguous amino acids of IglA (SEQ ID NO: 2); a fusion protein comprising at least 8 contiguous amino acids of IglB (SEQ ID NO: 3); and/or a fusion protein comprising at least 8 contiguous amino acids of IglC (SEQ ID NO: 4); and/or (b) the F. tularensis promoter expresses the fusion protein in an amount at least 1.5 fold greater than the expression of endogenous IglA protein, endogenous IglB protein and/or endogenous IglC protein expressed in the LVS.
3. The immunogenic composition of claim 2, wherein the at least 8 contiguous amino acids of IglA, the at least 8 contiguous amino acids of IglB, and the at least 8 contiguous amino acids of IglC are covalently linked together in a single fusion protein.
4. The immunogenic composition of claim 2, wherein the LVS expresses: a major histocompatibility class I or a major histocompatibility class II epitope present in amino acid residues 33-132 of IglA (SEQ ID NO: 2); a major histocompatibility class I or a major histocompatibility class II epitope present in amino acid residues 446-506 of IglB (SEQ ID NO: 3); a major histocompatibility class I or a major histocompatibility class II epitope present in amino acid residues 29-149 of IglC (SEQ ID NO: 4).
5. The immunogenic composition of claim 1, wherein the antigenic polypeptide epitope is present in a Bacillus anthracis polypeptide selected from: lethal factor domain I (SEQ ID NO: 5); the extended domain IV of protective antigen (SEQ ID NO: 6); and AhpC (SEQ ID NO: 7).
6. The immunogenic composition of claim 1, wherein the antigenic polypeptide epitope is present in a Burkholderia pseudomallei, or Burkholderia mallei polypeptide selected from: Burkholderia pseudomallei Hcp-1 (SEQ ID NO: 35), Hcp-2 (SEQ ID NO: 36), Hcp-3 (SEQ ID NO: 37), Hcp-4 (SEQ ID NO: 38), Hcp-6 (SEQ ID NO: 39), LolC (SEQ ID NO: 40), TypA (SEQ ID NO: 41), BipB (SEQ ID NO: 42), BipC (SEQ ID NO: 43), BipD (SEQ ID NO: 44), Omp3 (SEQ ID NO: 45), Omp7 (SEQ ID NO: 46), Omp85 (SEQ ID NO: 47), OmpW (SEQ ID NO: 48), PotF (SEQ ID NO: 49), OppA (SEQ ID NO: 50), BopA (SEQ ID NO: 51), BimA (SEQ ID NO: 52), BPSL1897 (SEQ ID NO: 53), BPSL3369 (SEQ ID NO: 54), BPSL2287 (SEQ ID NO: 55), BPSL2765 (SEQ ID NO: 56), and VgrG5 (SEQ ID NO: 57). Burkholderia mallei BMA_A0768 (SEQ ID NO: 58), BMA_2821 (SEQ ID NO: 59), BMA_0816 (SEQ ID NO: 60), and GroEL (SEQ ID NO: 61).
7. The immunogenic composition of claim 1, wherein the antigenic polypeptide epitope is present in a Yersinia pestis polypeptide selected from: F1 (SEQ ID NO: 8); LcrV (SEQ ID NO: 9); YscF (SEQ ID NO: 10); YopE (SEQ ID NO: 11); and Psn (SEQ ID NO: 12).
8. The immunogenic composition of claim 1, wherein the F. tularensis promoter comprises a bfr promoter (SEQ ID NO: 13) and/or an omp promoter (SEQ ID NO: 14).
9. The immunogenic composition of claim 1, wherein the LVS expresses two or more antigenic polypeptide epitopes present in a Francisella tularensis, Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei or Burkholderia mallei polypeptide.
10. A method of generating an immune response in a mammal comprising administering the immunogenic composition of claim 1 to the mammal so that an immune response is generated to the antigenic polypeptide epitope present in a Francisella tularensis, Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei or Burkholderia mallei polypeptide.
11. The method of claim 10, wherein the method comprises administering the immunogenic composition of claim 1 in a primary vaccination; and administering the immunogenic composition of claim 1 in a subsequent homologous booster vaccination.
12. The method of claim 10, wherein the method consists essentially of administering the immunogenic composition of claim 1 in a primary vaccination; and administering the immunogenic composition of claim 1 in a subsequent homologous booster vaccination.
13. The method of claim 11, wherein the method comprises administering the immunogenic composition to the mammal less than 4 times.
14. The method of claim 10, wherein the method comprises administering the composition of claim 1 in a primary vaccination; and administering a second heterologous immunogenic composition comprising the antigenic polypeptide epitope present in a Francisella tularensis, Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei, or Burkholderia mallei polypeptide in a subsequent booster vaccination.
15. The method of claim 14, wherein the second immunogenic composition comprises an attenuated strain of Listeria monocytogenes expressing the antigenic polypeptide epitope.
16. The method of claim 15, wherein the attenuated Listeria monocytogenes expresses: a fusion protein comprising at least 8 contiguous amino acids of IglA (SEQ ID NO: 2); a fusion protein comprising at least 8 contiguous amino acids of IglB (SEQ ID NO: 3); and/or a fusion protein comprising at least 8 contiguous amino acids of IglC (SEQ ID NO: 4).
17. The method of claim 16, wherein the amino acid residues of IglA, the amino acid residues of IglB, and the amino acid residues of IglC are covalently linked in a single fusion protein.
18. The method of claim 17, wherein the fusion protein is less than 50 kilodaltons.
19. The method of claim 15, wherein the method comprises administering the composition of claim 1 and the second immunogenic composition to the mammal less than a total of four times.
20. The method of claim 19 wherein method comprises administering a single dose of the composition of claim 1, and one or more doses of the second immunogenic composition.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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[0044] twice at weeks 0 and 4 with 10.sup.6 rLVS capB/bfr-iglABC (Groups D & E, resp.) (panel a); primed i.d. once at week 0 with rLVS capB/bfr-iglABC (Groups F & G) or LVS capB (Groups I & J) and boosted intramuscularly (i.m.) twice at weeks 4 and 6 with rLmprfA* Vector (Lm actA inlB prfA*) (Groups F & I) or rLmprfA*/hly-iglABC (rLm actA inlB prfA*/hly-iglABC, Groups G & J); or immunized simultaneously i.d.
[0045] with rLVS capB/hfr-iglABC and i.m. with rLmprfA*/hly-iglABC (Group K) (panel b). All the mice were challenged i.n. with 10 CFU (3 LD.sub.50) F. tularensis Schu S4 at week 10, and monitored for signs of illness, weight change and death for up to 21 days post challenge. Survival curves were compared between vaccine groups and the sham group by log-rank test (Mantel-cox, Prism); the P value for the comparison of a specific vaccine vs. Sham is color-coded to the color of the vaccine symbol.
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DETAILED DESCRIPTION OF THE INVENTION
[0051] In the description of embodiments, reference may be made to the accompanying figures which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention.
[0052] All publications mentioned herein are incorporated by reference to disclose and describe aspects, methods and/or materials in connection with the cited publications. For example, U.S. Pat. No. 8,481,024, titled VACCINES AGAINST TULAREMIA, filed by Marcus A. Horwitz et al. and U.S. Pat. No. 8,206,700, titled METHODS AND COMPOSITIONS FOR TREATING TULAREMIA, filed by Marcus A. Horwitz et al., are incorporated by reference in their entirety herein.
[0053] Many of the techniques and procedures described or referenced herein are well understood and commonly employed by those skilled in the art. Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.
[0054] As previously described, there is a need for a safe and effective tularemia vaccine, anthrax vaccine, and plague vaccine. The only currently available tularemia vaccine is an unlicensed toxic vaccine known as Live Vaccine Strain (LVS). An improved heterologous prime-boost vaccine against tularemia is described herein where the prime is LVS capB expressing or not expressing a F. tularensis protein (IglA or IglC) or fusion protein (IglABC), and the booster vaccine is a heterologous vector, attenuated L. monocytogenes expressing a F. tularensis protein (IglA or IglC) or fusion protein (IglABC). This heterologous prime-boost vaccine against tularemia is safer and more potent than the current unlicensed and toxic LVS vaccine. The prime LVS capB based vaccines described herein are >10,000 fold less virulent than LVS and hence much safer. The heterologous prime-boost vaccine is also more potent than LVS. See also the Illustrative embodiments of multiantigenic live attenuated prime and booster vaccines against Tularemia section below.
[0055] With respect to individual anthrax vaccines, the acellular AVA vaccine contains various amounts of PA and small amounts of LF and other secreted proteins; PA plays the major role in the protective immunity. The major drawback of AVA is its requirement for extensive repetitive vaccinations, suggesting that PA delivered via adjuvant is limited in its capacity to induce-long-lasting protective immunity. The only commercially available anthrax vaccine (AVA) requires six immunizations plus frequent boosters and is poorly effective. Currently, two major approaches are being used to improve PA-based anthrax vaccines: a) delivering and presenting PA more effectively, e.g. via live attenuated heterologous vectors such as viral vectors (however, pre-existing immunity adversely affects their efficacy) (15-18), Bacillus subtilis (19), or Salmonella typhimurium (20, 21); and b) complementation of PA with additional bacterial components of protective value (e.g., LF and EF). A fusion protein of LFD1-PAD4 adsorbed to alum has been shown to induce 100% protection against Ba spore challenge in mice (12). Hence, in the present invention, live attenuated vaccine vectors have been constructed expressing this LFD1-PAD4 fusion protein. Since this approach not only induces strong antibody responses but also T cell responses, this approach is more likely to induce long-lasting immunity. The heterologous prime-boost and homologous prime-boost vaccines against anthrax described herein are safe and found to be much more effective than AVA in animal studies.
[0056] With respect to individual plague vaccines, several new vaccines have been described based primarily on the antigens expressed herein (22-25) but also others (26). However, the only currently available plague vaccine is unlicensed and toxic (EV76). The heterologous prime-boost vaccine against plague described herein is safe and effective.
[0057] In aspects of the invention, the prime-boost vaccine described herein comprises a prime vaccine and a booster vaccine. With respect to the prime vaccine, the vector LVS capB and recombinant LVS capB overexpressing individual proteins of F. tularensis including IglA and IglC have been previously described. In an illustrative embodiment of a prime vaccine against F. tularensis, the present invention provides a LVS capB vector expressing a fusion protein (IglABC) of three immunodominant epitopes of three different proteins- IglA, IglB, and IglC. These vaccines are safer than the unlicensed and toxic Live Vaccine Strain (LVS) and highly immunogenic. In other embodiments, the same vector is used for expressing immunoprotective antigens of B. anthracis and Y. pestis. This vector may also be used to express antigens of other pathogens including other Tier 1 Select Agents, as well as agents and toxins listed in Table 1 and known in the art such as those identified by the National Institute of Allergy and Infectious Diseases as emerging infectious diseases/pathogens and/or those identified by the Center for Disease Control as USDA Federal select agents and toxins.
[0058] With respect to the booster vaccine, an attenuated recombinant Listeria monocytogenes vaccine expressing individual proteins of F. tularensis including IglA and IglC has been previously described. In an illustrative embodiment of a booster vaccine against F. tularensis, the present invention provides an attenuated Listeria monocytogenes vaccine expressing a fusion protein (IglABC) of three immunodominant epitopes of three different proteinsIglA, IglB, and IglC. In other embodiments, the same vector is used for expressing immunoprotective antigens of B. anthracis and Y. pestis. This vector may also be used to express antigens of other pathogens including other Tier 1 Select Agents, as well as agents and toxins listed in Table 1 and known in the art such as those identified by the National Institute of Allergy and Infectious Diseases as emerging infectious diseases/pathogens and/or those identified by the Center for Disease Control as USDA Federal select agents and toxins.
[0059] Typically, the vaccine is administered to humans or animals by injection intradermally or by another route, e.g. subcutaneously, intramuscularly, orally, intranasally, or by inhalation.
[0060] In another aspect, the present invention provides the first known single platform vaccine against multiple Tier 1 Select Agents, for instance F. tularensis (tularemia), B. anthracis (anthrax), and Y. pestis (plague). As noted above, a single platform simplifies manufacture, regulatory approval, clinical evaluation, and vaccine administration, and would be more acceptable to people than multiple individual vaccines, and be less costly. This single vaccine platform may be designed to express the antigens of multiple pathogens, for instance one, two or all three illustrative pathogens (i.e. F. tularensis, B. anthracis, and Y. pestis). That is, the LVS capB vector, which already contains antigens of F. tularensis, may be designed to also express B. anthracis and/or Y. pestis antigens and the L. monocytogenes vector may similarly be designed to express antigens of one, two or all three illustrative pathogens. The same platform may be used to express additional immunoprotective antigens of Tier 1 Select Agents or other pathogens.
[0061] As noted above, the invention disclosed herein has a number of embodiments. An exemplary embodiment of the invention is a method of immunizing a susceptible host against an infection from at least one pathogen from Table 1 (e.g. Francisella tularensis, Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei or Burkholderia mallei), comprising administering to the host a vaccine comprising an attenuated recombinant live vaccine strain lacking a polynucleotide encoding CapB (LVS capB), wherein the LVS capB expresses an antigen of at least one pathogen from Table 1; or alternatively administering to the host a vaccine comprising an attenuated Listeria monocytogenes expressing the antigen of the pathogen from Table 1; or alternatively administering to the host a prime vaccine comprising an attenuated recombinant live vaccine strain lacking a polynucleotide encoding capB (LVS capB), wherein the LVS capB expresses an antigen of at least one pathogen from Table 1; and a heterologous booster vaccine comprising an attenuated Listeria monocytogenes expressing the antigen of the pathogen from Table 1. Optionally, the LVS capB and attenuated Listeria monocytogenes express antigens of two or more pathogens selected from the group consisting of Francisella tularensis, Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei, and Burkholderia mallei. Typically, the prime vaccine and/or heterologous booster vaccine are each administered intradermally, subcutaneously, intramuscularly, intranasally, by inhalation or orally. In an illustrative embodiment, the pathogen is Francisella tularensis (F. tularensis) and the LVS capB over-expresses a fusion protein comprising IglA, IglB, and IglC (IglABC).
[0062] Embodiments of the invention include an immunogenic composition comprising a recombinant attenuated Francisella tularensis subspecies holarctica live vaccine Strain (LVS) having a deleted capB gene (SEQ ID NO: 1) which comprises a heterologous promoter that expresses a fusion protein comprising an antigenic polypeptide epitope present in a Francisella tularensis, Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei or Burkholderia mallei polypeptide. While it is desirable to include large segments of Francisella tularensis, Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei or Burkholderia mallei polypeptides in this invention in order to present a large number of immunoreactive epitopes to the mammalian immune system, for example, one that is up to 250 killidatltons in size. Surprisingly, certain larger fusion proteins are observed to make the immunogenic composition unstable. For this unexpected reason, in some embodiments of the invention, the fusion protein expressed by the heterologous promoter is less than 100 (or 90 or 80 or 70 or 60) kilodaltons in size. This size delineation therefore facilitates the vaccines in this embodiment ability to elicit an immune response in a mammalian host when the immunogenic composition is administered orally, intradermally (i.d.), subcutaneously (s.q.), intramuscularly (i.m.), or intranasally (i.n.) to the mammalian host. Optionally the LVS expresses two or more antigenic polypeptide epitopes present in a Francisella tularensis, Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei or Burkholderia mallei polypeptide.
[0063] In an exemplary working example of this embodiment of the immunogenic composition, the heterologous promoter (e.g. a heterologous promoter that comprises a bfr promoter (SEQ ID NO: 13) and/or an omp promoter (SEQ ID NO: 14)) expresses a fusion protein comprising at least 8 contiguous amino acids of IglA (SEQ ID NO: 2); a fusion protein comprising at least 8 contiguous amino acids of IglB (SEQ ID NO: 3); and/or a fusion protein comprising at least 8 contiguous amino acids of IglC (SEQ ID NO: 4). In a very specific desirable working embodiment, the heterologous promoter is designed to express the fusion protein in an amount at least 1.5, 2.0 or 2.5 fold greater than the expression of endogenous IglA protein, endogenous IglB protein and/or endogenous IglC protein expressed in the LVS (see e.g.
[0064] MHC class I molecules bind short peptides, whose N- and C-terminal ends are anchored into the pockets located at the ends of the peptide binding groove. While the majority of the peptides are of length 9, longer peptides can be accommodated by the bulging of their central portion, resulting in binding peptides of length 8 to 15. Peptides binding to class II proteins are not constrained in size and can vary from 11 to 30 amino acids long. In embodiments of the invention, while 8 amino acids or more are sufficient to elicit immunological reaction of MHC Class I (which bind peptides of 8-15 amino acids), typically larger stretches of amino acids are used. For example, 11 or more amino acids are sufficient to elicit immunological reaction of MHC Class II (which binds peptides of 11-30 amino acids). See e.g., Meydan et al., BMC Bioinformatics 201314(Suppl 2): S13. Optionally the at least 8 contiguous amino acids of a polypeptide such as IglA, the at least 8 contiguous amino acids of a polypeptide such as IglB, and the at least 8 contiguous amino acids of a polypeptide such as IglC are covalently linked together in a single fusion protein. In a typical example, the LVS expresses a major histocompatibility class I or a major histocompatibility class II epitope present in amino acid residues 33-132 of IglA (SEQ ID NO: 2), a major histocompatibility class I or a major histocompatibility class II epitope present in amino acid residues 446-506 of IglB (SEQ ID NO: 3), and/or a major histocompatibility class I or a major histocompatibility class II epitope present in amino acid residues 29-149 of IglC (SEQ ID NO: 4).
[0065] In another of the immunogenic composition embodiments, the antigenic polypeptide epitope is present in a Bacillus anthracis polypeptide selected from lethal factor domain I (SEQ ID NO: 7), the extended domain IV of protective antigen (SEQ ID NO: 8); and AhpC (SEQ ID NO: 9). In another of the immunogenic composition embodiments, the antigenic polypeptide epitope is present in a Yersinia pestis polypeptide selected from Fl (SEQ ID NO: 8), LcrV (SEQ ID NO: 9), YscF (SEQ ID NO: 10), YopE (SEQ ID NO: 11), and Psn (SEQ ID NO: 12). In another of the immunogenic composition embodiments, the antigenic polypeptide epitope is present in a Burkholderia pseudomallei polypeptide selected from Hcp-1 (SEQ ID NO: 35), Hcp-2 (SEQ ID NO: 36), Hcp-3 (SEQ ID NO: 37), Hcp-4 (SEQ ID NO: 38), Hcp-6 (SEQ ID NO: 39), Lo1C (SEQ ID NO: 40), TypA (SEQ ID NO: 41), BipB (SEQ ID NO: 42), BipC (SEQ ID NO: 43), BipD (SEQ ID NO: 44), Omp3 (SEQ ID NO: 45), Omp7 (SEQ ID NO: 46), Omp85 (SEQ ID NO: 47), OmpW (SEQ ID NO: 48), PotF (SEQ ID NO: 49), OppA (SEQ ID NO: 50), BopA (SEQ ID NO: 51), BimA (SEQ ID NO: 52), BPSL1897 (SEQ ID NO: 53), BPSL3369 (SEQ ID NO: 54), BPSL2287 (SEQ ID NO: 55), BPSL2765 (SEQ ID NO: 56), and VgrG5 (SEQ ID NO: 57). In another of the immunogenic composition embodiments, the antigenic polypeptide epitope is present in a Burkholderia mallei polypeptide selected from BMA_A0768 (SEQ ID NO: 58), BMA_2821 (SEQ ID NO: 59), BMA 0816 (SEQ ID NO: 60), and GroEL (SEQ ID NO: 61).
[0066] Another embodiment of the invention is a method of generating an immune response in a mammal comprising administering one or more of immunogenic compositions disclosed herein to the mammal so that an immune response is generated to the antigenic polypeptide epitope present in a Francisella tularensis, Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei or Burkholderia mallei polypeptide. In one such embodiment, the method comprises administering a LVS immunogenic composition disclosed herein in a primary vaccination; and administering the same immunogenic composition of LVS immunogenic composition disclosed herein in a subsequent homologous booster vaccination. Typically, the method consists essentially of administering the immunogenic composition of an LVS immunogenic composition disclosed herein in a primary vaccination; and administering the immunogenic composition of LVS immunogenic composition disclosed herein in a subsequent homologous booster vaccination. Optionally, the method comprises administering the immunogenic composition to the mammal less than 4 times.
[0067] In another embodiment of the invention, the method comprises administering an LVS composition as disclosed herein in a primary vaccination; and administering a second heterologous immunogenic composition comprising the antigenic polypeptide epitope present in a Francisella tularensis, Bacillus anthracis, Yersinia pestis, Burkholderiapseudomallei, or Burkholderia mallei polypeptide in a subsequent booster vaccination. Optionally, the second immunogenic composition comprises an attenuated strain of Listeria monocytogenes expressing the antigenic polypeptide epitope. For example, in an illustrative embodiment of the invention, the attenuated Listeria monocytogenes expresses a fusion protein comprising at least 8 contiguous amino acids of IglA (SEQ ID NO: 2), a fusion protein comprising at least 8 contiguous amino acids of IglB (SEQ ID NO: 3); and/or a fusion protein comprising at least 8 contiguous amino acids of IglC (SEQ ID NO: 4). Optionally, the amino acid residues of IglA, the amino acid residues of IglB, and the amino acid residues of IglC are covalently linked in a single fusion protein, one that is less than 50, 60, 70 or kilodaltons in size. In certain embodiments, the method comprises administering LVS immunogenic composition disclosed herein and a second immunogenic composition to the mammal less than a total of four times. Optionally for example, the method comprises administering a single dose of a LVS immunogenic composition disclosed herein, and one or more doses of the second immunogenic composition.
[0068] As already noted, F. tularensis, B. anthracis and Y. pestis are Tier I bioterrorist threats and a vaccine is needed to protect against use of these agents as a bioweapon. In illustrative implementations, this vaccine may be used by the military to protect its personnel. Health care workers upon whom the public is relying for assistance in a bioterrorist attack would also be high priority candidates for a vaccine such as this. Finally, if the vaccine is sufficiently benign on human testing, the general public worldwide could benefit from this vaccine to protect them from a bioterrorist attack.
[0069] It should be emphasized that a major drawback to widespread use of the currently available but unlicensed LVS and EV76 vaccines is toxicity. Since the vaccines provided herein are non-toxic or of exceedingly low toxicity, but comparably efficacious or more efficacious, these vaccines would likely be much better accepted than the LVS vaccine for widespread use among the military, first responders, and the public. Furthermore, the AVA vaccine requires at least six administrations and is not highly efficacious. Since the vaccines provided herein are more efficacious and require only two or three vaccinations, these vaccines would likely be much better accepted than the AVA vaccine for widespread use among the military, first responders, and the public.
Illustrative Embodiments of Multiantigenic Live Attenuated Prime and Booster Vaccines Against Tularemia
[0070] In one or more illustrative embodiments, a multivalent prime and booster vaccine for preventing tularemia in humans and animals is provided. The invention is useful for preventing infection caused by Francisella tularensis, the agent of tularemia, and a potential agent of bioterrorism. F. tularensis is a Tier I bioterrorism agent that has previously been stock-piled as a germ-warfare agent and may have been used as such in World War II. Especially when spread by the air-borne route, F. tularensis can cause a highly fatal pneumonia. The present vaccine is designed to prevent military and non-military personnel from disease caused by F. tularensis.
[0071] A safe, well-characterized, stable and effective vaccine against F. tularensis is needed to protect against potential use of this agent as a bioterrorist weapon. A vaccine against F. tularensis was developed a half-century ago, but it has not been approved for general use. This vaccine, called Live Vaccine Strain, or LVS, is an attenuated form of Francisella tularensis subspecies holarctica, a much less virulent subspecies of F. tularensis than the highly virulent subspecies of concern as a bioterrorist agent, F. tularensis subspecies tularensis. The LVS vaccine is unstable in that different colonial morphology types emerge on culture, and somewhat toxic to humans vaccinated with it. Moreover, it may not protect against the high doses of F. tularensis subspecies tularensis that might be released in an airborne bioterrorism attack. Hence, a vaccine that is safer, more stable, and more effective against airborne highly virulent F. tularensis is needed.
[0072] Previously, mutants of the LVS vaccine that lack the 0- Antigen have been investigated, i.e. the LPS molecule. These LPS-deficient mutants (LVS wbtDEF and LVS wzy) were highly attenuated vs. LVS. Immunization of mice with these mutants conferred some protective immunity, but very little. The LVS strain was several orders of magnitude more potent. LVS mutants with deletions in the capA, B, and/or C genes have also been previously described (27, 28). These mutants were attenuated compared with LVS. However, each of these transposon insertion mutants contains the sequence for transposons and kanamycin resistant genes in their chromosome. Moreover, the capacity of the mutants to induce protective immunity against F. tularensis challenge was not evaluated.
[0073] A first generation vaccine and vaccine vector comprised of an un-marked attenuated deletional mutant of the LVS vaccine has also been previously described. The deletion is in a gene called capB. The LVS capB mutant, like the previously evaluated LVS wbtDEF mutant, is highly attenuated compared with LVS. Surprisingly, however, the LVS capB mutant induces protective immunity against F. tularensis intranasal challenge comparable to LVS. Recombinant versions of LVS capB expressing single key F. tularensis immunoprotective proteins including IglA or IglC have also been described. Additionally, a heterologous prime-boost vaccination strategy where the prime was LVS capB expressing one F. tularensis protein, and the booster vaccine was a heterologous vector, attenuated L. monocytogenes expressing one F. tularensis proteins has been described. Intradermal immunization with the heterologous prime-boost vaccine induced protection greater than that of the LVS vaccine. Boosting also included immunizing with the F. tularensis protein or proteins in adjuvant.
[0074] The present invention describes improved versions of both the aforementioned prime and booster vaccines. The improved prime vaccine (rLVS capB/bfr-iglABCSee Table 3 for complete definition) and booster vaccine (rLm actA inlB prfA*/hly-iglABC) express a fusion protein (IglABC) comprising immunogenic portions of three major F. tularensis proteins vs. one protein for the predecessor vaccines. This prime vaccine (rLVS capB/bfr-iglABC) has demonstrated improved immunogenicity and immunoprotection compared with the first generation rLVS capB vaccines. The booster vaccine (rLm actA inlB prfA*/hly-iglABC) has also demonstrated improved immunogenicity compared with the first generation rLm vaccines.
[0075] The invention consists of two types of vaccines. One vaccine is a recombinant LVS capB overexpressing a fusion protein consisting of immunodominant epitopes of F. tularensis Francisella Pathogenicity Island (FPI) and Type VI Secretion System (T6SS) proteins IglA, IglB, and IglC (IglABC). The second vaccine is a heterologous booster vaccine: an attenuated Listeria monocytogenes expressing the fusion protein IglABC. Each vaccine is administered intradermally or by another route, e.g. subcutaneously, intramuscularly, intranasally, inhaled, or even orally to a mammalian host. The vaccine can be administered as part of a homologous or heterologous prime-boost vaccination strategy. The vaccine induces a strong cell-mediated immune response to pathogen antigens in the vaccine and a strong antibody response.
[0076] With respect to the prime vaccine, the vector LVS capB and recombinant LVS capB overexpressing individual proteins of F. tularensis including IglA and IglC have been previously described. These vaccines were safer than the unlicensed and toxic Live Vaccine Strain (LVS) and highly immunogenic. Here, the vaccine is improved in two ways. First, a fusion protein of three different proteinsIglABCis overexpressed. Second, the fusion protein is expressed from a stronger promoterthe bfr promoter and omp promoter instead of the previously used groEL promoter.
[0077] With respect to the booster vaccine, an attenuated recombinant Listeria monocytogenes vaccine (rLm) expressing individual proteins of F. tularensis including IglA and IglC has been previously described. Here, the vaccine is improved by expressing a fusion protein of three different proteinsIglABC.
[0078] There is a need for a safe and effective tularemia vaccine. The only currently available vaccine is an unlicensed toxic vaccine known as Live Vaccine Strain (LVS). The prime LVS capB based vaccines described herein are >10,000 fold less virulent than LVS and hence much safer. In the case of first generation rLVS capB vaccines expressing a single F. tularensis protein from a weak promoter (e.g. groEL), intranasal immunization yielded high level protection, equivalent to LVS and intradermal immunization yielded protection comparable to or slightly less than LVS. The multiantigenic high-expressing vaccine described herein is more immunogenic and protective than the first generation vaccine.
[0079] In the case of a first generation booster vaccine, rLm expressing single F. tularensis proteins, when used in a heterologous prime-boost vaccination strategy with rLVS capB vaccines as the prime, was more potent than LVS by the intradermal route. Thus, the first-generation prime-boost vaccination strategy provided a vaccine that is safer and more potent than the LVS vaccine. The multiantigenic version of the rLm booster vaccine is more immunogenic and therefore more protective than the previous vaccine as well in a prime-boost vaccination strategy.
[0080] As already noted, F. tularensis is a Tier I bioterrorist threat and a vaccine is needed to protect against use of this agent as a bioweapon. In illustrative implementations, this vaccine may be used by the military to protect its personnel. Health care workers upon whom the public is relying for assistance in a bioterrorist attack would also be high priority candidates for a vaccine such as this. Finally, if the vaccine is sufficiently benign on human testing, the general public worldwide could benefit from this vaccine to protect them from a bioterrorist attack. It should be emphasized that a major drawback to widespread use of the currently available but unlicensed vaccine, LVS, is its toxicity. Since the present vaccines are much less toxic, but comparably efficacious, the present vaccine would likely be much better accepted than the LVS vaccine for widespread use among the military, first responders, and the public.
Illustrative Aspects and Embodiments of the Invention
[0081] The invention disclosed herein has a number of embodiments. One embodiment is a method of immunizing a susceptible host against an infection from at least one pathogen from Table 1. The method comprises administering to the host a prime vaccine and a heterologous booster vaccine. The prime vaccine comprises an attenuated recombinant live vaccine strain lacking a polynucleotide encoding CapB (LVS capB), wherein the LVS capB expresses an antigen of at least one pathogen from Table 1. The heterologous booster vaccine comprises an attenuated Listeria monocytogenes expressing the antigen of the pathogen from Table 1. Typically, each vaccine is administered intradermally, subcutaneously, intramuscularly, intranasally, by inhalation or orally. Each vaccine is administered to the host in an amount sufficient to induce an immune response in the host to the pathogen antigen. In certain implementations, the host is administered a single dose of the prime vaccine and one or more doses of the booster vaccine.
[0082] In one or more embodiments, the pathogen is selected from the group of Tier 1 Select Agents consisting of Francisella tularensis, Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei, and Burkholderia mallei. In certain embodiments, the LVS capB and attenuated Listeria monocytogenes express antigens of two or more pathogens selected from the group consisting of Francisella tularensis, Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei, and Burkholderia mallei. In one instance, the LVS capB and attenuated Listeria monocytogenes express antigens of Francisella tularensis, Bacillus anthracis, and Yersinia pestis.
[0083] In one illustrative embodiment, the pathogen is Francisella tularensis (F. tularensis) and the LVS capB over-expresses a fusion protein comprising the immunodominant epitopes of IglA, IglB, and IglC (IglABC). In specific instances, the fusion protein is expressed by rLVS capB under the control of a bfr promoter and/or omp promoter. The attenuated Listeria monocytogenes also expresses the fusion protein comprising the immunodominant epitopes of IglA, IglB, and IglC (IglABC). In specific instances, the fusion protein is expressed by rLm under the control of a actA promoter and/or hly promoter.
EXAMPLES
Example 1
Construction and Characterization of LVS capB and Recombinant Listeria monocytogenes Expressing B. anthracis and Y. pestis Antigens and Demonstration of their Immunogenicity and Efficacy
[0084] Concept of Using Attenuated Francisella as a Prime and booster Vaccine in a Homologous Prime Boost Vaccination Strategy; or Listeria monocytogenes as a Prime and Booster Vaccine in a Homologous Prime-Boost Vaccination Strategy; or Francisella as a Prime Vaccine and Listeria monocytogenes as a Booster Vaccine in a Heterologous Prime-Boost Vaccination Strategy as Delivery Platforms for B. anthracis and Y. pestis Immunoprotective Antigens
[0085] Bacillus anthracis (Ba), Yersinia pestis (Yp), and Francisella tularensis (Ft), causing anthrax, plague, and tularemia, respectively, are classified as Tier 1 Select Agents of bioterrorism as they have extraordinarily high infectivity, cause high morbidity and mortality- especially when inhaled, are relatively easily cultured and dispersed, and have previously been weaponized. As post-exposure prophylaxis is not a practical public health alternative for countering an outbreak of pneumonic tularemia, anthrax or plague, a safe and effective pre-exposure vaccine is needed. Currently, there are no licensed vaccines against pneumonic tularemia and plague and the only currently available human anthrax vaccine is suboptimal as it requires 6 doses followed by annual boosters and its duration of efficacy is unknown. Practically speaking, a multivalent vaccine is needed as it would simplify manufacture, regulatory approval, clinical evaluation, and vaccine administration, and be more acceptable to people than multiple individual vaccines. Our strategy for a multivalent vaccine platform is to use LVS capB as a single vector platform to express B. anthracis, Y. pestis and F. tularensis immunoprotective proteins in a homologous prime-boost vaccination strategy; or to use an attenuated Listeria monocytogenes (Lm) as a single vector platform to express B. anthracis, Y. pestis and F. tularensis immunoprotective proteins in a homologous prime-boost vaccination strategy; or to use LVS capB expressing recombinant B. anthracis, Y. pestis, and F. tularensis immunoprotective proteins as the prime vaccine and an attenuated Listeria monocytogenes (Lm) vector expressing recombinant Ba, Yp, and Ft immunoprotective proteins as the booster vaccine in a heterologous prime-boost vaccination strategy. The same vectors can be used to express antigens of other pathogens including other Tier 1 Select Agents.
[0086] In preliminary studies, we have developed vaccines using LVS capB as a vector to express F. tularensis (IglABC) B. anthracis (Ba), or Y. pestis (Yp) antigens and rLm actA inlB prfA*(G155S) (rLmprfA*) as a vector to express Ft, Ba or Yp antigens, and shown that these vaccines are highly immunogenic. In a recent study in mice, two doses of rLVS capB/bfr-iglABC yielded greater protection than one dose against intranasal (i.n.) challenge with highly virulent Ft SchuS4; protection was equivalent to (i.d.) or better than (i.n.) that from LVS, indicating that homologous prime-boost vaccination is sufficient for high-level protection against virulent Ft SchuS4 (
[0087] Ultimately, we envision construction of a trivalent Ft rLVS capB (prime) vaccine expressing Ba+Yp antigens downstream of a strong Ft promoter, bacterioferritin (bfr, FTL_0617) or outer membrane protein 26 (omp, FTN_1451) and a trivalent rLm actA inlB prfA* booster vaccine expressing Ba+Yp+Ft antigens downstream of a strong Lm promoter (actA/hly) [or, alternatively, a bivalent rLm actA inlB prfA*/Ba+Yp used in concert with our rLm actA inlB prfA*/IglABC vaccine]. Both the Ft LVS and Lm actA inlB parental strains have been tested in humans. LVS capB is a fully defined further attenuated LVS with three major attenuating deletions (capB, pilA, and FTT 0918). rLm actA inlB prfA* comprises Lm actA inlB with an additional mutation in prfA*(G155S)shown not to affect the virulence of this strain (7, 8). Thus, both the prime and boost vectors are anticipated to be exceedingly safe. Moreover, we have shown that both vectors are capable of inducing strong humoral and cellular immune responses to expressed antigens (9, 10); such responses play key roles in immunoprotection against these pathogens. These vectors can express immunoprotective antigens of Ba and Yp. Ba and Yp antigens expressed by these vectors in our studies include the immunogenic domains of the protective antigen (PA) and lethal factor (LF) for Ba and fraction 1 capsular antigen (F1) and low-calcium-response V antigen (LcrV) for Yp.
[0088] The translated amino acid sequences of the fusion protein of B. anthracis lethal factor domain 1 and the extended Protective Antigen domain 4 separated by GGSG (SEQ ID NO: 62) (designated L1) or GSSGGSSG (SEQ ID NO: 65) (L2) and the fusion protein of Y. pestis F1, LcrV, and/or YscF either linked directly (designated D) or separated by a GGSG (SEQ ID NO: 62) (L1) or (GGGS)3 (SEQ ID NO: 66) (L3) linker are listed below:
TABLE-US-00001 A.B.anthracisLFPA(L1)(LFD1-GGSG-PAD4) (SEQIDNO:16) MAGGHGDVGMHVKEKEKNKDENKRKDEERNKTQEEHLKEIMKHIVKIEVKGEEAVKKEAAEKLLEKVPS DVLEMYKAIGGKIYIVDGDITKHISLEALSEDKKKIKDIYGKDALLHEHYVYAKEGYEPVLVIQSSEDY VENTEKALNVYYEIGKILSRDILSKINQPYQKFLDVLNTIKNASDSDGQDLLFINQLKEHPIDFSVEFL EQNSNEVQEVFAKAFAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEINLGGSGNFDQQTSQNIKNQLAEL NATNIYTVLDKIKLNAKMNILIRDKRFHYDRNNIAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKI LSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYAVIKENT IINPSENGDISINGIKKILIFSKKGYEIG B.B.anthracisLFPA(L2)(LFD1-GSSGGSSG-PAD4) (SEQIDNO:17) MAGGHGDVGMHVKEKEKNKDENKRKDEERNKTQEEHLKEIMKHIVKIEVKGEEAVKKEAAEKLLEKVPS DVLEMYKAIGGKIYIVDGDITKHISLEALSEDKKKIKDIYGKDALLHEHYVYAKEGYEPVLVIQSSEDY VENTEKALNVYYEIGKILSRDILSKINQPYQKFLDVLNTIKNASDSDGQDLLFINQLKEHPIDFSVEFL EQNSNEVQEVFAKAFAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEINLGSSGGSSGNFDQQTSQNIKNQ LAELNATNIYTVLDKIKLNAKMNILIRDKRFHYDRNNIAVGADESVVKEAHREVINSSTEGLLLNIDKD IRKILSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYAVIK ENTIINPSENGDISINGIKKILIFSKKGYEIG C.Y.pestisF1V(D)(F1-LcrV) (SEQIDNO:18) MKKISSVIAIALFGTIATANAADLTASTTATATLVEPARITLTYKEGAPITIMDNGNIDTELLVGTLTL GGYKTGTTSTSVNFTDAAGDPMYLTFTSQDGNNHQFTTKVIGKDSRDFDISPKVNGENLVGDDVVLATG SQDFFVRSIGSKGGKLAAGKYTDAVTVTVSNQMIRAYEQNPQHFIEDLEKVRVEQLTGHGSSVLEELVQ LVKDKNIDISIKYDPRKDSEVFANRVITDDIELLKKILAYFLPEDAILKGGHYDNQLQNGIKRVKEFLE SSPNTQWELRAFMAVMHFSLTADRIDDDILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEIN KHLSSSGTINIHDKSINLMDKNLYGYTDEEIFKASAEYKILEKMPQTTIQVDGSEKKIVSIKDFLGSE NKRTGALGNLKNSYSYNKDNNELSHFATTCSDKSRPLNDLVSQKTTQLSDITSRFNSAIEALNRFIQK YDSVMQRLLDDTSGK D.Y.pestisF1V(L1)(F1-GGSG-LcrV) (SEQIDNO:19) MKKISSVIAIALFGTIATANAADLTASTTATATLVEPARITLTYKEGAPITIMDNGNIDTELLVGTLTL GGYKTGTTSTSVNFTDAAGDPMYLTFTSQDGNNHQFTTKVIGKDSRDFDISPKVNGENLVGDDVVLATG SQDFFVRSIGSKGGKLAAGKYTDAVTVTVSNQGGSGMIRAYEQNPQHFIEDLEKVRVEQLTGHGSSVLE ELVQLVKDKNIDISIKYDPRKDSEVFANRVITDDIELLKKILAYFLPEDAILKGGHYDNQLQNGIKRVK EFLESSPNTQWELRAFMAVMHFSLTADRIDDDILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQ AEINKHLSSSGTINIHDKSINLMDKNLYGYTDEEIFKASAEYKILEKMPQTTIQVDGSEKKIVSIKDF LGSENKRTGALGNLKNSYSYNKDNNELSHFATTCSDKSRPLNDLVSQKTTQLSDITSRFNSAIEALNR FIQKYDSVMQRLLDDTSGK E.Y.pestisVF1YscF(L3)(LcrV-[GGGS]3-F1-[GGGS]3-YscF) (SEQIDNO:20) MIRAYEQNPQHFIEDLEKVRVEQLTGHGSSVLEELVQLVKDKNIDISIKYDPRKDSEVFANRVITDDIE LLKKILAYFLPEDAILKGGHYDNQLQNGIKRVKEFLESSPNTQWELRAFMAVMHFSLTADRIDDDILKV IVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDKSINLMDKNLYGYTDEEIF KASAEYKILEKMPQTTIQVDGSEKKIVSIKDFLGSENKRTGALGNLKNSYSYNKDNNELSHFATTCSDK SRPLNDLVSQKTTQLSDITSRFNSAIEALNRFIQKYDSVMQRLLDDTSGKGGGGSGGGGSGGGGSADLT ASTTATATLVEPARITLTYKEGAPITIMDNGNIDTELLVGTLTLGGYKTGTTSTSVNFTDAAGDPMYLT FTSQDGNNHQFTTKVIGKDSRDFDISPKVNGENLVGDDVVLATGSQDFFVRSIGSKGGKLAAGKYTDAV TVTVSNQGGGGSGGGGSGGGGSMSNFSGFTKGTDIADLDAVAQTLKKPADDANKAVNDSIAALKDKPDN PALLADLQHSINKWSVIYNINSTIVRSMKDLMQGILQKFP
Construction and Verification of Bivalent rLVS capB Prime Vaccines Expressing the Fustion Protein of B. anthracis Immunogenic Domains of the Lethal Factor (LF) and Protective Antigen (PA) and the Fusion Protein of Y. pestis Fraction 1 Capsular Antigen (F1), Low-Calcium-Response V Antigen (LcrV), and/or the Type III Secretion System Needle Protein YscF
[0089] We have constructed the following Francisella-vectored vaccine candidates expressing B. anthracis and Y. pestis antigens (Table 2): 1) rLVS capB expressing a fusion protein comprising the B. anthracis lef-encoded LF amino terminal domain [domain 1 (LFD1), aa 1-254] (11) and pagA-encoded PA carboxy-terminal host cell receptor-binding domain [domain 4 (PAD4), aa 552-735] (12) separated by a GGSG (SEQ ID NO: 62) (L1) or GSSGGSSG (SEQ ID NO: 65) (L2) flexible linker, resulting in rLVS capB/BaLFPA(L1) and rLVS capB/BaLFPA(L2); 2) rLVS capB expressing the fusion protein comprising the B. anthracis LFD1-GGSG-PAD4 ligated in frame with the carboxy-terminal of the F. tularensis KatG signal sequence (KatGss) or the F. novicida PepO signal sequence (PepOss), resulting in rLVS capB/katGss-BaLFPA(L1) and rLVS capB/PepOss-BaLFPA(L1); 3) rLVS capB expressing the Y. pestis cafI-encoded Fl antigen and lcrV-encoded LcrV antigen either linked directly (D) or separated by a flexible linker GGSG (SEQ ID NO: 62) (L1), downstream of the Ft omp promoter, resulting in rLVS capB/YpF1V(D) and rLVS capB/YpF1V(L1); 4) rLVS capB expressing the Y. pestis F1V(L1) ligated in frame with the C-terminal of F. novicida PepPOss, resulting in rLVS capB/PepOss-YpF1V(L1); 5) rLVS capB expressing the Y. pestis lcr V-encoded LcrV antigen, cafI-encoded F1 antigen, and yscF-encoded YscF separated by a flexible linker GGGSGGGSGGGS (SEQ ID NO: 66) (L3) and downstream of the Ft bfr promoter, resulting in rLVS capB/YpVF1YscF(L3).
Construction and Verification of Monovalent rLm Booster Vaccines Expressing Ba Fustion Protein LFD1-GGSG-PAD4 and Yp Fusion Protein F1-GGSG-LcrV
[0090] We have constructed: 1) rLm expressing Ba LFD1-L-PAD4 downstream of Lm hly or actA promoter and fused with the listeriolysin O signal sequence (LLOs.s.) or the ActA N-terminal 100 amino acids (ActAN100) using Lm actA and Lm actA inlB prfA*Lm vectors, resulting in rLm actA/actA-BaLFPA(L1), rLm actA inlB prfA*/actA-BaLFPA(L1), rLm actA/hly-BaLFPA(L1), and rLm actA inlB prfA*/hly-BaLFPA(L1); and 2) rLm expressing Yp F1-LcrV under the control of Lm hly and actA promoters by using two different version of Lm vectors, resulting in rLm actA/actA-YpF1V(L1), rLm actA inlB prfA*Lm/actA-YpF1V(L1), rLm actA/hly-YpF1V(L1), and rLm actA inlB prfA*/hly-YpF1V(L1). See Table 2 for a complete list of the rLm vaccines constructed.
Demonstration that B. anthracis and Y. pestis Fustion Proteins are Expressed by F. tularensis and Lm Platforms
[0091] rLVS capB and rLm vaccines express Ba and Yp fusion proteins in broth (
Demonstration that B. anthracis and Y. pestis Fustion Proteins are Expressed by rLVS capB from Infected Macrophase-Like THP-1 Cells
[0092] To examine whether the F. tularensis rLVS capB platform can express the B. anthracis and Y. pestis fusion proteins from infected macrophage-like cells, we seeded THP-1 cells at 310.sup.5 cells/well on 24-well plates and differentiated them in the presence of PMA for 3 days. Vaccine vector (LVS capB) and vaccines were grown on Chocolate agar supplemented without or with kanamycin (7.5 g/ml) for 3 days. Differentiated THP-1 cells were infected with 0.5 ml of vaccines or vaccine vector opsonized with human serum for 10 min at 37 C., spun at 1000g for 30 min at 4 C., and incubated at 37 C. for 1 h. The cells were then washed with RPMI three times, incubated with complete RPMI supplemented with gentamycin (0.1 g/ml) to inhibit extracellular bacterial growth. At 5 and 24 h post infection, medium was removed from wells and cells were lysed in 0.125 ml SDS buffer and boiled for 5 min. The cell lysates were loaded onto 4-15% SDS gels and proteins separated by electrophoresis; the proteins transferred to a nitrocellulose membrane; and the proteins probed with antibodies to B. anthracis PA antigen (mAb to B. anthracis PA) plus goat polyclonal antibody to Y. pestis LcrV antigen (
Demonstration that F. tularensis rLVS capB Expressing Fusion Proteins of B. anthracis and Y. pestis Grow SImilarly to the Parental LVS capB in Infected THP-1 Cells
[0093] To examine the growth kinetics of the rLVS capB vaccine candidates expressing B. anthracis and Y. pestis antigens, we infected THP-1 cells as described above for
Demonstration that Immunization with rLVS capB- and/or rLm actA inlB prfA*Vectored Ba and Yp Vaccines Includes Antigen-Specific Functional Humoral and Strong LFSpecific T-cell Mediated Immune Responses
[0094] Mice immunized with rLVS capB/BaLF-PA (rLVS capB/BaLFPA[L1]) or prime-boosted with rLVS capB/BaLF-PA and rLmprfA*/BaLF-PA (rLm actA inlB prfA*/hly-BaLFPA(L1)) produced significantly greater amounts of Ba (LF and PA) and Ft (HI-LVS) antigen specific serum IgG antibody than sham-immunized mice, dominated by subtype IgG2a, and with appreciable amounts of IgG1 and IgG2b, and minimal amounts of IgG3 to LF/PA (
Demonstration that Immunization with Francisella- and Lm-Vectored B. anthracis Vaccines Induces Antigen specific T-cell Mediated Immune Responses
[0095] To examine whether immunization with B. anthracis vaccines delivered by Francisella and Lm platforms induces T-cell immune responses, we immunized groups of mice as described above, isolated their spleen and lung cells, and assayed T-cell mediated immune responses by measuring cytokine secretion and intracellular cytokine staining. Our results show that in response to PA antigen, spleen cells from mice immunized with the AVA vaccine subcutaneously twice, rLVS capB/BaLFPA(L1) intranasally twice, or primed with rLVS capB/BaLFPA(L1) intranasally and boosted twice with rLmprfA */hly-BaLFPA intramuscularly produced greater amounts of interferon gamma (IFN-) than sham-immunized mice, mice immunized only once with rLVS capB/BaLFPA(L1) intranasally, or mice primed with rLVS capB/BaLFPA(L1) intranasally and boosted twice with rLmprfA*/hly-BaLFPA intranasally, although the difference did not reach statistical significance (
Demonstration that Systemic Homologous Vaccination with rLVS capB/Ba LFPA(L1) and Both Systemic and i.n. Heterologous Prime-Boost Vaccination with rLVS capB/BaLFPA(L1) and rLm actA inlB prfA*/hly-BaLFPA Induces Strong Protective Immunity Against Respiratory Challenge wih Ba and protection is Superior to Systemic Homologous Boosting with the AVA Vaccine
[0096] Mice were sham-immunized, or immunized subcutaneously twice with AVA (Biothrax, 0.025m1), i.d. or i.n. twice with rLVS capB/Ba (rLVS capBIBaLFPA(L1)) or prime-boosted with rLVS capB/Ba once and rLm/Ba (rLm actA inlB prfA*/hly-BaLFPA) once systemically (i.d./i.m.) or mucosally (i.n./i.n.) (10.sup.6 of each) at Week 0 and 4, as indicated in
Demonstration that Systemic Heterologous Prime-Boost Vaccination with rLVS capB/YpF1V and rLm actA inlB prfA*/hly-YpF1V Protects Against Respiratory Challenge with Yp (CO92)
[0097] Mice were sham-immunized, or immunized subcutaneously with EV76 (10.sup.6), i.d. or i.n. twice with rLVS capB/Yp (rLVS capB/YpF1V[L1]) or prime-boosted with rLVS capB/Yp once and rLm/Yp (actA inlB prfA*/hly-YpF1V) once systemically (i.d./i.m.) or mucosally (i.n./i.n.) (10.sup.6 of each) at Week 0 and 4, as indicated in
Demonstration that Homologous Prime-Boost Vaccination with rLVS capB/BaLFPA(L1) or with rLm actA inlB prfA*/hly-BaLFPA(L1) and Heterologous Prime-Boost Vaccination with rLVS capB/BaLFPA(L1)rLm actA inlB prfA*hly-BaLFP A(L1) via Systemic and Mucosal Routes Induce Antigen-Specific Functional Humoral Immune Responses
[0098] As described and indicated in the legend to
Demonstration that Both Systemic and Mucosal Homologous Prime-Boost Vaccination with rLVS capB/BaLFPA or rLm actA inlB prfA*/hly-BaLFPA and Heterologous Prime-Boost Vaccination with rLVS capB/BaLFPArLm actA inlB prfA*/hly-BaLFPA Induces Strong Protective Immunity Against Respiratory Challenge with Ba and Protection is Superior to Systemic Homologous Prime-Boosting with the AVA Vaccine
[0099] Mice were immunized as described above and as indicated in
Demonstration that Systemic and Mucosal Homologous Prime-Boost Vaccination with rLVS capB/YpF1V and Systemic Heterologous Prime-Boost Vaccination with rLVS capB/YpF1VrLm actA inlB prfA*/hly-YpF1V Induce Antigen-Specific Humoral Immune Responses
[0100] Mice were sham-immunized, or immunized subcutaneously (s.c.) with EV76 (10.sup.6) once, i.n. or i.d. with rLVS capB/YpF1V or rLm actA inlB prfA*/hly-YpF1V three times at weeks 0, 4, and 8, or primed i.d. with rLVS capB/YpF1V once at week 0 and boosted i.m. with rLm actA inlB prfA*/hly-YpF1V (10.sup.6 of each) once at week 4 or twice at weeks 4 and 8, as indicated in
Demonstration that Systemic Homologous Prime-Boost Vaccination with rLVS capB/YpF1V Induces Strong Protective Immunity against Yp CO92 Respiratory Challenge
[0101] Mice were immunized as described above and in the legend to
Example 2
Construction and Characterization of LVS capB and Recombinant Listeria monocytogenes Overexpressing/Expressing the Fusion Protein of IglA, IglB, and IglC in vitro and in vivo and Demonstration of their Improved Immunogenicity as a Vaccine Against F. tularensis
[0102] A. Recombinant LVS capB Overexpressing the Fusion Protein of IglA, IglB, and IglC
Rationale and Construction of LVS capB Overexpressing the Fusion Protein of IglA, IglB, and IglC as a Vaccine Candidate against F. tularensis
[0103] Previously, we have constructed recombinant LVS capB (rLVS capB) overexpressing a single immunogenic protein of Francisella tularensis at a time, including proteins expressed by the genes located in the Francisella pathogenicity Island (FPI)intracellular growth locus A (iglA, FTT1714/1359) (27), iglB (FTT1713/1358), iglC (FTT1712/1357), and vgrG (1702/1347) from a shuttle plasmid under the control of the Francisella tularensis groEL (FTL_1715) promoter (28)rLVS capBlgro-iglA, rLVS capBlgro-iglB, rLVS capBlgro-iglC and rLVS capB/gro-vgrG. IglA, IglB, IglC, and VgrG are major components of the Francisella Type VI Secretion System (T6SS) apparatus that is required by F. tularensis to escape from its phagosome and multiply intracellularly in host cells; the heterodimers of IglA/IglB assemble to form the Francisella T6SS outer sheath (29), which upon contraction, thrusts an inner tube likely comprising IglC through the bacterial wall and into the target phagosomal membrane. We have shown that mice immunized with rLVS capBlgro-iglA and rLVS capBlgro-iglC survive longer than sham-immunized mice and mice immunized with the parental rLVS capB vaccine against lethal aerosol challenge with virulent F. tularensis Schu S4 strain (27). To improve the potency of the rLVS capB vaccine, we sought to improve the activity of the transcription promoter for antigen expression in the shuttle plasmid and to increase the F. tularensis antigen pool. To improve the transcription promoter activity, we constructed rLVS capB strains expressing FPI proteins IglA, IglB, IglC or the fusion protein of IglA, IglB, and IglC (IglABC) under the control of the groEL promoter and two additional Francisella promoters, the promoter of the F. tularensis bacterioferritin (bfr, FTL 0617), which is about 10 times more potent than the groEL promoter (30), and the promoter of F. novicida outer membrane protein 26 (omp, FTN_1451) (31). To increase the antigen pool without compromising the stability of the shuttle plasmid, we constructed rLVS capB expressing the fusion protein of the immunodominant epitopes of IglA (residues 33-132), IglB (residues 446-506) and IglC (residues 29-149), either directly fused in-frame [IglABC(D) or separated by a flexible linker GGSG (SEQ ID NO: 62) [IglABC(GGSG)] or GGSGGGSG (SEQ ID NO: 63) [IglABC(2GGSG)] downstream of the bfr or omp promoter and the Shine-Dalgarno sequence (
[0104] The translated amino acid sequences of IglABC(D), IglABC(GGSG), and IglABC(2GGSG) are listed below:
TABLE-US-00002 A.IglABC(D) (SEQIDNO:21) MLVVGDLSKGRSVDAKKEFADREVRRVNNGVDRVLEEMNISFDFEAPNFV SKDRSNLKVNYRIESVKDFRPDAVAKKVPEIRALLEMKEILASFAKDIEN NQLPLEMARYPFRNVSIEVETIPGKPGWYSCKINVIPHIQFEGMNITMTI DIRLEPELFGINNNTGNCRLFIDSLTIAGEKLDKNIVAIDGGEDVIKADS ATAAASVIRLSITPGSINPTISITLGVLIKSNVRTKIEEKVSSILQASAT DMKIKLGNSNKKQEYKTDEAWGIMIDLSNLELYPI B.IglABC(GGSG) (SEQIDNO:22) MLVVGDLSKGRSVDAKKEFADREVRRVNNGVDRVLEEMNISFDFEAPNFV SKDRSNLKVNYRIESVKDFRPDAVAKKVPEIRALLEMKEILASFAKDIEN NQLGGSGPLEMARYPFRNVSIEVETIPGKPGWYSCKINVIPHIQFEGMNI TMTIDIRLEPELFGINNNGGSGTGNCRLFIDSLTIAGEKDKNIVAIDGGE DVIKADSATAAASVIRLSITPGSINPTISITLGVLIKSNVRTKIEEKVSS ILQASATDMKIKLGNSNKKQEYKTDEAWGIMIDLSNLELYPI C.IglABC(2GGSG) (SEQIDNO:23) MLVVGDLSKGRSVDAKKEFADREVRRVNNGVDRVLEEMNISFDFEAPNFV SKDRSNLKVNYRIESVKDFRPDAVAKKVPEIRALLEMKEILASFAKDIEN NQLGGSGGGSGPLEMARYPFRNVSIEVETIPGKPGWYSCKINVIPHIQFE GMNITMTIDIRLEPELFGINNNGGSGGGSGTGNCRLFIDSLTIAGEKLDK NIVAIDGGEDVIKADSATAAASVIRLSITPGSINPTISITLGVLIKSNVR TKIEEKVSSILQASATDMKIKLGNSNKKQEYKTDEAWGIMIDLSNLELYPI
Demonstration that rLVS capB Overexpresses IglC and IglIABC in Broth and in Infected Human Macrophage-like THP-1 Cells
[0105] To examine the protein expression by rLVS capB, we cultured each vaccine strain in TSBC (Tryptic Soy Broth with 0.1% Cysteine), collected the bacteria, and performed Western blotting. Western blotting analyses using polyclonal antibodies specific to IglA (BEI Resources) or IglC (prepared by our laboratory) or monoclonal antibody to IglB (BEI Resources) showed that IglA, IglB, and IglC expression by rLVS capB/bfr-iglA, rLVS capB/bfr-iglB, and rLVS capB/bfr-iglC was 1.5-1.6-fold higher than that of the parental LVS capB (
Demonstration that rLVS capB Overexpressing IglABC (rLVS capB/bfr-iglABC) Grows Similarly to the Parental LVS capB in Broth Culture and Intracellularly in Infected Human Macrophage-like THP-1 Cells
[0106] To examine whether the protein expression cassette driven by the groE, bfr or omp promoter in the shuttle plasmid affects the growth kinetics of rLVS capB vaccine candidates in broth, we cultured the vaccines on chocolate agar, inoculated them into Chamberlain defined medium (CDM) or 3% Tryptic Soy Broth supplemented with 0.1% L-cysteine (TSBC) medium, and followed their growth kinetics by measuring optical density at 540 nm. As shown in
Demonstration that a) rLVS capB Overexpressing IglA, IglB, IglC are Safe in Mice; b) rLVS capB Overexpressing IglA, IglB, IglC Disseminate and are Cleared Similarly to the Parental rLVS capB Strain in Mice; and c) the Shuttle Plasmid for Overexpressing IglA, IglB, or IglC is Stable in Mice
[0107] To evaluate the safety, dissemination and clearance of rLVS capB/iglA, iglB, iglC, and iglABC, we infected BALB/c mice i.n. or i.d. with LVS, LVS capB, or rLVS capB/bfr-iglA, iglB, iglC, or iglABC and monitored them for signs of illness for 14 days. At 1, 4, 7, and 14 days post-immunization, we assayed the bacterial burden in various organs. As shown in
[0108] To examine whether the shuttle plasmid with the bfr promoter is stable in vivo in the absence of antibiotic selection, we harvested animal organs at various days post-vaccination, cultured organ homogenates on chocolate agar in the presence or absence of kanamycin for 3-5 days, and tested the colonies by colony PCR, amplifying the F. tularensis antigen expression cassette in the shuttle plasmid. We found that the shuttle plasmids for IglA and IglC were more stable than the one for IglB (Tables 4 & 5). That the expression cassette for the large protein IglB was slightly less stable than the others may indicate that stability depends to some degree on the size of the antigen expression cassette.
Demonstration that Higher Doses of rLVS capB Overexpressing IglABC are well Tolerated by Mice after Intranasal Vaccination
[0109] To test the safety of rLVS capB/bfr-iglABC at relatively high doses, we immunized BALB/c mice intranasally, 4 per group, with six different doses of rLVS capB/bfr-iglABC, ranging from 110.sup.7 to 410.sup.8 CFU/mouse. Mice immunized with 1-2.510.sup.7 CFU of rLVS capB/bfr-iglABC lost 6% of their weight at 3-4 days post immunization and showed signs of mild illness (ruffled fur); mice immunized with rLVS capB/bfr-iglABC 5-2010.sup.7 CFU lost 6-12% of their weight at 3-4 days post immunization (
Demonstration that Immunization with rLVS capB Overexpressing FPI T6SS Proteins Induces Antigen-Specific Cytokine Production and Th1-Type Antibody Responses
[0110] To examine T cell immune responses generated by the rLVS capB vaccines, we immunized BALB/c mice i.d. with various vaccines at Week 0, sacrificed them at Week 4, and assayed T-cell mediated immune responses and serum antibody. In response to in vitro stimulation with IglC protein, splenocytes from mice immunized with rLVS capB/iglC or iglABC secreted greater amounts of IL-17A (
Demonstration that Immunization with rLVS capB/bfr-iglABC Induces Improved Protective Immunity Against Respiratory Challenge with F. tularensis Schu S4
[0111] To evaluate rLVS capB/bfr-iglA, iglB, iglC, or iglABC for efficacy in mice against respiratory challenge with virulent F. tularensis Schu S4, we immunized mice i.d. with PBS (Sham), LVS, LVS capB, or rLVS capB, challenged them 7 weeks later i.n. with a high lethal dose ofF. tularensis Schu S4 (16 CFU, equivalent to 5LD.sub.50), and observed the mice closely for signs of illness and death. Mice immunized with rLVS capB/bfr-iglABC survived longer (mean survival time 9.1 days) than sham-immunized mice (mean survival time 4.5 days) (p<0.0001) and mice immunized with the parental LVS capB or rLVS capB/bfr-iglA, iglB, or iglC; (mean survival time 6.6-8.0 days; difference not statistically significant (
[0112] To verify further the efficacy of rLVS capB vaccines against challenge with F. tularensis Schu S4, we repeated the above experiment and challenged mice i.n. 6 weeks later with two higher lethal doses (31 and 310 CFU) of F. tularensis Schu S4, equivalent to approximately 10 and 100 LD.sub.50, respectively. Consistently, after i.n. challenge with 31 CFU of Schu S4, mice immunized with rLVS capB/bfr-iglA , iglB, iglC, or iglABC survived longer than sham-immunized mice (P<0.0001) and generally longer than LVS capB-immunized mice (differences not significant). In this experiment, mice immunized with rLVS capB/bfr-iglA were especially well protectedmean survival time 11.6 days vs. 4 days for sham- immunized mice and 7.1 days for LVS capB-immunized mice- but not as well protected as LVS-immunized mice (mean survival time 15.8 days), but this difference was not statistically significant (
[0113] To explore further the efficacy of rLVS capB/bfr-iglABC by the i.n. route, we immunized mice i.n. and challenged them 6 weeks later with 26 CFU of F. tularensis SchuS4. As shown in
Demonstration that Immediate Pre- or Post-Exposure Prophylaxis with rLVS capB/bfr-iglABC but not LVS Induces Partial Protective Immunity Against Respiratory Challenge with F. tularensis Schu S4 Strain and Protection is Correlated with the Induction of a Strong Innate Immune Response
[0114] To evaluate the efficacy of rLVS capB/bfr-iglABC as a pre- or post-exposure vaccine, we immunized mice with PBS (Sham), 100 CFU LVS, or 110.sup.6 or 510.sup.6 CFU rLVS capB/bfr-iglABC two days before (-2 days), the same day as (0 day), or one day (1 day) or two days (2 days) after Schu S4 challenge and monitored mice closely for signs of illness and weight change. We evaluated mice for illness using a Clinical Score (CS) of 0-4 as follows: 0, normal; 1, questionable illness; 2, mild but definitive illness; 3, moderate to severe illness (euthanized if poorly responsive); 4, severe illness, moribund and euthanized. All sham- and LVS- immunized mice became ill by 3 or 4 days post-challenge and became moribund and were euthanized at day 5 post-challenge (
[0115] To explore the efficacy of a higher dose of rLVS capB/bfr-iglABC as immediate pre- and post-exposure prophylaxis against Schu S4 challenge, we repeated the experiment described above with an immunizing dose of 510.sup.7 CFU (tested as safe,
[0116] To explore the mechanism of protection provided by immediate pre-exposure prophylaxis with rLVS capB/bfr-iglABC, we immunized mice in groups of 3 i.n. with PBS (Sham), 10.sup.2 CFU LVS, or 110.sup.6 or 510.sup.7 CFU rLVS capB/bfr-iglABC; euthanized them 2 days later; and assayed lung lysates and sera for cytokine/chemokine production using a mouse 32-Plex cytokine/chemokine kit. We found that mice immunized with 110.sup.6 or 510.sup.7 rLVS capB/bfr-iglABC had significantly higher levels of pro-inflammatory cytokines/chemokines in their lungs (21 out of 32 cytokines/chemokines assayed) and sera (7 out of the 32) than sham-immunized mice and mice immunized with LVS (
B. Recombinant Listeria monocytogenes Expressing the Fusion Protein of IglA, IglB, and IglC; Heterologous Prime-Boost Vaccination
Construction of Attenuated Recombinant Listeria monocytogenes Vaccine Candidates Expressing Immunodominant Epitopes of F. tularensis IglA, IglB, and IglC Antigens
[0117] Previously, we constructed attenuated recombinant Listeria monocytogenes stably expressing each of the seven F. tularensis proteins, including IglC (32). We showed that mice immunized intradermally with rLm/iglC develop cellular immune responses to F. tularensis IglC as evidenced by lymphocyte proliferation and CD4+ and CD8+ T-cell intracellular expression of interferon-gamma. Moreover, mice immunized with rLm/iglC are protected against lethal challenge with F. tularensis LVS administered by the intranasal route, a route chosen to mimic airborne infection, and, most importantly, against aerosol challenge with the highly virulent Type A F. tularensis SchuS4 strain. To develop a more potent vaccine without sacrificing safety, we utilized a heterologous prime-boost vaccination strategy with LVS capB or LVS capB overexpressing F. tularensis IglA or IglC as the prime vaccine and rLm/iglC as the booster vaccine. We show that the LVS capB - rLm/iglC prime-boost vaccine induces strong cellular immune responses and confers protective immunity against F. tularensis Schu S4 aerosol challenge that is comparable to or greater than that conferred by LVS (27).
[0118] To expand the F. tularensis immunogenic antigen pool in the Listeria monocytogenes booster vaccines, we now have constructed 4 different versions of attenuated rLm expressing the immunodominant epitopes of F. tularensis IglA, IglB, and IglC antigens, IglABC, by using Lm actA or Lm actA inlB prfA* as a vector to express F. tularensis IglABC fused with the Lm LLO signal sequence and downstream of the hly promotor or fused with the amino- terminal 100 amino acids including the signal sequence of Lm ActA (ActAN100) downstream of the actA promoter, resulting in four vaccine candidates: rLm actA/hly-iglABC, rLm actA/actA-iglABC, rLm actA inlB prfA*/hly-iglABC, and rLm actA inlB prfA*/actA-iglABC. We have shown that all four rLm vaccine candidates express the F. tularensis IglABC (
Demonstration that Priming with rLVS capB/bfr-iglABC and Boosting with rLmprfA*/hly-iglABC Induces Antigen Specific T Cell-Mediated and Humoral Immune Responses
[0119] To examine the immune responses induced by priming with rLVS capB/bfr-iglABC and boosting with rLmprfA*/hly-iglABC (rLm actA inlB prfA*/hly-iglABC), we immunized mice i.d. at week 0 with rLVS capB/bfr-iglABC and boosted them at week 4 with rLm/hly-iglABC. Sham-immunized mice and mice immunized i.d. once at week 0 with rLVS capB/bfr-iglC and mice immunized i.d. once at week 4 with rLm/hly-iglABC served as controls. At week 5, we anesthetized the mice, bled and euthanized them, isolated their splenocytes and lung cells, stimulated the cells with IglC peptide (TDEAWGIMIDLSNLE (SEQ ID NO: 64)), peptide pools of IglA or IglB, IglC protein, or heat-inactivated LVS and assayed antigen-specific T-cell immune response by intracellular cytokine staining. In response to stimulation with IglB peptides, IglC peptides, or IglC protein, mice primed with rLVS capB/bfr-iglABC and boosted with rLmprfA*/hly-iglABC produced significantly greater frequencies of CD4+ T cells expressing IFN-, TNF, or IL-2 in their spleens (
[0120] After analysis of the 7 possible combinations of CD4+ T cells producing one or more cytokines among IFN-, IL-2 and TNF, we found that in response to stimulation with IglB peptide, IglC peptide, IglC protein, or HI-LVS, the largest subsets of T cells were CD4+ T cells producing only IFN-, TNF, and IL-2 or multi-functional CD4+ T cells producing both IFN-y and TNF (data not shown). These results show that priming with rLVS capB/bfr-iglABC and boosting with rLmprfA*/hly-iglABC produced T-cell-mediated immune responses. We assayed humoral immune response on the sera collected from the immunized mice. Mice prime-boosted with rLVS capB/bfr-iglCrLm/hly-iglABC produced HI-LVS specific Ig1G antibody at a significantly higher level than sham-immunized mice and mice immunized with the only the booster vaccine, comparable to mice immunized with the prime vaccine only or mice prime-boosted with rLVS capB/bfr-iglCrLm vector (
Demonstration that Priming with rLVS capB/bfr-iglABC and Boosting with rLmprfA*/hly-iglABC Induces Strong Protective Immunity to Respiratory Challenge with F. tularensis SchuS4. To explore the capacity of a heterologous prime boost vaccine comprising a rLVS capB/bfr-iglABC prime and rLmprfA*/hly-iglABC boost to induce protective immunity to F. tularensis Schu S4 challenge, we immunized mice intradermally once with rLVS capB/bfr-iglABC (110.sup.6 CFU) at Week 0 and boosted them once at Week 4 or twice at Week 4 and 6 with rLm vaccines (110.sup.6 CFU) expressing IglABC where the genes encoding IglABC are expressed by the LmprfA* vector (Lm actA inlB prfAG155S) and downstream of hemolysin promoter (hly) fused with the LLO (listeriolysin O, encoded by hly) signal peptide (hly-iglABC) or downstream of the actA promoter fused with the N-terminal 100 amino acids of ActA (including the signal peptide) (actA-iglABC) as indicated in Table 6. Control mice were sham-immunized, not boosted, or boosted with the LmprfA* vector (110.sup.6 CFU). The mice were challenged intranasally with 29 CFU of F. tularensis Schu S4 (equivalent to 10LD.sub.50) and monitored for survival for 21 days. Sham-immunized mice had a mean survival time (MST) of 4 days. Whereas mice not boosted had a MST of 7.9 days, mice boosted with the vector alone had a MST of 6.4 days, and mice boosted once with rLmprfA* vaccine expressing IglABC from hly-iglABC or actA-iglABC had a MST of 7.6 and 7.1 days, respectively, the mice prime-boosted twice with rLmprfA* expressing IglABC from hly-iglABC or actA-iglABC had the longest survival timesboth with MST of 9.4 days.
C. Homologous Prime-Boosting with rLVS capB/bfr-iglABC and Heterologous Prime-Boosting with rLVS capB/bfr-iglABC as the Prime Vaccine and rLm/iglABC as the Booster Vaccine
Demonstration that Homologous Prime-Boosting with rLVS capB/bfr-iglABC or Heterologous Prime-Boosting with rLVS capB/bfr-iglABC as the Prime Vaccine and rLmprfA*/hly-iglABC as the Booster Vaccine Induces Strong Protective Imunity to Respiratory Challenge with F. tularensis SchuS4.
[0121] To further evaluate homologous prime-boosting with rLVS capB/hfr-iglABC or heterologous prime-boosting with rLVS capB/bfr-iglABC as the prime vaccine and rLmprfA*/hly-iglABC as the booster vaccine for efficacy in mice against respiratory challenge with virulent F. tularensis Schu S4, we immunized BALB/c mice, 8/group, i.d. once at week 0 with PBS (Sham, group A), 10.sup.4 CFU LVS (Group B), 10.sup.6 CFU LVS capB vector (Group H), or 10.sup.6 rLVS capB/bfr-iglABC (Group C); immunized them i.d. or i.n. twice at weeks 0 and 4 with 10.sup.6 rLVS capB/bfr-iglABC (Groups D & E, resp.); primed them i.d. once at week 0 with rLVS capB/bfr-iglABC (Groups F & G) or LVS capB (Groups I & J) and boosted them intramuscularly (i.m.) twice at weeks 4 and 6 with rLmprfA* Vector (Lm actA inlB prfA*) (Groups F & I) or rLmprfA*/hly-iglABC (Groups G & J); or immunized them simultaneously i.d. with rLVS capB/bfr-iglABC and i.m. with rLmprfA*/hly-iglABC once at week 6 (Group K). We challenged the mice with 10 CFU F. tularensis Schu S4 at week 10 and monitored them for signs of illness and survival for 3 weeks. As shown in
Aspects of Working Embodiments with Antigenic Polypeptide Epitopes Present in a Francisella tularensis IglA, IglB, and IglC Epitopoes
[0122] We hypothesized that proteins abundantly secreted by Ft or upregulated by Ft inside mononuclear phagocytes may be immunogenic antigens. Starting with 7 proteins, we found that Ft IglC (intracellular growth factor subunit C, FTT1712/ 1357) is a potent immunoprotective antigen, consistent with other studies showing that IglC is one of the most prominent Ft immunogenic antigens in murine tularemia models and in human immune sera. IglC is a 23-kDa protein encoded by the iglABCD operon in the Francisella Pathogenicity Island (FPI). It has no homology to other proteins, which is important because vaccines targeting IglC are less likely to cause a non-specific immune response to host proteins. IglC is one of the most upregulated Ft proteins during macrophage intracellular infection, required for intracellular survival, growth, and phagosome escape, and based on studies of iglC mutants in Ft subsp. novicida, holarctica, and tularensis, essential for virulence (6).
[0123] Other proteins upregulated by Ft in macrophages and therefore of interest to us as immunogens are IglA (FTT1714/1359), IglB (FTT1713/1358), and VgrG (valine glycine repeat protein G, FTT1702/1347) encoded by genes located within the FPI. Similar to every other gene located in the FPI, iglA, iglB and vgrG are required for intramacrophage growth and virulence in mice. More importantly, IglA, IglB and VgrG have been found to be immunogenic in murine models and in human tularemia [
Fustion Protein Promoters
[0124] We cloned the GFP expression cassette driven by the groE or bfr promoter into the pFNLTP6-derived plasmid. The resulting plasmids, pFNLTP6/pgroE-GFP and pFNLTP6/pbfr-GFP, were electroporated into LVS; transformants selected on chocolate agar supplemented with kanamycin; and transformants verified by colony PCR. LVS/pbfr-GFP and LVS/pgro-GFP were grown in CDM for 24 h and protein expression analyzed. GFP was expressed at a higher level by LVS/pbfr-GFP than that by LVS/pgro-GFP. This study prompted us to construct rLVS capB strains expressing Ft-Ag downstream of the bfr promoter.
[0125] Demonstration that IglC driven by the Ft bfr and omp promoter was expressed at a level higher than IglC driven by the groE promoter. To improve the expression of IglC by the rLVS capB strains, we engineered the IglC coding sequence downstream of the Ft bfr or outer membrane protein (omp26) promoter. Consistent with the GFP expression, expression of IglC driven by the bfr or omp promoter was greater than that driven by the groE promoter (
Route of Immunization (e.g., Intradermal [I.D.] vs. Intranasal [I.N.])
[0126] We have tested systemic routes (intradermal [i.d.] or intramuscular [i.m.]) vs. mucosal route of immunization (i.n.).
[0127] B. anthracis: As shown in
[0128] Y. pestis: As shown in
[0129] F. tularensis: Mucosal route (intranasal) is generally superior to systemic (intradermal) route. However, two doses of rLVS capB/bfr-iglABC is comparable to LVS at the safe dose by the intradermal route (
TABLES
[0130]
TABLE-US-00003 TABLE 1 Tier 1 Select Agents Francisella tularensis (Tularemia) Bacillus anthracis (Anthrax) Yersinia pestis (Plague) Burkholderia pseudomallei (melioidosis) Burkholderia mallei (glanders) Category B Pathogens Coxiella burnetii (Q Fever) Brucella species (Brucellosis) Chlamydia psittaci (Psittacosis) Rickettsia prowazekii (Typhus) Listeria monocytogenes (Listeriosis) Campylobacter jejuni (Gastroenteritis) Yersinia enterocolitica (Yersiniosis) Additional Pathogens Chlamydia pneumonia Chlamydia trachomatis Mycoplasma pneumonia Legionella pneumophila Staphylococcus aureus Streptococcus pneumoniae
TABLE-US-00004 TABLE 2 Plasmids, recombinant F. tularensis subsp. holarctica, Listeria monocytogenes for B. anthracis and Y. pestis vaccines Plasmids Name Description Reference pFNLTP6 groE- E. coli-Francisella shuttle vector with gfp driven (13) gfp by Francisella groE promoter; Amp.sup.r Kan.sup.r pBC11 pFNLTP6-derived E. coli-Francisella shuttle This Pomp-Ypcaf1-D- vector for expressing the fusion protein of Y. pestis study lcrV F1 (encoded by caf1) and LcrV (encoded by lcrV) linked directly and downstream of F. tularensis omp promoter pBC12/ pFNLTP6-derived E. coli-Francisella shuttle This Pomp-Ypcaf1- vector for expressing the fusion protein of Y. pestis study GGSG-lcrV F1 (encoded by caf1) and LcrV (encoded by lcrV) separated by a GGSG linker (SEQ ID NO: 62) and downstream of F. tularensis omp promoter pSG39/ pFNLTP6-derived E. coli-Francisella shuttle This Pomp-pepOss- vector for expressing the fusion protein of Y. pestis study Ypcaf1-GGSG- F1 (encoded by caf1) and LcrV (encoded lcrV by lcrV) separated by a GGSG linker (SEQ ID NO: 62) and downstream of F. tularensis omp promoter and PepO signal peptide pBC14/ pFNLTP6-derived E. coli-Francisella shuttle This Pbfr-BalefD1- vector for expressing B. anthracis lethal factor study GGSG-pagAD4 (encoded by lef) domain 1 and Protective antigen (encoded by pagA) domain 4 separated by a GGSG linker (SEQ ID NO: 62) and downstream of F. tularensis bfr promoter pSG37/ pFNLTP6-derived E. coli-Francisella shuttle This Pomp-pepOss- vector for expressing the fusion protein of B. anthracis study Balef1D1-GGSG- lethal factor (encoded by lef) domain 1 pagAD4 and Protective antigen (encoded by pagA) domain 4 with a GGSG linker (SEQ ID NO: 62) and downstream of F. tularensis omp promoter and PepO signal peptide pSG38/ pFNLTP6-derived E. coli-Francisella shuttle This Pomp-katGss vector for expressing the fusion protein of B. anthracis study Balef1D1-GGSG- lethal factor (encoded by lef) domain 1 pagAD4 and Protective antigen (encoded by pagA) domain 4 with a GGSG linker (SEQ ID NO: 62) and downstream of F. tularensis omp promoter and KatG signal peptide pSG28/ pPL2-derived conjugation vector for integration (14) & ActAN100-Ypcaf1- of the antigen expression cassette ActAN100- This GGSG-lcrV Ypcaf1-GGSG-lcrV into the tRNA.sup.arg locus of the study rLm chromosome pSG29/ pPL2-derived conjugation vector pPL2-derived (14) & hly-Ypcaf1-GGSG- conjugation vector for integration of the antigen This lcrV expression cassette hly-Ypcaf1-GGSG-lcrV into study the tRNA.sup.arg locus of the rLm chromosome pSG40/ pPL2-derived conjugation vector for integration (14) & ActAN100-Balef1- of the antigen expression cassette ActAN100- This GGSG-pagA4 Balef1-GGSG-pa4 into the tRNA.sup.arg locus of the study rLm chromosome pSG41/ pPL2-derived conjugation vector for integration (14) & hly-Balef1-GGSG- of the antigen expression cassette hly-Balef1- This pagA4 GGSG-pa4 into the tRNA.sup.arg locus of the rLm study chromosome pQJ137/ pFNLTP6-derived E. coli-Francisella shuttle This Phfr-BalefD1- vector for optimized expression in Ft LVS of B. anthracis study (GSSG)2-pagAD4 lethal factor (encoded by lef) domain 1 and Protective antigen (encoded by pagA) extended domain 4, separated by a GSSGGSSG linker (SEQ ID NO: 65) and downstream of F. tularensis bfr promoter pQJ138/ pFNLTP6-derived E. coli-Francisella shuttle This Pbfr-YplcrV- vector for optimized expression in Ft LVS of the study (GGGS)3-caf1- fusion protein of Y. pestis LcrV (encoded by (GGGS)3-YscF lcrV), F1 (encoded by caf1), and YscF (encoded by yscF), separated by GGGSGGGSGGGS linker (SEQ ID NO: 66) and downstream of F. tularensis bfr promoter Strain Description Reference Attenuated recombinant F. tularensis subsp. holarctica LVS Live vaccine strain CDC LVS capB Unmarked LVS with deletion of capB (10) rLVS capB/ LVS capB carrying pBC11/Pomp-Ypcaf1-D- This YpF1V(D) lcrV study rLVS LVS capB carrying pBC12/Pomp-Ypcaf1- This capB/YpF1V(L1) GGSG-lcrV study rLVS capB/ LVS capB carrying pBC14/Pbfr-BalefD1- This BaLFPA(L1) GGSG-pagAD4 study rLVS capB/ LVS capB carrying pSG39/Pomp-pepOss: This pepOss- Ypcaf1-GGSG-lcrV study YpF1V(L1) rLVS capB/ LVS capB carrying pSG38/Pomp-katGss- This katGss- Balef1D1-GGSG-pagAD4 study BaLFPA(L1) rLVS capB/ LVS capB carrying pSG37/Pomp-pepOss- This pepOss- Balef1D1-GGSG-pagAD4 study BaLFPA(L1) rLVS capB/ LVS capB carrying pQJ137/Pbfr-BalefD1- This BaLFPA(L2) (GSSG)2-pagAD4 optimized for expression in Ft study LVS rLVS capB/ LVS capB carrying pQJ138/Pbfr-YplcrV- This YpVF1YscF(L3) (GGGS)3-caf1-(GGGS)3-YscF optimized for study expression in Ft LVS Attenuated recombinant Listeria monocytogenes Lm actA inlB Lm actA inlB uvrAB prfA*(G155S) [7] prfA* rLm actA/actA- rLm actA integrated with the actAN100-Balef1- This BaLFPA(L1) GGSG-pa4 antigen expression cassette at the study tRNA.sup.arg locus rLm actA inlB rLm actA inlB uvrAB prfA*(G155S) This prfA*/actA- integrated with the actA-Balef1-GGSG-pagA4 study BaLFPA antigen expression cassette at the tRNA.sup.arg locus rLm actA/ rLm actA integrated with the hly-Balef1-GGSG- This hly-BaLF-PA pagA4 antigen expression cassette at the tRNA.sup.arg study locus rLm actA inlB rLm actA inlB uvrAB prfA*(G155S) This prfA*/hly- integrated with the hly-Balef1-GGSG-pa4 antigen study BaLFPA expression cassette at the tRNA.sup.arg locus rLm actA/actA- rLm actA integrated with the actAN100-Ypcaf1- This YpF1V GGSG-lcrV antigen expression cassette at the study tRNA.sup.arg locus rLm actA inlB rLm actA inlB uvrAB prfA*(G155S) This prfA*/actA- integrated with the actAN100-Ypcaf1-GGSG-lcrV study YpF1V antigen expression cassette at the tRNA.sup.arg locus rLm actA/hly- rLm actA integrated with the hly-Ypcaf1-GGSG- This YpF1V lcrV antigen expression cassette at the tRNA.sup.arg study locus rLm actA inlB rLm actA inlB uvrAB prfA*(G155S) This prfA*/hly-YpF1V integrated with the hly-Ypcaf1-GGSG-lcrV study antigen expression cassette at the tRNA arg locus
TABLE-US-00005 TABLE3 Plasmids,recombinantF.tularensissubsp.holarctica,Listeria monocytogenesforF.tularensisvaccines Plasmids Name Description Reference pFNLTP6groE-gfp E.coli-Francisellashuttlevector [2] withgfpdrivenbyFrancisella groEpromoter;Amp.sup.rKan.sup.r pFNL/bfr-iglA E.coli-Francisellashuttlevector [34] forover-expressingIglA downstreamofbfrpromoter pFNL/bfr-iglB E.coli-Francisellashuttlevector [34] forover-expressingIglB downstreamofbfrpromoter pFNL/bfr-iglC E.coli-Francisellashuttlevector [34] forover-expressingIglC downstreamofbfrpromoter pFNL/bfr-iglABC(D) E.coli-Francisellashuttlevector [34] forover-expressingIglABC downstreamofbfrpromoter pFNL/bfr-iglABC(GGSG) E.coli-Francisellashuttlevector [34] forover-expressingIglABCwitha GGSGlinker(SEQIDNO:62)and downstreamofbfrpromoter pFNL/bfr-iglABC(GGSG2) E.coli-Francisellashuttlevector [34] forover-expressingIglABCwith GGSG2linker(SEQIDNO:63) anddownstreamofbfrpromoter pFNL/omp-iglA E.coli-Francisellashuttlevector [34] forover-expressingIglA downstreamofomppromoter pFNL/omp-iglB E.coli-Francisellashuttlevector [34] forover-expressingIglB downstreamofomppromoter pFNL/omp-iglC E.coli-Francisellashuttlevector [34] forover-expressingIgIC downstreamofomppromoter pFNL/omp-iglABC(D) E.coli-Francisellashuttlevector [34] forover-expressingIglABC downstreamofomppromoter pFNL/omp-iglABC(GGSG) E.coli-Francisellashuttlevector [34] forover-expressingIglABCwitha GGSGlinker(SEQIDNO:62)and downstreamofomppromoter pFNL/omp-iglABC(GGSG2) E.coli-Francisellashuttlevector [34] forover-expressingIglABCwith GGSG2linker(SEQIDNO:63) anddownstreamofomp promoter F.tularensissubsp.holarctica Strain Description Reference LVS Livevaccinestrain CDC LVScapB UnmarkedLVSwithdeletionof [10] capB rLVScapB/gro-iglA LVScapBcarryingpFNL/gro- [27] iglA rLVScapB/gro-iglC LVScapBcarryingpFNL/gro- [27] iglC rLVScapB/gro-iglABC LVScapBcarryingpFNL/gro- [34] iglABC rLVScapB/bfr-iglA LVScapBcarryingpFNL/bfr-iglA [34] rLVScapB/bfr-iglB LVScapBcarryingpFNL/bfr-iglB [34] rLVScapB/bfr-iglC LVScapBcarryingpFNL/bfr-iglC [34] rLVScapB/bfr-iglABC(D) LVScapBcarryingpFNL/bfr- [34] iglABC(D) rLVScapB/bfr-iglABC LVScapBcarryingpFNL/bfr- [34] iglABC(GGSG) rLVScapB/bfr- LVScapBcarryingpFNL/bfr-iglABC(GGSG2) [34] iglABC(GGSG2) rLVScapB/omp-iglA LVScapBcarryingpFNL/omp-iglA [34] rLVScapB/omp- LVScapBcarryingpFNL/omp-iglABC(D) [34] iglABC(D) rLVScapB/omp- LVScapBcarryingpFNL/omp-iglABC(GGSG) [34] iglABC(GGSG) rLVScapB/omp- LVScapBcarryingpFNL/omp-iglABC(GGSG2) [34] iglABC(GGSG2) AttenuatedrecombinantListeriamonocytogenes rLm/iglC LmactAintegratedwiththehly-iglCantigen [9] expressioncassetteatthetRNA.sup.arglocus rLmactA/hly- LmactAintegratedwiththehly-iglABCantigen [34] iglABC expressioncassetteatthetRNA.sup.arglocus rLmactA/actA- LmactAintegratedwiththeactA-iglABCantigen [34] iglABC expressioncassetteatthetRNA.sup.arglocus rLmactAinlB LmactAinlBuvrABprfA*(G155S)integrated [34] prfA*/hly-iglABC withthehly-iglABCantigenexpressioncassette atthetRNA.sup.arglocus rLmactAinlB LmactAinlBuvrABprfA*(G155S)integrated [34] prfA*/actA-iglABC withtheactA-iglABCantigenexpressioncassette atthetRNA.sup.arglocus Primers Description Sequence(5-3)* [34] SnaB_iglA33_F agatgctacgtaatgctagttgttggcgatttatca(SEQIDNO: [34] 25) iglA132_R attattttcaatgtccttagcaa(SEQIDNO:26) [34] iglA_Mfe_iglB446_F gacattgaaaataatcaattgcctttagaaatggcgagatatcctttc [34] (SEQIDNO:27) iglB506_R gttattatttgtaccgaataattc(SEQIDNO:28) [34] iglB_Age_iglC29_F ggtacaaataataacaccggtaattgtagattatttattgattcttta [34] (SEQIDNO:29) BamH_iglC149_R agatgcggatccctattatattggatataactctaaattaga(SEQ [34] IDNO:30) MfeI_GGSG_iglB446_F aagtaacaattgggtggttctggtcctttagaaatggcgagatatcctt [34] tc(SEQIDNO:31) AgeI_GGSG_iglB506_R aagtaaaccggttccagaaccaccgttattatttgtaccgaataattct [34] gg(SEQIDNO:32) MfeI_GGSG2_iglB446_F Aagtaacaattgggtggttctggtggtggttctggtcctttagaaatgg [34] cgagatatcctttc(SEQIDNO:33) AgeI_GGSG2_iglB506_R Aagtaaaccggttccagaaccaccaccagaaccaccgttattatttgta [34] ccgaataattctgg(SEQIDNO:34) *Restriction sites underlined in the primers.
TABLE-US-00006 TABLE 4 Plasmid stability in vivo (I) Percentage of Kanamycin resistance (# of KanR/# of total clones) on days post infection 1.sup.a) 4.sup.b) 7.sup.c) 14.sup.d) LVS 0 (0/15) 0 (0/15) 0 (0/15) 0 (0/24) LVS capB 0 (0/2) 0 (0/15) 0 (0/10) 0 (0/15) LVS 100 (46/46) 100 (30/30) 100 (30/30) 100 (36/36) capB/bfr-iglA LVS 100 (40/40) 100 (30/30) 93 (26/28) 71 (5/7) capB/bfr-iglB LVS 100 (48/48) 100 (30/30) 100 (30/30) 100 (26/26) capB/bfr-iglC .sup.a)Day 1 post infection: Colonies were patched from chocolate agars plated with lung lysates of a randomly chosen single animal. .sup.b)Day 4 post infection: colonies were from patched from chocolate agars plated with lung lysates of a randomly chosen single animals. .sup.c)Day 7 post infection: colonies were from patched from chocolate agars plated with lung and/or spleen lysates of a single randomly chosen animal. .sup.d)Day 14 post infection: colonies were from patched from chocolate agars plated with lung, spleen, liver, and lymph node lysates of 1-2 animals that still had colonies.
TABLE-US-00007 TABLE 5 Plasmid stability in vivo (II) Percentage of plasmid persistence [# of PCR (+) clones/# of Total clones tested] 1 4 7 14 LVS LVS capB LVS capB/bfr-iglA 100 (8/8) 100 (8/8) 100 (8/8) 100 (4/4) LVS capB/bfr-iglB 100 (8/8) 100 (8/8) 87.5 (7/8) 100 (4/4) LVS capB/bfr-iglC 100 (8/8) 100 (8/8) 100 (8/8) 100 (3/3)
TABLE-US-00008 TABLE 6 Efficacy of Heterologous Prime-Boost Vaccine Expressing IgIABC Mean Survival Vaccine Vaccine (week Challenge (week Time (Days) (week 0) 4) Boost (week 6) 10) (weeks 10-13) PBS PBS ID 29 CFU SchuS4 4.0 rLVS 29 CFU SchuS4 7.9 capB/bfr- iglABC rLVS rLm prfA* rLm prfA* 29 CFU SchuS4 6.4 capB/bfr- (vector) (vector) iglABC rLVS rLm prfA*/hly- 29 CFU SchuS4 7.6 capB/bfr- iglABC iglABC rLVS rLm prfA*/hly- rLm prfA*/hly- 29 CFU SchuS4 9.4 capB/bfr- iglABC iglABC iglABC rLVS rLm 29 CFU SchuS4 7.1 capB/bfr- prfA*/ActA- iglABC iglABC rLVS rLm rLm prfA*/ActA- 29 CFU SchuS4 9.4 capB/bfr- prfA*/ActA- iglABC iglABC iglABC
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TABLE-US-00009 SEQUENCES B.anthracispolypeptidesequences a.Anthraxprotectiveantigenprecursor AAA22637.1protectiveantigenprecursor[Bacillusanthracis] MKKRKVLIPLMALSTILVSSTGNLEVIQAEVKQENRLLNESESSSQGLLGYYFSDLNFQAPMVVTSSTTG DLSIPSSELENIPSENQYFQSAIWSGFIKVKKSDEYTFATSADNHVTMWVDDQEVINKASNSNKIRLEKG RLYQIKIQYQRENPTEKGLDFKLYWTDSQNKKEVISSDNLQLPELKQKSSNSRKKRSTSAGPTVPDRDND GIPDSLEVEGYTVDVKNKRTFLSPWISNIHEKKGLTKYKSSPEKWSTASDPYSDFEKVTGRIDKNVSPEA RHPLVAAYPIVHVDMENIILSKNEDQSTQNTDSETRTISKNTSTSRTHTSEVHGNAEVHASFFDIGGSVS AGESNSNSSTVAIDHSLSLAGERTWAETMGLNTADTARLNANIRYVNTGTAPIYNVLPTTSLVLGKNQTL ATIKAKENQLSQILAPNNYYPSKNLAPIALNAQDDFSSTPITMNYNQFLELEKTKQLRLDTDQVYGNIAT YNFENGRVRVDTGSNWSEVLPQIQETTARIIENGKDLNLVERRIAAVNPSDPLETTKPDMILKEALKIAF GFNEPNGNLQYQGKDITEFDFNFDQQTSQNIKNQLAELNATNIYTVLDKIKLNAKMNILIRDKRFHYDRN NIAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDRYDMLNISSLRQD GKTFIDFKKYNDKLPLYISNPNYKVNVYAVIKENTIINPSENGDISTNGIKKILIFSKKGYEIG(SEQ IDNO:6) b.Anthraxtoxinlethalfactorprecursor;lef,plasmidpXO1, ProteinID:NP_052803.1 NP_052803.1pXO1-107(plasmid)[Bacillusanthracis] MNIKKEFIKVISMSCLVTAITLSGPVFIPLVQGAGGHGDVGMHVKEKEKNKDENKRKDEERNKTQEEHLK EIMKHIVKIEVKGEEAVKKEAAEKLLEKVPSDVLEMYKAIGGKIYIVDGDITKHISLEALSEDKKKIKDI YGKDALLHEHYVYAKEGYEPVLVIQSSEDYVENTEKALNVYYEIGKILSRDILSKINQPYQKFLDVLNTI KNASDSDGQDLLFINQLKEHPIDFSVEFLEQNSNEVQEVFAKAFAYYIEPQHRDVLQLYAPEAFNYMDKF NEQEINLSLEELKDQRMLSRYEKWEKIKQHYQHWSDSLSEEGRGLLKKLQIPIEPKKDDIIHSLSQEEKE LLKRIQIDSSDFLSTEEKEFLKKLQIDIRDSLSEEEKELLNRIQVDSSNPLSEKEKEFLKKLKLDIQPYD INQRLQDTGGLIDSPSINLDVRKQYKRDIQNIDALLHQSIGSTLYNKIYLYENMNINNLTATLGADLVDS IDNIKINRGIFNEFKKNFKYSISSNYMIVDINERPALDNERLKWRIQLSPDTRAGYLENGKLILQRNIGL EIKDVQIIKQSEKEYIRIDAKVVPKSKIDTKIQEAQLNINQEWNKALGLPKYTKLITENVHNRYASNIVE SAYLILNEWKNNIQSDLIKKVINYLVDGNGRFVFIDITLPNIAEQYTHQDEIYEQVHSKGLYVPESRSIL LHGPSKGVELRNDSEGFIHEFGHAVDDYAGYLLDKNQSDLVINSKKFIDIFKEEGSNLTSYGRTNEAEFF AEAFRLMHSTDHAERLKVQKNAPKTFQFINDQIKFIINS(SEQIDNO:5) c.AhpC,AlkylhydroperoxidereductasesubunitC[Bacillusanthracis str.Ames] NP_842892.1alkylhydroperoxidereductasesubunitC[Bacillus anthracisstr.Ames] MLLIGTEVKPFKANAYHNGEFIQVIDESLKGKWSVVCFYPADFIFVCPTELEDLQNQYAILKELGVEVYS VSIDTHFTHKAWHDSSETIGKIEYIMIGDPIRTITTNFNVLMEEEGLAARGIFIIDPDGVIQSMEINADG IGRDASILVNKIKAAQYVRNNPGEVCPAKWQEGSATLKPSLDLVGKI(SEQIDNO:7) Yersiniapestispolypeptidesequences a.F1capsuleantigen NP_395430.1F1capsuleantigen(plasmid)[YersiniapestisCO92] MKKISSVIAIALFGTIATANAADLTASTTATATLVEPARITLTYKEGAPITIMDNGNIDTELLVGILTLG GYKTGITSTSVNFTDAAGDPMYLIFTSQDGNNHQFTTKVIGKDSRDFDISPKVNGENLVGDDVVLATGSQ DFFVRSIGSKGGKLAAGKYTDAVIVIVSNQ(SEQIDNO:8) b.Vantigen(LcrV)protein NP_395165.1secretedeffectorprotein(plasmid)[Yersiniapestis CO92] MIRAYEQNPQHFIEDLEKVRVEQLIGHGSSVLEELVQLVKDKNIDISIKYDPRKDSEVFANRVITDDIEL LKKILAYFLPEDAILKGGHYDNQLQNGIKRVKEFLESSPNTQWELRAFMAVMHFSLTADRIDDDILKVIV DSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDKSINLMDKNLYGYTDEEIFKAS AEYKILEKMPQTTIQVDGSEKKIVSIKDFLGSENKRTGALGNLKNSYSYNKDNNELSHFATTCSDKSRPL NDLVSQKTTQLSDITSRFNSAIEALNRFIQKYDSVMQRLLDDTSGK(SEQIDNO:9) c.YscF NP_395189.1needlecomplexmajorsubunit(plasmid)[Yersiniapestis CO92] MSNFSGETKGTDIADLDAVAQTLKKPADDANKAVNDSIAALKDKPDNPALLADLQHSINKWSVIYNINST IVRSMKDLMQGILQKFP(SEQIDNO:10) d.YopE AJJ86307.1outermembranevirulenceproteinyopE(plasmid)[Yersinia pestisCO92] MKISSFISTSLPLPTSVSGSSSVGEMSGRSVSQQTSDQYANNLAGRTESPQGSSLASRIIERLSSVAHSV IGFIQRMFSEGSHKPVVTPAPTPAQMPSPTSFSDSIKQLAAETLPKYMQQLNSLDAEMLQKNHDQFATGS GPLRGSITQCQGLMQFCGGELQAEASAILNTPVCGIPFSQWGTIGGAASAYVASGVDLTQAANEIKGLAQ QMQKLLSLM(SEQIDNO:11) e.Ybt/pesticinreceptorPsn[Yersiniapestis] AAC69592.1Ybt/pesticinreceptorPsn[Yersiniapestis] MKMTRLYPLALGGLLLPAIANAQTSQQDESTLVVTASKQSSRSASANNVSSTVVSAPELSDAGVTASDKL PRVLPGLNIENSGNMLFSTISLRGVSSAQDFYNPAVTLYVDGVPQLSTNTIQALTDVQSVELLRGPQGTL YGKSAQGGIINIVTQQPDSTPRGYIEGGVSSRDSYRSKFNLSGPIQDGLLYGSVTLLRQVDDGDMINPAT GSDDLGGTRASIGNVKLRLAPDDQPWEMGFAASRECTRATQDAYVGWNDIKGRKLSISDGSPDPYMRRCT DSQTLSGKYTTDDWVFNLISAWQQQHYSRTFPSGSLIVNMPQRWNQDVQELRAATLGDARTVDMVFGLYR QNTREKLNSAYDMPTMPYLSSTGYTTAETLAAYSDLTWHLTDRFDIGGGVRFSHDKSSTQYHGSMLGNPF GDQGKSNDDQVLGQLSAGYMLTDDWRVYTRVAQGYKPSGYNIVPTAGLDAKPFVAEKSINYELGTRYETA DVTLQAATFYTHTKDMQLYSGPVRMQTLSNAGKADATGVELEAKWRFAPGWSWDINGNVIRSEFTNDSEL YHGNRVPFVPRYGAGSSVNGVIDTRYGALMPRLAVNLVGPHYFDGDNQLRQGTYATLDSSLGWQATERMN ISVYVDNLFDRRYRTYGYMNGSSAVAQVNMGRTVGINTRIDFF(SEQIDNO:12) F.tularensispolypeptidesequences a.IglA(intracellulargrowthlocus,subunitA)[Francisella tularensissubsp.tularensisSCHUS4] CAG45992.1intracellulargrowthlocus,subunitA[Francisella tularensissubsp.tularensisSCHUS4] MLIRCCEKKDNKMAKNKIPNSRLMINYETNVDGVLKKKELPYRVLVVGDLSKGRSVDAKKEFADREVRRV NNGVDRVLEEMNISFDFEAPNFVSKDRSNLKVNYRIESVKDFRPDAVAKKVPEIRALLEMKEILASFAKD IENNRNLKKTIDMIFSDSNELESLKSKIPALTNYTIKDSCDAAESQDLSNQQVDGK(SEQIDNO: 2) b.IglB(intracellulargrowthlocus,subunitB)[Francisella tularensissubsp.tularensisSCHUS4] CAG45991.1intracellulargrowthlocus,subunitB[Francisella tularensissubsp.tularensisSCHUS4] MVSREDFIMTINKLSLIDELLNNFGGSTEVDSVLKNIDFDVSDDASKVLSLSIDYNARNLMALSLVLANN DNINNYNQKYIQKVITVIDKLIDLQVNSIISNDEFRALEQEWLKVQEVCQEDYDNVEVSILDVKKEELQY DFERNLYDISSSDFFKKVYVSEFDQYGGEPYGAILGLYNFENTINDIIWLIGMGMVAKNSHAPFIASIDK SFFGVKDLSEITHIKSFEALLEHPRYKEWNDFRNLDVAAYIGLTVGDFMLRQPYNPENNPVQYKLMEGFN EFVDYDKNESYLWGPASIHLVKNMMRSYDKTRWFQYIRGVESGGYVKNLVACVYDNKGILETKSPLNVLF ADYMELSLANIGLIPFVSEKGISNACFFSVNSAKKVEEFVDGFDSANSRLIANLSYTMCISRISHYIKCV IRDKIGSIVDVESIQKILSDWISEFVTIVYQPIPLEMARYPFRNVSIEVETIPGKPGWYSCKINVIPHIQ FEGMNITMTIDIRLEPELFGINNN(SEQIDNO:3) c.IglC(intracellulargrowthlocus,subunitC)[Francisella tularensissubsp.tularensisSCHUS4] CAG45990.1intracellulargrowthlocus,subunitC[Francisella tularensissubsp.tularensisSCHUS4] MIMSEMITRQQVISGETIHVRTDPTACIGSHPNCRLFIDSLTIAGEKLDKNIVAIDGGEDVIKADSATAA ASVIRLSITPGSINPTISITLGVLIKSNVRTKIEEKVSSILQASATDMKIKLGNSNKKQEYKTDEAWGIM IDLSNLELYPISAKAFSISIEPTELMGVSKDGMRYHIISIDGLITSQGSLPVCCAASTDKGVAKIGYIAA A(SEQIDNO:4) d.VgrG,conservedhypotheticalprotein[Francisellatularensis subsp.tularensisSCHUS4] CAG45980.1conservedhypotheticalprotein[Francisellatularensis subsp.tularensisSCHUS4] MSKADHIFNLEEQGLLIDIKDDSKGCTTKLESSGKITHNATESIESSADKQIIENVKDSKISITEKEIL LATKKSSIMLSEDKIVIKIGNSLIILDDSNISLESATINIKSSANINIQASQNIDIKSLNNSIKADVNL NAEGLDVNIKGSVTASIKGSTATMVG(SEQIDNO:15) e.capsulebiosynthesisproteinCapB[Francisellatularensis] MTTLDFWLIVVVFVILCVYLIIENIVHNNSIKSIPIRIHNGTRGKSSVARLIAAGVRAGGYRTVAKTTG TLARYIDVDGSETPVFRIGSNIAEQVKIMFKARRAKADAIVIECMALQPLLQSLCELKLIKATHGVLTN ARPDHLDVMGPTERDVAKALAATVPVGAKYFTAEDIHLDFFEYACKDRGELIAATAQDAEKISDEEINK FVYSEFKINVALALKVIDDLGIPREIALKGMWEATPDPGAMTEYNFNIKAEINFANAFAANDPVSTKML WDKLCAKYSGCDKKVLVVNCRDDREDRSKQMAEAALGWQKQDLIVLIGTGTEVFTSFYKYAKSLNKPMT KVIVCEEMTPIQILEKTVDSNPANSYILVGVGNIKDIGMELVDYCDTSHKKKHNL(SEQIDNO: 1) Francisellatularensispromotersequences a.PromoterforFrancisellatularensissubsp.holarcticaFTL_0617 (bacterioferritin,bfr) CP009694.1:1332650-1332938Francisellatularensissubsp.holarctica LVS,completegenome TTGTTACCTCCATTATTTAAAACTCTAATCATTATAATCAACATACTAACTAGTATAATTCATATTAGCA ATCAAGTTAGCATACAAAAGAAAATTTAATTCTTTATAAGTTATCAATTTAGTATCTAATTATATATCAA AATATCTAAGAATTCAACCTAGATATTTTAATAAAAATGATATTATGCTATTTTTAGATAAGTTAAATTT ACTATTTTTAATAATAATATTTAAGAAAAATAAATGAAGAAAATTAATTTTAATATTGTGATGATGGCAA TAGTAACCA(SEQIDNO:13) b.PromoterforFrancisellatularensissubsp.novicidaFTN_1451 (omp26,omp) CP009633.1:589316-589474Francisellatularensissubsp.novicidaU112, completegenome TTTGGGTTGTCACTCATCGTATTTGGTTTATAATTTTAAGCTAATAACCTAATTATAACTAATTAATAGT TTTGTATCTTGAAAAAATAGCTATAAAACTTATTTAAATAACGAAGATTTTTGTGTATAAAATATTTATA ACAAAAAAAGGAGACTAAA(SEQIDNO:14) Burkholderiapseudomalleipolypeptidesequences Hcp-1 typeVIsecretionsystem[Burkholderiapseudomallei] GenBank:CRY35672.1 >CRY35672.1typeVIsecretionsystem[Burkholderiapseudomallei] MLHMHLKFGSPAIKGESADKDHEGWIELKSWDHSIVQPRSATASTAGGHTATRCEHGDMVFTKEIDSSSP LLYQHASGGTTFDEVTIDFLRADGEGQRVKYLEIKLKYVIISSIAPSVHTEGLPVETFSLKYAAVQWKQT QQKIGGNQGGNTQGAWSLTKNDKTYAV(SEQIDNO:35) Hcp-2(T6SS) typeVIsecretionsystem[Burkholderiapseudomallei] GenBank:CPN31289.1 >CPN31289.1typeVIsecretionsystem[Burkholderiapseudomallei] MSHDIFLKINGIDGEAEDATHKGEIEVLSWSWNVSQQSNMHLGSGGGAGKATIDDLQFEHYIDRASPNLV QYCLLGKHIDEARLVVRKAGGSPLEYIKLTMSDVLVTQVSPAGVAQDESRPRELVRLSFSRLKQEYVVQN PQGGSGGAITATFDIKKNAA(SEQIDNO:36) Hcp-3(T6SS) typeVIsecretionsystem[Burkholderiapseudomallei] GenBank:CRY29196.1 >CRY29196.1typeVIsecretionsystem[Burkholderiapseudomallei] MAQDIFLKIDGINGESLDDSHKDEIEVLNWNWEIQQESTMHIGSGGGAGKASVKDLIFEHAIDRASPNLM KYALIGKHVDQAVLVMRKAGGNPLEYLKLIMSDVIITRVRPSGSRDDTERSRETVSLSFAKVKQEYVVQN AQGGSGGAVITSFDIKGNKEA(SEQIDNO:37) Hcp-4(T6SS) typeVIsecretionsystem[Burkholderiapseudomallei] GenBank:CRY33495.1 >CRY33495.1typeVIsecretionsystem[Burkholderiapseudomallei] MANALVDYFLQIDGVEGESTDQQYPGLIQIQSWQWAEENSGRWGFGSGGGAGKVEMKDFEFRMVSNKASP KLFLMCATGEHIQNAKLICRKSGKGQQEFLTISFASGLVSSFRILGNMPISQLGHASGEVDGVLPTDQIR INFAQIEFEYREQRNDGTMGAVIKAGYDLKQNAPI(SEQIDNO:38) Hcp-6(T6SS) typeVIsecretionsystem[Burkholderiapseudomallei] GenBank:CRY19699.1 >CRY19699.1typeVIsecretionsystem[Burkholderiapseudomallei] MGVAMFMKVDGVTGESADAQHKGWIDIQSFSWGASQPGAMASGSGGNAGKASFNDLVVAAYMDKGATAII KNCASGKHLPTVEISACKTGGSQIEFMRVILQEVLVISAQIAGVDPGDAADRLMMQYGFQAAKVKKQYWQ QNDNGGKGAEVSVGWNIKENTEM(SEQIDNO:39) LolC(ATPbindingcassettesystem) lipoprotein-releasingsystemtransmembranesubunitLolC[Burkholderia pseudomallei] NCBIReferenceSequence:WP_050865936.1 >WP_050865936.1lipoprotein-releasingsystemtransmembranesubunit LolC[Burkholderiapseudomallei] MKLPYEWQIGWRYTRAGKRAIGNGFISFIALVSMLGIALGVAALIVVLSVMNGFQKEVRDRMLSVLAHVE IFSPIGSMPDWQLTAKEARLNRSVIGAAPYVDAQALLTRQDAVSGVMLRGVEPSLEPQVSDIGKDMKAGA LTALAPGQFGIVLGNALAGNLGVGVGDKVILVAPEGTITPAGMMPRLKQFTVVGIFESGHYEYDSTLAMI DIQDAQALFRLPAPTGVRLRLTDMQKAPQVARELAHTLSGDLYIRDWIQQNKTWFSAVQIEKRMMFIILT LIIAVAAFNLVSSLVMTVINKQADIAILRILGAQPGSIMKIFVVQGVTIGFVGTATGVALGCLIAWSIPW LIPMIEHAFGVQFLPPSVYFISELPSELVAGDVIKIGVIAFALSALATLYPSWRGAKVRPAEALRYE (SEQIDNO:40) TypA GTP-bindingproteinTypA[Burkholderiapseudomallei] GenBank:CRY12842.1 >CRY12842.1GTP-bindingproteinTypA[Burkholderiapseudomallei] MTRALRNIAIIAHVDHGKITLVDQLLRQSGTFRENQQVAERVMDSNDIEKERGITILAKNCAVEYEGTHI NIVDTPGHADFGGEVERVLSMVDSVLLLVDAVEGPMPQTREVIKKALALGLKPIVVINKIDRPGARIDWV INQTFDLFDKLGATEEQLDFPIVYASGLNGYASLDPAARDGDMRPLFEAILQHVPVRPADPDAPLQLQIT SLDYSTYVGRIGVGRITRGRIKPGQPVVMRFGPEGDVLNRKINQVLSFQGLERVQVDSAEAGDIVLINGI EDVGIGATICAVEAPEALPMITVDEPTLIMNFLVNSSPLAGREGKEVISRQIRDRLMKELNHNVALRVKD TGDETVFEVSGRGELHLTILVENMRREGYELAVSRPRVVMQEIDGVKHEPYELLTVDLEDEHQGGVMEEL GRRKGEMLDMVSDGRGRIRLEYRIPARGLIGFQSEFLILTRGTGLMSHIFDSYAPVKEGSVGERRNGVLI SQDDGAAVAYALWKLQDRGRMFVKPGDALYEGMIIGIHSRDNDLVVNPIKGKQLINVRASGTDEAVRLVP PIQMSLEYAVEFIDDDELVEVTPQSIRLRKRHLKEHERRRASREAEAG(SEQIDNO:41) BipB BipB[Burkholderiapseudomallei] GenBank:AB026356.1 >AB026356.1BipB[Burkholderiapseudomallei] MSSGVQGGPAAHANAYQTHPLRDAASALGTLSPQAYVDVVSAAQRNFLERMSQLASEQCDAQPAAHDARL DDKPALRAPQERDAPPLGASDTGSRASGAAKLTELLGVLMSVISASSLDELKQRSDIWNQMSKAAQDNLS RLSDAFQRATDEAKAAADAAEQAAAAAKQAGADAKAADAAVDAAQKRYDDAVKQGLPDDRLQSLKAALEQ ARQQAGDAHGRADALQADATKKLDAASALATQARACEQQVDDAVNQATQQYGASASLRTPQSPRLSGAAE LTAVLGKLQELISSGNVKELESKQKLFTEMQAKREAELQKKSDEYQAQVKKAEEMQKTMGCIGKIVGWVI TAVSFAAAAFTGGASLALAAVGLALAVGDEISRATTGVSFMDKLMQPVMDAILKPLMEMISSLITKALVA CGVDQQKAELAGAILGAVVTGVALVAAAFVGASAVKAVASKVIDAMAGQLTKLMDSAIGKMLVQLIEKFS EKSGLQALGSRTATAMTRMRRAIGVEAKEDGMLLANRFEKAGTVMNVGNQVSQAAGGIVVGVERAKAMGL LADVKEAMYDIKLLGDLLKQAVDAFAEHNRVLAQLMQQMSDAGEMQTSTGKLILRNARAV(SEQID NO:42) BipC BipC[Burkholderiapseudomallei] GenBank:AB026357.1 >AB026357.1BipC[Burkholderiapseudomallei] MSIGVQSSGINISHAELSRLVDAGKSEQGDKAVRDDGRALARADAALAAVVGERVAARRDAVAGSGAQRV ELARPKPDAQTRATDRRTVSGLEREHKRLAASQTPRVTGMHDALVQRHVSLDGAKAAHGEGVKRAAGDAP RAAADAPQRFAFADDKAFDAMLALGAAMQKNVQSDLAMQGKLTMLAHDAMMSAAAQDRSIGAAQMTAAIA GGALQATTSLGGAMQQMKSLSTKSMSIEKELKPQAELKQFHAEQALELRGINKPVLSNDEVSHVKIKRDT GETVRHEIDHGGERMSDEHASVLAQEAPARQHRIDMHGMRHEENLVKASRQQMKGDLLQSGGQIGKNQID GASAQQQGADRAEQKEDENAQQTAMAAASTRDEAAHRSREAAQKAIDAAKSQVANDNAVAAQVAGNLRT (SEQIDNO:43) BipD BipD[Burkholderiapseudomallei] GenBank:ABL67521.1 >ABL67521.1BipD[Burkholderiapseudomallei] MNMHVDMGRALTVRDWPALEALAKTMPADAGAREMTDDDLRAAGVDRRVPEQKLGAAIDEFASLRLPDRI DGRFVDGRRANLTVFDDARVAVRGHARAQRNLLERLETELLGGTLDTAGDEGGIQPDPILQGLVDVIGQG KSDIDAYATIVEGLTKYFQSVADVMSKLQDYISAKDDKNMKIDGGKIKALIQQVIDHLPTMQLPKGADIA RWRKELGDAVSISDSGVVTINPDKLIKMRDSLPPDGTVWDTARYQAWNTAFSGQKDNIQNDVQTLVEKYS HQNSNFDNLVKMLSGAISTLTDAAKSYLQI(SEQIDNO:44) Omp3 OmpAfamilyprotein[Burkholderiapseudomallei] GenBank:ACN64870.1 >ACN64870.1OmpAfamilyprotein[Burkholderiapseudomallei] MNKLSKLAFIAATAVMAASASAQSVPASRQAVNDNWVNGTGEWVWMNGTNELCWRDAFWTPATANAKCDG ALVAQAPAPAPVAPVAPAITSQKITYQADTLFDFDKAVLKPAGKQKLDELAAKIQGMNVEVVVATGYTDR IGSDKYNDRLSLRRAQAVKSYLVSKGVPANKVYTEGKGKRNPVTGNTCKQKNRKQLIACLAPDRRVEVEV VGTQEVQKTTVPAQ(SEQIDNO:45) Omp7 OmpAfamilylipoprotein[Burkholderiapseudomallei] GenBank:ACN64871.1 >ACN64871.1OmpAfamilylipoprotein[Burkholderiapseudomallei] MTTRRVTMMSKKLRLAFAMLMIGALAACKSGVKLDEHANQGDAVSTQPNPENVAQVTVDPLNDPNSPLAK RSVYFDFDSYSVQDQYQALLQQHAQYLKSHPQRHILIQGNTDERGTSEYNLALGQKRAEAVRRALSLLGV GDAQMEAVSLGKEKPVALGHDEASWAQNRRADLVYQQ(SEQIDNO:46) Omp85 putativeoutermembraneprotein[BurkholderiapseudomalleiK96243] GenBank:CAH36153.1 >CAH36153.1putativeoutermembraneprotein[Burkholderia pseudomalleiK96243] MLFKPHREVPKTVAAAALAAHGLAAHATAPFVVQDIKIEGLQRVEAGSVFAYLPIKQGDTFTDDKASEAI RALYATGFFNDVRIATQGGVVIVQVQERPAIASIDFTGIKEFDKDNLNKALKAVGLSQGRYYDKALVDKA EQELKRQYLTRGFYAAEVSTTVTPVDANRVSILFAVAEGPSAKIRQINFIGNKAFKTSTLRDEMQLSTPN WFSWYTKNDLYSKEKLTGDLENVRSYYLNRGYLEFNIESTQVSISPDKKDMYLTVALHEGEPYTVSSVKL AGNLLDRQAELEKLVKIKPGDRFSAEKLQQTTKAIVDKLGQYGYAFATVNAQPEIDQATHKVGLTLVVDP SRRVYVRRINIVGNTRTRDEVVRREMRQLESSWFDSSRLALSKDRVNRLGYFTDVDVTTVPVEGTNDQVD VNVKVAEKPTGAITLGAGESSTDKVVLSAGISQDNVEGSGTSLAVNVNTAKSYRTLTVTQVDPYFTVDGI KRITDVEYRTYQPLYYSTNSSFRIITAGGNLKEGIPFSETDTVYFGAGFEQNRLDVDSNTPQSYQDYVNE FGRVSNTVPLTIGWSRDARDSALIPSRGYFTQANAEYGVPVGKIQYYKMDVQGQYYYSFARGFILGLNFQ AGYGNGIGNPYPIFKNYYAGGIGSVRGYEPSSLGPRDTKTNDPIGGSKMVVGNIELTFPLPGTGYDRTLR VFTFLDGGNVWGNAPGGTSTGANGLRYGYGIGLAWISPIGPLKLSLGFPLQKHEGDQYQKFQFQIGTAF (SEQIDNO:47) OmpW outermembraneproteinW[Burkholderiapseudomallei] GenBank:A1173329.1 >ALJ73329.1outermembraneproteinW[Burkholderiapseudomallei] MGDKHRRRRARRDWRAPVSCWLGATLLACAWSAHAQDSGAARWRDGADGIGFFPGGDAPGFDARAWGPVP GDARRAAASDARNGVAASAGSEATAAAPAADANAAPARKLTEERITLGERVAPVADAARRVRADGDDGIG FADAPGGPPAGGATPSAACDDGACVPDGGDAGRAPRRPPAGATPRFIAGVRYDRMPYELHPIDPERLPDL PEAQGPTLLEQLQGDDSNMIGVGWHYVLSTGRSTPVTTSTAALGIGSFANPGSAVSISNTNTPAFTFTHF FGEHVAAEIVAGIPPELTMRGHGSIGLPFDKIFPGVQGRLPLVDLGNTQSNPLGTTRAWLGSAVFKYYLG KREDRLRPYVGLGLSYTRFTNTNLNPVFAHKLASLGGLLSAGISLGDLQSLLTDSGALDRLLQAGANLIL PNGVRATADVKSAWTPVFVVGANYQLTRQLSLSTALSYIPLKAAITVNINDTKGILASNTTTLSANVLLC TMLLNFRF(SEQIDNO:48) PotF spermidine/putrescineABCtransportersubstrate-bindingproteinPotF[Burkholderia pseudomallei] GenBank:ANW57327.1 >ANW57327.1spermidine/putrescineABCtransportersubstrate-binding proteinPotF[Burkholderiapseudomallei] MKRIAWLAAVLASLACAAARAAGGNVLNIYNWAEYFAPDTIAGFEKETGIKVRLDVYDSNEALQTKLTTG NSGYDLVFPSNDFLARQIQAGLYRKLDKSRLPNLTNLDPAIVARAAEVDPGNQYSVPYMQGTFGLGLNVA KVKQALGGPLPANTLELIFNPAYAAKLERCGIAFNDAGSEVFPLALRYIGRDPNTTDPRDYEAALDMMKK IRPTIRQFIATPVMNDLATGDVCVVTGYSGAVLVAARRAAEAKNGQQIVYSLPSAGAPFWFDSMAIPKGA AHADHALRFIDYILRPDVVAKISNKVMYPNPNRVATPLVDRRLTANPAIYPDAATMRTLWVKRPMPPQAM RMQTRYWTRFKTGY(SEQIDNO:49) OppA oligopeptideABCtransporterperiplasmicoligopeptide-bindingprotein[Burkholderia pseudomallei] GenBank:CRY20041.1 >CRY20041.1oligopeptideABCtransporterperiplasmicoligopeptide- bindingprotein[Burkholderiapseudomallei] MKHTHAFAAVLAALALTIAPSAPAVTVASNVTLADQQDLTRQVPAEVESLDPAHIESWTGNTIGLDLFEG LARIDASGAVVPGVAQAWEHKAPDTWIFKLRRDAKWSNGQPVTAADFVYAWQRLADPKTGSKYTILVEFV KNASAIIAGKQPPGDLGIRAIDPYTIEVKTEVPVSYFPELTAMAPLTPVNKDAVAKFGDAWTRPKNIVSN GPYTLVDWQPNNRIVMAKSDKYWNARNVVIRKVTYLPIENDETALRMYQAGQIDYTYSIPAGGFGQISKQ FGKELRPGLQLATYYYYLKNSDPALKDKRVREALAMVLDREILTSKITQAGEVPMYGLMPKGVKGVQRPF TPDWASWPMARRVDYAKNLLKQAGHGDANPLTFTLTYNTNDLHKKVALFAASEWRTKLGVTAKLENVEFK VLMKQRHDGKVQVARDGWFADYNDAMTFFDLIRCGSSQNTVGYCNPKVDSLVAEANQKLDDGARAALLTQ AHDLAMNDYPMVPLFQYSADRLVKSYVGGYTLTNYIDMRASQDMYLIKH(SEQIDNO:50) BopA(TypeIIIEffector) BopAprotein[Burkholderiapseudomallei] GenBank:CRY24966.1 >CRY24966.1BopAprotein[Burkholderiapseudomallei] MINVGAFVASARSGARVVVGGDARGPVVSAARLGMKERLFAFLAHVPLLKHCDAVRRYAEQVRMENRRSL EVFVLALSKRYGPEGAKAAFDYGARRDGAPLDQRRVRNMVSIAEHFHGTGDAKPLARQMVERSWECRGLD HPGHASLTIKNQADADAGRHVYEHVSWWPNQRLGSKEHFDRIEPKTLDGYRIDKRSEISSATEQRLREGD AARRKILADGFKYANQDERHDARFFPRAGQKLDKDAEWGLSARKVYFPAIGFNHDRRDTDRPRAFVLFGL NEAAMLRDARTVKEGAKSGELKYRMISKKENCASMALRVLRAGGAEHFVPYTAAWISEDPNHAHAYALAV QARIDALNQRRADVERRCERLRDSASVRQAWRAFSEAGGASASPLAEDAGRGRASAHMRQARLDEHAREV ERIGAYFAELSAGRSGKHRDRADAALADAMKRCAPSARDDVAALTRKASVLVETLGRHLDAPPPSDSSAL RRLAAHAMIGRIEAFMAAAIAA(SEQIDNO:51) BimA(Autotransporterprotein) BimA[Burkholderiapseudomallei] GenBank:ACF94992.1 >ACF94992.1BimA[Burkholderiapseudomallei] MHAKASSSHAPDAPKPSSIATTLCRALASLSLGLSMDAEANPPEPPGGTNIPVPPPMPGGGANIPVPPPM PGGGANIPPPPPPPGGIGGATPSPPPLTPVNGNPGASTPTKTGLLKTLNRLSAELQNNPRVTEDVVDNVD AVIRNAVNLAPDANGDFSGRSAMPIEMAANAALRSLKKNPGDAGHAAPAYLPAERIGQLREKVRRTIEAL ESNRPPKPQPRSTPPQSTPPKPTQHPTAPNPNVPDASTPDASTPDASTPDASTPDASTPSRPAPAPRAGT GAPAASAATRAPAFANRVRKPNPAMPAASSHAIASDFASSNAFAIGDDSTAVGAQATAFSEQSIAIGSRA IAAGARSIAVGTDATAAAPDSVALGSGSIAEREGTVSVGRDGHERQITHVASGTEPTDAVNVTQLRAAMS NANAYTNQRIGDLQQSITDTARDAYSGVAAATALTMIPDVDRDKRVSIGVGGAVYKGHRAVALGGTARIN ENLKVRAGVAMSAGGNAVGIGMSWQW(SEQIDNO:52) BPSL1897 hypotheticalproteinBP[BurkholderiapseudomalleiK96243] NCBIReferenceSequence:YP_108497.1 >YP_108497.1hypotheticalproteinBPSL1897[Burkholderiapseudomallei K96243] MTRALGRLRSPRRQRGATAIEFAILFPMFFLILYGIITYGMIFAAQQSLTLAATEGARAALNYQVAQTQS AALGLRAAAACTAANNLTGWLSGATCTTSTNYTCSYDSTMYCIQVTLTYPYAANPLVPAVALFDAVLPTT LTSRATVQINPTNII(SEQIDNO:53) BPSL3369 acetaldehydedehydrogenase[BurkholderiapseudomalleiK96243] GenBank:CAH37382.1 >CAH37382.1acetaldehydedehydrogenase[Burkholderiapseudomallei K96243] MNHADMQHLNIEFPYRKQYGNFIGGEWVAPVGGEYFDNVSPVTGRPFTAIPRSREADIELALDAAHAAKA GWAAKGAAERANVLLRIADRMEANLTRLAVAETIDNGKPLRETTAADVPLAIDHFRYFAGCIRAQEGSIA DIGGDMVAYHFHEPLGVVGQIIPWNFPLLMAAWKLAPALAAGNCVVLKPAEQTPASILVFAELIQDLLPP GVLNIVNGFGLEAGKPLASSKRIAKIAFTGETSTGRLIMQYASENLIPVTLELGGKSPNIFFADVMDRDD SYFDKALEGFAMFALNQGEVCTCPSRALVEESIYDRFIERALKRVEAIKQGHPLDSQTMIGAQASAEQLE KILSYIDIGRGEGAQCLTGGERNVLGGELAEGYYVKPTVFRGHNKMRIFQEEIFGPVLAVTTFKTEEEAL EIANDTLYGLGAGVWTRDGNRAYRFGRGIQAGRVWTNCYHAYPAHAAFGGYKQSGIGRETHKMMLDHYQQ TKNLLVSYSEKPLGFF(SEQIDNO:54) BPSL2287 HesBfamilyprotein[BurkholderiapseudomalleiK96243] GenBank:CAH36290.1 >CAH36290.1HesBfamilyprotein[BurkholderiapseudomalleiK96243] MAITLTEKAAQHVQKYLARRGKGLGLRLGVRTTGCSGLAYKLEYVDELTPEDQMFESHGVKVFVDPKSLA YIDGTELDFAREGLNEGFKFNNPNVKDECGCGESFRV(SEQIDNO:55) BPSL2765 putativeOmpAfamilylipoprotein[BurkholderiapseudomalleiK96243] GenBank:CAH36773.1 >CAH36773.1putativeOmpAfamilylipoprotein[Burkholderia pseudomalleiK96243] MMSKKLRLAFAMLMIGALAACKSGVKLDEHANQGDAVSTQPNPENVAQVTVDPLNDPNSPLAKRSVYFDF DSYSVQDQYQALLQQHAQYLKSHPQRHILIQGNTDERGTSEYNLALGQKRAEAVRRALSLLGVGDAQMEA VSLGKEKPVALGHDEASWAQNRRADLVYQQ(SEQIDNO:56) VgrG5 RhselementVgrprotein[Burkholderiapseudomallei1710b] GenBank:ABA53677.1 >ABA53677.1RhselementVgrprotein[Burkholderiapseudomallei1710b] MRLIELRSPLLDPDAVALSFVVHENLSQEPSYQLDLLSHDSNLDFDALLGSTLSADIDLGEGDIRTENTH VFGGYDTGQMSGQYTYTLELRSWLSFLAENRNSRIFQDLSVPQIVEQVFQGHQRNGYRFELEGTYEPREY CVQFQETDLNFVKRLLEDEGIYFWVEHEPDRHVVVISDTQRFEDLPLPNDTLEYLPDGEESRAIQGREGV QRLQRTRRIKSNNVALRDFDYHAPSKQLDSDAQVEQQSLGGIPLEYYDYAAGYRDPEQGERLARLRLEAI QADAHALGGEANARALAVGRAFTLVGHPALSRNRRYYVTNSELTFIQDGPDSTSQGRNVAVKFRALADDQ PFRPLLVTKRPRVPGIQSATVVGPEMSEVHTDKLGRIRVHFHWDRYKTTEADASCWIRVTQAWAGKGWGV LAMPRVGQEVIVVYVDGDLDRPLATGIVYNGENPTPYDLPKDIRYTGLVTRSIKRAGGIPNASQLTFDDQ HGAERVMIHAERDLQQTVERNSSTSIAQDLNLSVKGTSTSVVGISVSFTGISVSYTGLSVSFTGVSARFT GVSTSFTGVSTSFTGVSTSFTGVDTSFTGVSTGFKGVDTSFTGVATSMVGVSTSITGSSNSVTGVSNSMT GISSSWKDVSMSTTGQSESITGVSLSYTGTSNSMTGTSTSVTGTSTSITGTSMSNTGSSTSITGTSMSTT GSSVSTTGSSMSATGSSVGTTGSSVSTTGSKMSVTGFSFSYTGASYEDVGVDLKKLGMQTKN (SEQIDNO:57) Burkholderiamalleipolypeptidesequences BMA_A0768mannitoldehydrogenasefamilyprotein mannitoldehydrogenasefamilyprotein[BurkholderiamalleiATCC23344] GenBank:AAU46944.1 >AAU46944.1mannitoldehydrogenasefamilyprotein[Burkholderia malleiATCC23344] MPLLSSDHCRALPPEVSRPRYDRRALRTGIVHLGLGAFHRAHQACYTETLVERGDLRWGIAGVELRRRHT VERLAAQDHLYSVTERAGDAARTRVVGAVHRTLFAPQALATLLGLIADPSVSIVSLTVTEKGYYRRPGGG GLDLDDPAIRRDLAQPHAPSTTLGVLAAGIRLRAAHAPLSVLSCDNMPSNGDTLRALLAQYAEQTDGALA RRIRCDVAFPNTMVDRIVPAATPESLDWVQSRIGVRDEAAIVCEPFAQWVFEDRFAGARPRWEDAGALVA ADVRPYEKMKLRLLNGSHSAIAYAGQLRGRRTVSDAMADPLIDALARGVMTRELLATLDVPAGYDVRAYC ASLIERFRNPALAHRTAQIATDGTQKVPLRWLPALAESAAAGVERPFLERSLAMWLHYVEVARDESGRPL VLEDPGAQALAARLHGAPGATDAVRAALGLIASRDAARWPEALTARVGAHLETVRTRGTDALLRPLLDA (SEQIDNO:58) BMA_2821ABCTransporterATPbindingprotein glutathioneABCtransporterATP-bindingprotein[Burkholderiamallei] GenBank:KKM47108.1 >KKM47108.1glutathioneABCtransporterATP-bindingprotein [Burkholderiamallei] MSASRAAPSLPDARVLAVDGLTVTFRREDAAFVAVRDLSFHVDRGETLAIVGESGSGKSVTSLALMRLVE HGGGAIAGGAIALRRRGGAVLDLARATPSTLRTVRGADVAMIFQEPMTSLNPVETVGDQISEAIALHQHK SAGEARAETLRLLDLVRIPEARRVFARHPHQLSGGMRQRVMIAMALSCRPALLIADEPTTALDVTIQAQI LQLIRGLQDEMDMGVIFITHDMGVVAEVADRVLVMYRGEKVEEGACDAIFAAPSHPYTKALLAAVPRLGS MRGIDAPAKFPLLRFDPAAGDALVVAGGDATAASGDAARESVLFVDSDAAAASAASTASTASAASAASAA PTACARPAIDAGAPPLLRVRELVIRFPVKSGVFGRVSQYVHAVERVSFELRAGETLALVGESGCGKSITG RSLLRLVERVSGSIEFEGREIGALKGRELQALRRNIQFIFQDPFASLNPRLTVGFSIMEPLLVHGVASGR QAQARVDWLLERVGLPADAARRYPHEFSGGQRQRIAIARALALNPKVVVADESVSALDVSVQAQIVNLML DLQRELGVAYLFISHDMAVVERISHRVAVMYLGQIVEIGPRRAVFETPRHPYIKKLMSAVPIADPACRHA PRTLPADELPSPIRALGDEPEVAPLVAVGPAHFVAEHRVGGAY(SEQIDNO:59) BMA_0816maltooligosyltrehalosesynthase maltooligosyltrehalosesynthase,putative[BurkholderiamalleiATCC23344] GenBank:AAU49513.1 >AAU49513.1maltooligosyltrehalosesynthase,putative[Burkholderia malleiATCC23344] MKPRATLRLQLHAGFTFDDAAAHVGYFARLGVSHLYLSPITAAEPGSRHGYDVIDYSTVNPELGGEAAFV RLIDALRRRGMGAIVDIVPNHMGVGGSSNRWWNDVLEWGARSRFARHFDIDWHASDPALQRKVLLPCLGR PYGEALAAGDIALRADAAHGRFAIACAGRILPVQIGAYPDILRAANRSDLNALAERFDAPGARPSNHARL DAAHAALRDYAAARGPGALDAVLHGFDPRIARSREMLHRLLEQQHYRLAWWRIATDEINWRRFFDISTLA CMRIEDAAVFDDVHALLWRLYAAGLVDGVRIDHVDGLADPRGYCRQLRGRLAALRDGEPYIVVEKILAPD ERLPEDWRVDGTTGYDFMNDVSALLHDAAGAAPLAALWADMTGAETTFAREALDGKRRVLARQFAAEHER VARAMHRLARASRDARDFALNPIRRAVAELAIRLPVYRLYPSAGAPQRTDRALLAGAWQAARSAIAPADR AALDYVAATLGLPGVARAVAGLGDPARLAARVGFAQLTAPLAAKGVEDTACYRYGRLLSRNEVGAHADAL SLAPGAFHTRNRRRRRTFPGALLATATHDHKRGEDARARLAVLSEAHRAWRAAALDWAAFNAPHHHGAPA AADRIPGPAAEAMLYQTLVGAWPPALAPDDAPGLAALTDRVERWQLKALREAKRDTDWLEPNLGYEAGCA AFLRAIMTPRGPDDFAHRLHRLVARIAPAGIVNSLSQAALRLLSPGVPDLYQGAQTWDHTLVDPDNRADV PFARYAAQRIDAPVAAYLRDWADGRVKHALIGRLLALRAAHPETFAAGAYVPLHVRGTRRGHALAFARRD ASTTIVVIATRLAYPLLGDAPARPCVEAACWADTAVGLAPGFAGPWRDMLNDGTLDAPSGMLPLAAALAH LPVAVLIREGGAADTPRRGA(SEQIDNO:60) GroEL molecularchaperoneGroEL[BurkholderiamalleiATCC23344] NCBIReferenceSequence:YP_103588.1 >YP_103588.1molecularchaperoneGroEL[BurkholderiamalleiATCC 23344] MAAKDVVEGDSARAKMVEGVNILANAVKVTLGPKGRNVVLERSEGGPTVTKDGVSVAKEIELKDKLQNMG AQMVKEVASKTSDNAGDGTTTATVLAQSIVREGMKYVASGMNPMDLKRGIDKAVAAAVEELKKISKPCTT NKEIAQVGAISANSDSSIGDRIAEAMDKVGKEGVITVEDGKSLADELDVVEGMQFDRGYLSPYFINNPDK QVAVLENPFVLLHDKKVSNIRDLLPVLEQVAKAGRPLLIIAEDVEGEALATLVVNNIRGILKTVAVKAPG FGDRRKAMLEDIAILTGGQVIAEETGLTLEKATLAELGQAKRIEVGKENTTIIDGAGEAMNIEARVKQIR TQIEEATSDYDREKLQERVAKLAGGVAVIKVGAATEVEMKEKKARVEDALHATRAAVEEGIVPGGGVALI RARTAIASLTGVNADQNAGIKIVLRAMEEPLRQIVTNGGEEASVVVAAVAAGKGNYGYNAATGEYVDMVE AGVVDPTKVTRTALQNAASVAGLLLTTDAAVAELPKEDAPMPGGMPGGMGGMGMGMGMDM (SEQIDNO:61)
CONCLUSION
[0169] This concludes the description of the preferred embodiment of the present invention. The foregoing description of one or more embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching.