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Patexia Research
Patent No. US 11224647
Issue Date Jan 18, 2022
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Patent 11224647 - Safe potent single platform vaccine against Tier 1 select agents and other pathogens > Description

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under Section 119(e) from U.S. Provisional Application Ser. No. 62/369,654, filed Aug. 1, 2016, entitled “SAFE POTENT SINGLE PLATFORM VACCINE AGAINST TIER 1 SELECT AGENTS AND OTHER PATHOGENS” the contents of which are incorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTION MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under Grant No. AI101189 awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 18, 2017, is named 30435_320-WO-U1_SL.txt and is 176,302 bytes in size.

TECHNICAL FIELD

The invention relates to single platform homologous and heterologous priming and boosting vaccines for preventing diseases caused by pathogens and in particular, Tier 1 Select Agents including tularemia, anthrax, and plague in humans and animals.

BACKGROUND OF THE INVENTION

When Francisella tularensis, Bacillus anthracis, and Yersinia pestis infect humans by the respiratory route, the route of greatest concern in an intentional bioterrorist attack, they cause highly fatal diseases—pneumonic tularemia, pulmonary anthrax, and pneumonic plague, resp. Pneumonic tularemia has a mortality of up to 40-−60% for infections with the highly virulent Type A Ft untreated and can be highly fatal even with appropriate antibiotic treatment (1). Pulmonary anthrax has a mortality as high as 100% untreated, and 45% with treatment (2); and pneumonic plague is rapidly lethal (˜50-90%) untreated (3). Because these pathogens are relatively easy to manufacture, environmentally hardy, and cause high mortality, they are considered the most likely pathogens to be employed by bioterrorists; indeed, they were developed as bioweapons during WWII and the Cold War (4, 5).

However, there are no licensed vaccines against F. tularensis and Y. pestis, and the licensed vaccine against B. anthracis is suboptimal. The currently available licensed human anthrax vaccines are the U.S. anthrax vaccine absorbed (AVA) and the U.K. anthrax vaccine precipitated (AVP); both are undefined acellular subunit vaccines, containing primarily the Ba Protective Antigen (PA) with a lesser amount of lethal factor (LF) and other proteins. AVA requires 3 prime and 3 boost vaccinations followed by annual boosters and its duration of efficacy is unknown. In addition, AVA causes adverse reactions such as local soreness, redness, itching and swelling at the site of injection. The complexity of the immunization schedule and adverse effects of AVA make it unattractive. There are currently no licensed vaccines against plague or tularemia. The Y. pestis EV76 strain was developed and used in humans in the former Soviet Union; however, it has significant toxicity and is not licensed in the U.S. (3). The F. tularensis Live Vaccine Strain (LVS) has been extensively studied in the U.S.; this unlicensed vaccine is relatively toxic and provides incomplete protection against aerosolized Ft (6).

Accordingly, there is a great need for vaccines and methods designed to protect military and non-military personnel from diseases caused by Tier 1 Select Agents as well as other pathogens and toxic agents. Furthermore, there is a need for a single platform vaccine that can provide protection from multiple pathogens and toxic agents.

SUMMARY OF THE INVENTION

The present invention is useful for preventing infection caused by pathogens and in particular, Tier 1 Select Agents of bioterrorism including F. tularensis, the agent of tularemia, B. anthracis, the agent of anthrax and Y. pestis, the agent of plague. Though the vaccines described herein are generally illustrated with the antigens for F. tularensis, B. anthracis, and/or Y. pestis, the same platform can be modified to accommodate antigens of other Tier 1 Select Agents, e.g. Burkholderia pseudomallei, as well as other pathogens and toxic agents 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.

Generally, the single platform vaccine comprises one or both of two types of vaccines. One vaccine is a recombinant LVS ΔcapB overexpressing antigens of F. tularensis and/or expressing antigens of B. anthracis and/or Y. pestis and/or other pathogens. The second vaccine is a recombinant attenuated Listeria monocytogenes expressing antigens of F. tularensis, B. anthracis, Y. pestis, and/or other pathogens. Each vaccine is administered intradermally (i.d.) or by another route, e.g. subcutaneously (s.q.), intramuscularly (i.m.), intranasally (i.n.), 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.

This 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. Currently, no single platform vaccine against Tier 1 Select Agents is available. Regarding manufacture, vaccines constructed from the same vectors can be manufactured under the same conditions. That is, the manufacture of the LVS ΔcapB vector will be the same regardless of which antigen it is expressing or overexpressing. Similarly, manufacture of the L. monocytogenes vector will be the same regardless of which antigen it is expressing.

Furthermore, a single platform vaccine also has the advantage of vaccines comprising any particular vector being able to be administered at the same time. That is, LVS ΔcapB overexpressing F. tularensis antigens and LVS ΔcapB expressing B. anthracis antigens and LVS ΔcapB expressing Y. pestis antigens can be administered together. Similarly, L. monocytogenes expressing F. tularensis antigens and L. monocytogenes expressing B. anthracis antigens and L. monocytogenes expressing Y. pestis antigens can be administered together. Thus, vaccination against all three pathogens would only require one prime vaccination and one or two booster vaccinations. Obviously, these same vectors expressing antigens of other pathogens, such as Tier 1 Select Agents, can be administered together as well.

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 homologous and/or heterologous booster vaccine. The prime vaccine comprises an attenuated recombinant live vaccine strain lacking a polynucleotide encoding CapB (LVS ΔcapB), wherein the LVS ΔcapB expresses an antigen of at least one pathogen from Table 1. The heterologous booster vaccine comprises an attenuated L. monocytogenes expressing the antigen of the same pathogen(s) from Table 1. Alternatively, using the homologous prime-boost vaccination strategy, the prime and boost vaccines are the same and can comprise either an attenuated recombinant live vaccine strain lacking a polynucleotide encoding CapB (LVS ΔcapB), wherein the LVS ΔcapB expresses an antigen of at least one pathogen from Table 1 or an attenuated recombinant L. monocytogenes expressing an antigen of at least one 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 homologous or heterologous booster vaccine.

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 ΔcapB 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 ΔcapB and attenuated Listeria monocytogenes express antigens of Francisella tularensis, Bacillus anthracis, and Yersinia pestis.

In one illustrative embodiment, the pathogen is Francisella tularensis (F. tularensis) and the LVS ΔcapB over-expresses a fusion protein comprising the immunodominant epitopes of IglA, IglB, and IglC (IglABC). In specific instances, the fusion protein is expressed by LVS ΔcapB under the control of a F. tularensis 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 L. monocytogenes under the control of a L. monocytogenes hly or actA promoter.

Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the present invention, are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Expression of fusion proteins Ba LFPA(L1), Ba LFPA(L2), Yp F1V(L1), and Yp VF1YscF(L3) by rLVS ΔcapB. rLVS ΔcapB/BaLFPA(L1) (A, lanes 3-6), rLVS ΔcapB/BaLFPA(L2) (C, lanes 3-6), rLVS ΔcapB/YpF1V(L1) (B, lanes 2-4), and rLVS ΔcapB/YpVF1YscF(L3) (D, lanes 3-7) were grown on agar plates and lysates analyzed by Western blotting using monoclonal antibody to Ba PA (A; C, top panel) or LF (C, middle panel), goat polyclonal antibody to Yp LcrV (B; D, top panel), or to Ft Bfr (C & D, bottom panels, serving as vector control). Lysate of LVS ΔcapB (A, lane 2; C, lane 1; D, lane 2), PA protein (A, lane 7; C, lane 8), LF (C, lane 9), monomer of F1-LcrV (B, lane 6), F1 (D, lane 9) or LcrV (D, lane 10) served as controls. B, lane 5, empty; C, lane 7, lysate of culture supernatant of rLVS ΔcapB/KatGS S-BaLFPA(L1).

FIG. 2. Expression of the fusion protein Ba LFPA(L1) and Yp F1V(L1) by recombinant L. monocytogenes. Culture filtrates of rLm ΔactA ΔinlB prfA* (A, top panel, Lanes 3 & 4) or rLm ΔactA (A, top panel, Lanes 5 & 6) expressing Ba fusion protein actA-BaLFPA(L1) (˜62 kDa, lanes 3, 5; ˜62 kDa band visible in lane 5 in over-exposed blot—not shown) and hly-BaLFPA(L1) (without/with LLOs.s of ˜51 & 54 kDa, resp., lanes 4, 6) or culture filtrates of rLm ΔactA ΔinlB prfA* expressing Yp fusion proteins hly-F1V(L1) (˜55 & 58 kDa) (B, lanes 2 & 3.) or actA-YpF1V(11) (˜66 kDa) (B, Lanes 4 & 5) were analyzed by Western blotting using monoclonal antibody to Ba PA (A, top panel) or polyclonal antibody to Lm P60 (A, bottom panel, loading control), or goat antibody to Yp LcrV (B). Lysate of Lm vector (A, lane 2) served as control.

FIG. 3. Expression of B. anthracis and Y. pestis fusion proteins by F. tularensis rLVS ΔcapB from infected macrophage-like THP-1 cells. Monocytic THP-1 cells were seeded at 3×105 cells/well on 24-well plates and differentiated in the presence of PMA for 3 days. Vaccine vector (LVS ΔcapB) (lane 3) and vaccines (lanes 4-6) were grown on Chocolate agars supplemented without or with kanamycin (7.5 μg/ml) for 3 days. The differentiated THP-1 cells were infected with the vaccines or vaccine vector opsonized with human serum 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 μg/ml) to inhibit extracellular bacterial growth. At 5 and 24 h post infection, medium was removed from wells; cells were lysed in 0.125 ml SDS buffer and boiled for 5 min. The cell lysates were analyzed by Western blotting using antibodies to B. anthracis PA antigen (mAb to B. anthracis PA) plus goat polyclonal antibody to Y. pestis LcrV antigen. These antibodies detected protein bands of ˜50- and 52-kDa in cells infected rLVS ΔcapB/BaLFPA(L1) (panels A & B: lane 4) and ˜50 kDa proteins in cells infected with rLVS ΔcapB/YpF1V(L1) (panels A & B: lanes 5 and 6 resp.).

FIG. 4. Growth kinetics of F. tularensis rLVS ΔcapB expressing B. anthracis and Y. pestis fusion proteins in infected macrophage-like THP-1 cells. Monocytic THP-1 cells were infected as described above in the legend for FIG. 3. At various times post infection, medium was removed from wells; the cells lysed with PBS containing 0.1% Saponin for 3-5 min at room temperature; and the lysates serially diluted and assayed for colony forming units (CFU).

FIG. 5. Humoral and cell-mediated immune responses induced by immunization with LVS ΔcapB- and Lm-vectored B. anthracis vaccines. As indicated by the colors and letters to right, mice (n=4/group) were vaccinated with PBS i.n. at week 0 (Sham, A), AVA subcutaneously (s.q.) twice at weeks 0 and 4 (B), rLVS ΔcapB/BaLFPA(L1) i.n. twice at weeks 0 and 4 (C) or once at week 4 (D), or primed with rLVS ΔcapB/BaLFPA(L1) i.n. at week 0 and boosted with rLmprfA*/hly-BaLFPA(L1) (rLm ΔactA ΔinlB prfA*hly-BaLFPA(L1)) twice at weeks 4 and 6 i.n (E) or i.m. (F). At week 7, all mice were bled and spleens and lungs removed. Antibody responses: Sera were assayed for IgG to Ba LF and PA proteins and heat inactivated Ft Live Vaccine Strain (HI-LVS) (a) or IgG subtypes IgG1, IgG2a, IgG2b, and IgG3 to these antigens as indicated (b to d). Cell-mediated Responses: Spleen and lung cells were stimulated with PA, LF or HI-LVS and their supernatants assayed for secretion of IFN-γ and IL-4 (e to h). Values are mean±SD.

FIG. 6. immunogenicity of LVS ΔcapB- and Lm-vectored Yp vaccines. Mice were vaccinated with PBS at week 0 (Sham, blue bars), rLVS ΔcapB/YpF1V(L1) intranasally (i.n.) once at weeks 0 (green bars) or twice at weeks 0 and 4 (red bars), or with rLmprfA*/YpF1V(L1) (rLm ΔactA ΔinlBprfA*/hly-YpF1V(L1)) intramuscularly (i.m.) twice at weeks 0 and 4 (black bars), or primed with rLVS ΔcapB/YpF1V(L1) i.n. at week 0 and boosted with rLmprfA*/YpF1V twice at weeks 4 and 6 i.n. (purple bars) or i.m. (orange bars). At week 7, all mice were bled and spleens and lungs removed. A. Antibody responses: Sera were assayed for IgG, subtypes IgG1 and IgG2a to Y. pestis antigen F1/V monomer protein (BE1 Resources). B. Cell-mediated Responses: Spleen and lung cells were stimulated with medium alone or medium supplemented with F1, LcrV, or F1-LcrV monomer protein and their supernatants assayed for secretion of IFN-γ. Values are mean±SD.

FIG. 7. Cytokine production induced by immunization with Francisella- and Lm-vectored B. anthracis vaccines. Mice (n=4/group) were vaccinated intranasally (i.n.) at week 0 with PBS (Sham, Group A), subcutaneously twice at weeks 0 and 4 with AVA (Group B), i.n. twice at weeks 0 and 4 (group C) or once at week 4 (Group D) with rLVS ΔcapB/BaLF-PA(L1), or primed intranasally with rLVS ΔcapB/BaLFPA(L1) at week 0 and boosted twice at weeks 4 and 6 i.n. (Group E) or intramuscularly (i.m.) (Group F) with rLmprfA*/hly-BaLFPA(L1) (rLm ΔactA ΔinlB prfA*/hly-BaLFPA(L1)). At week 7, all mice were bled, euthanized, their spleens (panels A, B, and C) and lung (Panels D, E, and F) cells isolated and stimulated with B. anthracis LF or PA antigen, or Francisella HI-LVS for 3 days. Supernatants of the spleen and lung cell culture were collected and assayed for interferon gamma (IFN-γ) secretion by ELISA. Values are mean±SD. **P<0.01; ***, P<0.001, ****, P<0.0001 by ANOVA (Prism 6.04).

FIG. 8. Immunization with LVS ΔcapB- and Lm-vectored B. anthracis vaccines induces elevated frequencies of spleen CD4+ T cells producing IFN-γ, IL-2, TNF, and IL17. Mice were immunized as described in legend to FIG. 5. Spleen cells were stimulated with PA, LF or HI-LVS, or PMA and assayed by intracellular cytokine staining for CD4+ T cells expressing IFN-γ (A), TNF (8), IL-2 (C), and IL-17A (D) as indicated. Values are means±SEM.

FIG. 9. Immunization with LVS ΔcapB- and Lm-vectored B. anthracis vaccines induces elevated frequencies of lung CD4+ T cells producing IFN-γ, IL-2, TNF, and IL-17. Mice were immunized as described in legend to FIG. 5. Spleen cells were stimulated with PA, LF or HI-LVS, or PMA and assayed by intracellular cytokine staining for CD4+ T cells expressing IFN-γ (A), TNF (B), IL-2 (C), and IL-17A (D) as indicated. Values are means±SEM.

FIG. 10. Immunization with LVS ΔcapB- and Lm-vectored B. anthracis vaccines induces elevated frequencies of multifunctional spleen CD4+ T cells producing IFN-γ, IL-2, TNF, and IL-17. Mice were immunized as described in legend to FIG. 5. Spleen cells were stimulated with PA (A), LF (B), HI-LVS (C), or PMA (D) and assayed by intracellular cytokine staining for 15 possible combinations of CD4+ T cells expressing IFN-γ, TNF, IL-2, and/or IL-17A. Values are means±SEM.

FIG. 11. Immunization with LVS ΔcapB- and Lm-vectored B. anthracis vaccines induces elevated frequencies of multifunctional lung CD4+ T cells producing IFN-γ, IL-2, TNF, and IL17. Mice were immunized as described in legend to FIG. 5. Lung cells were stimulated with PA (A), LF (B), HI-LVS (C), or PMA (D) and assayed by intracellular cytokine staining for 15 possible combinations of CD4+ T cells expressing IFN-γ, TNF, IL-2, and/or IL-17A. Values are means±SEM.

FIG. 12. Protective efficacy of rLVS ΔcapB/Ba & rLm/Ba against respiratory challenge with virulent Ba Ames spores. Mice, 8/group, were sham-immunized, or immunized subcutaneously (SC) twice with AVA (Biothrax, 0.025 ml), intradermally (ID) or intranasally (IN) twice with rLVS ΔcapB/Ba (rLVS ΔcapB/BaLFPA(L1)) or prime-boosted with rLVS ΔcapB/Ba once and rLm/Ba (rLm ΔactA ΔinlB prfA*/hly-BaLFPA(L1)) once systemically (ID/IM) or mucosally (IN/IN) (106 of each) at Week 0 and 4, as indicated in FIG. 8, challenged 4 weeks later with Ba Ames spores (5× LD50), and monitored for survival.

FIG. 13. Protective efficacy of rLVS ΔcapB/Yp & rLm/Yp against respiratory challenge with virulent Yp. Mice, 8/group, were sham-immunized, or immunized subcutaneously (SC) twice with EV76 (106), intradermally (ID) or intranasally (IN) twice with rLVS ΔcapB/Yp (rLVS ΔcapB/YpF1V(L1)) or prime-boosted with rLVS ΔcapB/Yp once and rLm/Yp (rLm ΔactA ΔinlB prfA*hly-YpF1V(L1)) once intramuscularly (ID/IM) or intranasally (IN/IN) (106 of each) at Week 0 and 4, as indicated on the right of the panels, challenged at week 9 with Yp (CO92), and monitored for survival for 14 days. Survival curves were compared by log-rank test (Mantel-cox, Prism).

FIG. 14. Construction of shuttle plasmids for antigen expression cassettes of IglABC(D), IglABC(GGSG), and IglABC(GGSG2) and expression of F. tularensis FPI T6SS proteins by the recombinant LVS ΔcapB strains. A. Antigen expression cassette for fusion protein of IglABC. The coding sequences for IglA (residues 33-132), IglB (residues 446-506), and IglC (residues 29-149) either fused directly in frame with each other [IglABC(D)] or linked by a flexible linker GGSG (SEQ ID NO: 62) [IglABC(GGSG)] or GGSGGGSG (SEQ ID NO: 63) [IglABC (GGSG2)] was amplified by using overlap PCRs from the genomic DNA of a recent clinical isolate of F. tularensis subsp. tularensis and primer pairs listed in Supplemental Table 1 as described in the supplemental methods. B. The F. tularensis IglA, IglC, IglC and IglABC are overexpressed by rLVS ΔcapB cultured in broth medium. Various rLVS ΔcapB glycerol stocks were grown in TSBC for overnight with agitation; the overnight culture was sub-cultured to mid-log phase in TSBC; cells collected by centrifugation and lysed in SDS buffer; cell lysates equivalent to 1×108 bacteria were analyzed by SDS-PAGE and Western blotting using polyclonal antibodies (pAb) to IglA (top), monoclonal antibody (mAb) to IglB (upper middle), pAb to IglC (lower middle) and pAb to Bacterioferritin (Bfr, bottom) (loading control). Note, the membrane probed with mAb to IglB was stripped and re-probed with pAbs to IglA and Bacterioferritin; a separated membrane applied with the same amount of cell lysates was probed with pAb to IglC. Lane 1, LVS ΔcapB; Lane 2, rLVS ΔcapB/bfr-iglA; Lane 3, rLVS ΔcapB/bfr-iglB; Lane 4, rLVS ΔcapB/bfr-iglC; Lane 5, rLVS ΔcapB/bfr-iglABC(D); Lane 6, rLVS ΔcapB/bfr-iglABC [by definition expressing IglABC(GGSG); see Table 3]; Lane 7, rLVS ΔcapB/bfr-iglABC(GGSG2). FIG. 14B discloses “GGSG” and “GGSG2” as SEQ ID NOS 62 and 63, respectively. C. Relative intensity of protein expression. The intensity of protein bands detected by various antibodies were analyzed by QuantityOne (Bio-Rad) and compared with the same protein expressed by the parental LVS ΔcapB (IglA, IglB, IglC, and Bfr, left panel) or with the endogenous protein from the same strain (IglA or IglC, right panel). Results were representative of multiple protein expression experiments tested in broth culture (CDM and TSBC) and in infected human (THP-1) and mouse macrophage-like cell lines. FIG. 14C discloses “GGSG” and “GGSG2” as SEQ ID NOS 62 and 63, respectively.

FIG. 15 Growth kinetics of rLVS ΔcapB candidate vaccines in broth and in human macrophage-like THP-1 cells. (A-F) Chocolate agar-grown rLVS ΔcapB vaccines expressing IglA, IglB, or IglC downstream of the groE, bfr, or outer membrane protein (omp) promoter were subcultured in tryptic soy broth-L-cysteine (TSBC, a-c) medium or Chamberlain Defined Medium (CDM, d-f) for up to 24 hours. At the indicated times post-infection, the optical density of each strain (a-f) was measured. (G-I) THP-1 cells were infected with chocolate agar-grown vaccines for 1 h at 37° C., washed, replaced with fresh medium, and cultured for 24, 48 or 72 hours. At the indicated times post infection, cells were harvested, lysed with 0.1% saponin/PBS, serially diluted, plated on Chocolate agar, and incubated for 3 days before CFU were enumerated.

FIG. 16. The rLVS ΔcapB vaccines are disseminated and cleared similarly to the parental strain after i.n. and i.d. vaccination. Mice (4/group) were immunized i.n. (top panels) with 102 CFU of LVS or 105 CFU of LVS ΔcapB or rLVS ΔcapB/bfr-iglA, iglB, iglC or iglABC vaccines, or immunized i.d. (middle and bottom panels) with 104 CFU of LVS or 106 CFU of LVS ΔcapB or rLVS ΔcapB/bfr-iglA, iglB, iglC, or iglABC vaccines; euthanized at various times post-vaccination, as indicated on the X-axis; and their organs removed and assayed for F. tularensis bacterial burden. Values are means±SE. Shown are the results combined from 3 independent experiments comprising 4-8 mice per group.

FIG. 17. Higher doses of rLVS ΔcapB/bfr-iglABC were well tolerated by BALB/c mice after intranasal vaccination. BALB/mice, 4 per group, were immunized intranasally with rLVS ΔcapB/bfr-iglABC at 6 different doses as shown in the Figure legend and monitored for signs of illness and weight change. Mice infected with virulent F. tularensis Schu S4 in a separate experiment were included as a control. Shown are mean of weight (top) or change of weight compared with the day of immunization (bottom).

FIG. 18. Immunization with rLVS ΔcapB overexpressing FPI T6SS proteins induces greater antigen-specific cytokine production and a Th1-type antibody response. Mice (3/group) were immunized i.d. with various vaccines; euthanized 4 weeks later; their splenocytes isolated and stimulated with IglC or HI-LVS for 3 days; and the culture supernatant assayed for IL-17A (A) or IFN-γ (B). Their sera were isolated and assayed for antibodies specific to HI-LVS (C). Values are means±SE. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001 by ANOVA (Prism 6.04).

FIG. 19. Immunization with rLVS ΔcapB vaccines induces elevated frequencies of CD4+ and CD8+ T cells producing IFN-γ or IFN-γ+ TNF. Mice (3/group) were immunized i.d. once with various vaccines; euthanized 4 weeks later; and their splenocytes isolated, stimulated with IglC or HI-LVS, and assayed for CD4+ (A) or CD8+ (B) T cells expressing IFN-γ or IFN-γ+ TNF as indicated. Values are means±SE. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001 by ANOVA (Prism 6.04).

FIG. 20. Mice immunized with attenuated rLVS ΔcapB/bfr-iglABC, comprising immunodominant epitopes of IglA, IglB, and IglC, survive longer than sham-immunized mice and mice immunized with the parental LVS ΔcapB. BALB/c mice (8/group) were immunized i.d. with various vaccines, challenged with (A) 16 CFU at 49 days post-immunization (A, Experiment I) or with 31 (B) or 310 (C) CFU F. tularensis Schu S4 at 42 days (B, Experiment II) and monitored for signs of illness and death for 21 days. Mean Survival Day was calculated by dividing the sum of the surviving days of all mice by the total number of mice examined, with animals surviving until the end of the experiment given a survival time of 21 days, when the experiment was terminated. Survival curves between different groups were compared by log-rank test (Mantel-Cox) (Prism 6.04).

FIG. 21. Mice immunized with attenuated rLVS ΔcapB over-expressing fusion protein IglABC, comprising immunodominant epitopes of IglA, IglB, and IglC, survive longer than sham-immunized mice and comparable to mice immunized with LVS and the parental LVS ΔcapB. BALB/c mice, 8/group, were immunized i.n. once with PBS (Sham), 100 CFU LVS, 1×106 CFU LVS ΔcapB, or 1×106 rLVS ΔcapB/bfr-iglABC, challenged i.n. with 26 CFU F. tularensis Schu S4 at week 6 post immunization, and monitored for signs of illness, weight change and death for up to 28 days post challenge. The survival curves were compared by the log-rank test (Prism 6.04).

FIG. 22. Immediate pre- or post-exposure prophylaxis with rLVS ΔcapB/bfr-iglABC but not LVS induces partial protective immunity against respiratory challenge with F. tularensis Schu S4. BALB/c mice were sham-immunized or immunized i.n. with 102 CFU of LVS (A, Experiment I, upper panel), 1×106 CFU or 5×106 CFU of rLVS ΔcapB/bfr-iglABC (A, Experiment I, lower panel), or 5×107 CFU of rLVS ΔcapB/bfr-iglABC (B, Experiment II) 2 days before (−2), 1 day before (−1), the day of (0), 1 day after (1), or 2 days after (2) challenge i.n. with 10 CFU F. tularensis Schu S4, and monitored for signs of illness and death for up to 21 days. Shown are the results from two independent experiments (one in A&B and the other in C). Mean Survival Day was calculated by dividing the sum of the surviving days of all mice by the total number of mice examined, with animals surviving until the end of the experiment given a survival time of 21 days, when the experiment was terminated. Survival curves between different groups were compared by log-rank test (Mantel-Cox) (Prism 6.04). In Experiment I, at day 3 post challenge, before any mice had died, the mean weight of mice immunized with LVS on day −2 was significantly lower than that of the sham-immunized mice (p<0.05), mice immunized with 1×106 rLVS ΔcapB/bfr-iglABC at day −2, 0, and 2, and mice immunized with 5×106 rLVS ΔcapB/bfr-iglABC at day 0 (A, left panels). At days 4 and 5 post challenge, the mean weight of mice immunized with rLVS ΔcapB/bfr-iglABC (Day −2, 1×106) was significantly greater than that of sham-immunized mice and mice immunized with rLVS ΔcapB/bfr-iglABC at day 0 and day 1 post challenge (A, lower left panel). In Experiment II, mice immunized with 5×107 rLVS ΔcapB/bfr-iglABC had transient weight loss that partially or fully recovered if they were immunized at day −2 or −1 before challenge (B, left panel).

FIG. 23. Immediate pre-exposure prophylaxis with rLVS ΔcapB/bfr-iglABC but not LVS induces strong innate immunity. Mice were immunized with PBS (Sham), 102 of LVS, or 1×106 or 5×107 of rLVS ΔcapB/bfr-iglABC (bfr-iglABC) as indicated and euthanized at day 2 post immunization. Their lung lysates (A-C) and sera (D) were assayed for cytokine/chemokine production by a mouse 32-Plex kit. Shown are means±SE (n=3 mice) for each cytokine/chemokine. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001 by Two-Way ANOVA with multiple comparisons (Prism 6.04).

FIG. 24. Expression of F. tularensis IglABC by rLm vector in culture supernatant of recombinant L. monocytogenes. Broth culture filtrates of each recombinant rLm strain (two clones per strain) were concentrated and a volume of filtrate equivalent to lml of the bacterial culture was analyzed by Western blotting using rabbit polyclonal antibody to IglC. On the right border of the blot are listed the proteins of interest and their predicted mass. On the left border are listed the sizes of the molecular mass standards. Note that proteins expressed by the Lm ΔactA ΔinlB prfA* vector (lanes 6-9) were generally more abundant than those expressed by the Lm ΔactA vector (lanes 2-5) and that proteins expressed downstream of the hly promoter (lanes 4, 5, 8, 9) were generally more abundant than those expressed downstream of the actA promoter (lanes 2, 3, 6, 7) in the broth culture supernatant.

FIG. 25. Priming with rLVS ΔcapB/bfr-iglABC and boosting with rLm/hly-iglABC induces antigen specific CD4+ T cells expressing Th1 type cytokines. Mice (n=4/group) were vaccinated, bled, euthanized, their spleen and lung cells stimulated with medium alone or with a IglC peptide (IglCpep, TDEAWGIMIDLSNLE (SEQ ID NO: 64)), peptide pools of IglA (IglApep) or IglB (IglBpep), IglC protein (IglCprot), or heat-inactivated LVS (HI-LVS), and assayed for intracellular cytokine staining. Values=mean±SD.

FIG. 26. Priming with rLVS ΔcapB/bfr-iglABC and boosting with rLm/hly-iglABC induces antigen (HI-LVS)-specific Th1/Th2-type antibody. Mice (n=4/group) were vaccinated, bled, euthanized and tested for serum antibody as described in the text. Values=mean±SD.

FIG. 27. Protective efficacy of homologous prime-boost vaccination with rLVS ΔcapB/bfr-iglABC and heterologous prime-boost vaccination with rLVS ΔcapB/bfr-iglABC—rLm/hly-iglABC against respiratory challenge with the virulent F. tularensis SchuS4 strain. BALB/c mice, 8/group, were immunized i.d. once at week 0 with PBS (Sham, Group A), 104 CFU LVS (Group B), 106 CFU LVS ΔcapB (Group H), or 106 rLVS ΔcapB/bfr-iglABC (Group C); immunized i.d. or i.n. twice at weeks 0 and 4 with 106 rLVS ΔcapB/bfr-iglABC (Groups D & E, resp.) (panel a); primed i.d. once at week 0 with rLVS ΔcapB/bfr-iglABC (Groups F & G) or LVS ΔcapB (Groups I & J) and boosted intramuscularly (i.m.) twice at weeks 4 and 6 with rLmprfA* Vector (Lm ΔactA ΔinlB prfA*) (Groups F & I) or rLmprfA*/hly-iglABC (rLm ΔactA ΔinlB prfA*/hly-iglABC, Groups G & J); or immunized simultaneously i.d. with rLVS ΔcapB/hfr-iglABC and i.m. with rLmprfA*/hly-iglABC (Group K) (panel b). All the mice were challenged i.n. with 10 CFU (3× LD50) 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.

FIG. 28. Humoral immune responses induced by immunization with LVS ΔcapB- and Lm-vectored B. anthracis vaccines. As indicated by the colors and letters to right, mice (n=8/group) were vaccinated i.d. with PBS once at week 0 (Sham, Group A), subcutaneously (s.c.) with AVA three times at weeks 0, 4, and 8 (Group B), i.n. or i.d. with rLVS/BaLFPA (rLVS ΔcapB/BaLFPA[L1]) three times at weeks 0, 4, and 8 (Groups C & D, resp.), three times i.n. or i.m. with rLm/BaLFPA (rLm ΔactA ΔinlB prfA*/hly-BaLFPA[L1]) (Groups E & F, resp.), primed i.n. (Groups G & H) with rLVS/BaLFPA once at week 0 and boosted i.n. once (Groups G) or twice (Group H), or primed i.d. with rLVS/BaLFPA once at week 0 (Groups I & J) and boosted i.m. once (Groups I) or twice (Group J) with rLm/BaLFPA. All the mice were bled at week 11, challenged at week 12, and monitored for 3 weeks post challenge (see FIG. 29 below). Sera were assayed for IgG or IgG subtypes IgG1 and IgG2a to Ba PA (A) and LF proteins (B). Values are mean±SE. Data were analyzed by two-way ANOVA with Tukey's post multiple comparisons test (Prism). Values that are significantly different from the Sham group are marked with asterisks over the comparison groups. *, P<0.05; **, P<0.01; ****, P<0.0001 by two-way ANOVA with Tukey's multiple comparisons test.

FIG. 29. Protective efficacy induced by immunization with LVS ΔcapB- and Lm-vectored B. anthracis vaccines against respiratory challenge with virulent B. anthracis. Mice (n=8/group) were immunized as described in the legend to FIG. 28 (the same experiment as the one for FIG. 28) and as indicated by colors, groups, and vaccines to the right, challenged at week 12 with Ba Ames spores (371,000 CFU), and monitored for survival. Survival curves between vaccinated groups and the sham group were compared by log-rank test (Mantel-cox, Prism); the P value for the comparison of a specific vaccine vs. Sham are color-coded to the color of the vaccine symbol.

FIG. 30. Humoral immune responses induced by immunization with LVS ΔcapB- and Lm-vectored Y. pestis vaccines. As indicated by the colors and letters to the right, mice (n=8/group) were vaccinated i.d. with PBS once at week 0 (Sham, Group A), subcutaneously (s.c.) with Y. pestis vaccine strain EV76 once at week 0 (Group B), i.n. or i.d. with rLVS ΔcapB/YpF1V (rLVS ΔcapB/YpF1V[L1]) three times at weeks 0, 4, and 8 (Groups C & D, resp.), i.m. with rLmprfA*/hly-YpF1V (rLm ΔactA ΔinlB prfA*/hly-YpF1V[L1]) three times at weeks 0, 4, and 8 (Group E), or primed i.d. with rLVS ΔcapB/YpF1V once at week 0 (Groups F & G) and boosted i.m. once (Group F) or twice (Group G) with rLmprfA*/hly-YpF1V. All the mice were bled at week 11, challenged with Yp CO92 at week 12, and monitored for 3 weeks for survival (see FIG. 31 below). Sera were assayed for IgG or IgG subtypes IgG1 and IgG2a to Yp F1 (A) and LcrV proteins (B). Values are mean±SE. Values that are significantly different from the Sham group are marked with asterisks over the comparison groups. **, P<0.01; ***, P<0.001; ****, P<0.0001 by two-way ANOVA with Tukey' s multiple comparisons test.

FIG. 31. Protective efficacy induced by immunization with LVS ΔcapB- and Lm-vectored Y. pestis vaccines against respiratory challenge with virulent Y. pestis. Mice (n=8/group) were immunized as described in legend to FIG. 30 and as indicated by colors, groups, and vaccines to the right, challenged at week 12 with Yp CO92 (1,800 CFU), and monitored for 21 days for survival. Survival curves between vaccinated groups and the Sham group were compared by log-rank test (Mantel-cox, Prism); the P value for the comparison of a specific vaccine vs. Sham are color-coded to the color of the vaccine symbol.

FIG. 32. Over-expression of IglC by rLVS ΔcapB strains in broth and in macrophages. rLVS ΔcapB strains carrying IglC protein expression cassette downstream of the Ft groE, bfr and omp promoter were cultured in Tryptic soy broth supplemented with L-cysteine (TSBC) or used to infected mouse macrophage-like J774 cells. Protein expression was analyzed using polyclonal antibodies specific for IglC, IglA, or Bfr (loading control). IglC fused with a 3×FLAG tag (3F-IglC) was expressed by the rLVS ΔcapB strains as a detection control for IglC. A. Protein expression in TSBC. B. Protein expression in J774 macrophages. C. Relative expression level of IglC in TSBC. D. Relative expression level of IglC in J774 macrophages.

DETAILED DESCRIPTION OF THE INVENTION

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.

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.

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.

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 ΔcapB 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 ΔcapB 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.

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.

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.

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 ΔcapB and recombinant LVS ΔcapB 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 ΔcapB 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.

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 proteins—IglA, 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.

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.

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 ΔcapB 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.

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 ΔcapB), wherein the LVS ΔcapB 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 ΔcapB), wherein the LVS ΔcapB 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 ΔcapB 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 ΔcapB over-expresses a fusion protein comprising IglA, IglB, and IglC (IglABC).

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.

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. FIG. 32).

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).

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 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). 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).

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.

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.

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.

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

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.

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.

Previously, mutants of the LVS vaccine that lack the O-Antigen have been investigated, i.e. the LPS molecule. These LPS-deficient mutants (LVS ΔwbtDEF and LVS Δwzy) 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.

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 ΔcapB mutant, like the previously evaluated LVS ΔwbtDEF mutant, is highly attenuated compared with LVS. Surprisingly, however, the LVS ΔcapB mutant induces protective immunity against F. tularensis intranasal challenge comparable to LVS. Recombinant versions of LVS ΔcapB 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 ΔcapB 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.

The present invention describes improved versions of both the aforementioned prime and booster vaccines. The improved prime vaccine (rLVS ΔcapB/bfr-iglABC—See Table 3 for complete definition) and booster vaccine (rLm ΔactA ΔinlB 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 ΔcapB/bfr-iglABC) has demonstrated improved immunogenicity and immunoprotection compared with the first generation rLVS ΔcapB vaccines. The booster vaccine (rLm ΔactA ΔinlB prfA*/hly-iglABC) has also demonstrated improved immunogenicity compared with the first generation rLm vaccines.

The invention consists of two types of vaccines. One vaccine is a recombinant LVS ΔcapB 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.

With respect to the prime vaccine, the vector LVS ΔcapB and recombinant LVS ΔcapB 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 proteins—IglA′B′C′—is overexpressed. Second, the fusion protein is expressed from a stronger promoter—the bfr promoter and omp promoter instead of the previously used groEL promoter.

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 proteins—IglABC.

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 ΔcapB based vaccines described herein are >10,000 fold less virulent than LVS and hence much safer. In the case of first generation rLVS ΔcapB 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.

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 ΔcapB 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.

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

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 ΔcapB), wherein the LVS ΔcapB 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.

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 ΔcapB 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 ΔcapB and attenuated Listeria monocytogenes express antigens of Francisella tularensis, Bacillus anthracis, and Yersinia pestis.

In one illustrative embodiment, the pathogen is Francisella tularensis (F. tularensis) and the LVS ΔcapB 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 ΔcapB 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.

EXAMPLESExample 1Construction and Characterization of LVS ΔcapB and Recombinant Listeria monocytogenes Expressing B. anthracis and Y. pestis Antigens and Demonstration of their Immunogenicity and Efficacy

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

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 ΔcapB 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 ΔcapB 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.

In preliminary studies, we have developed vaccines using LVS ΔcapB as a vector to express F. tularensis (IglABC) B. anthracis (Ba), or Y. pestis (Yp) antigens and rLm ΔactA ΔinlB 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 ΔcapB/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 (FIG. 27). Similarly, three doses of rLVS ΔcapB/BaLFPA given i.d. or i.n. provided protection superior to the AVA vaccine (FIG. 29); and three doses of rLVS ΔcapB/Yp provided excellent protection against respiratory challenge with virulent Yp CO92 (FIG. 31).

Ultimately, we envision construction of a trivalent Ft rLVS ΔcapB (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 ΔactA ΔinlB prfA* booster vaccine expressing Ba+Yp+Ft antigens downstream of a strong Lm promoter (actA/hly) [or, alternatively, a bivalent rLm ΔactA ΔinlB prfA*/Ba+Yp used in concert with our rLm ΔactA ΔinlB prfA*/IglABC vaccine]. Both the Ft LVS and Lm ΔactA ΔinlB “parental” strains have been tested in humans. LVS ΔcapB is a fully defined further attenuated LVS with three major attenuating deletions (capB, pilA, and FTT 0918). rLm ΔactA ΔinlB prfA* comprises Lm ΔactA ΔinlB 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.

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:

A. B. anthracis LFPA(L1) (LFD1-GGSG-PAD4)(SEQ ID NO: 16)MAGGHGDVGMHVKEKEKNKDENKRKDEERNKTQEEHLKEIMKHIVKIEVKGEEAVKKEAAEKLLEKVPSDVLEMYKAIGGKIYIVDGDITKHISLEALSEDKKKIKDIYGKDALLHEHYVYAKEGYEPVLVIQSSEDYVENTEKALNVYYEIGKILSRDILSKINQPYQKFLDVLNTIKNASDSDGQDLLFINQLKEHPIDFSVEFLEQNSNEVQEVFAKAFAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEINLGGSGNFDQQTSQNIKNQLAELNATNIYTVLDKIKLNAKMNILIRDKRFHYDRNNIAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYAVIKENTIINPSENGDISINGIKKILIFSKKGYEIGB. B. anthracis LFPA(L2) (LFD1-GSSGGSSG-PAD4)(SEQ ID NO: 17)MAGGHGDVGMHVKEKEKNKDENKRKDEERNKTQEEHLKEIMKHIVKIEVKGEEAVKKEAAEKLLEKVPSDVLEMYKAIGGKIYIVDGDITKHISLEALSEDKKKIKDIYGKDALLHEHYVYAKEGYEPVLVIQSSEDYVENTEKALNVYYEIGKILSRDILSKINQPYQKFLDVLNTIKNASDSDGQDLLFINQLKEHPIDFSVEFLEQNSNEVQEVFAKAFAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEINLGSSGGSSGNFDQQTSQNIKNQLAELNATNIYTVLDKIKLNAKMNILIRDKRFHYDRNNIAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYAVIKENTIINPSENGDISINGIKKILIFSKKGYEIGC. Y. pestis F1V(D) (F1-LcrV)(SEQ ID NO: 18)MKKISSVIAIALFGTIATANAADLTASTTATATLVEPARITLTYKEGAPITIMDNGNIDTELLVGTLTLGGYKTGTTSTSVNFTDAAGDPMYLTFTSQDGNNHQFTTKVIGKDSRDFDISPKVNGENLVGDDVVLATGSQDFFVRSIGSKGGKLAAGKYTDAVTVTVSNQMIRAYEQNPQHFIEDLEKVRVEQLTGHGSSVLEELVQLVKDKNIDISIKYDPRKDSEVFANRVITDDIELLKKILAYFLPEDAILKGGHYDNQLQNGIKRVKEFLESSPNTQWELRAFMAVMHFSLTADRIDDDILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDKSINLMDKNLYGYTDEEIFKASAEYKILEKMPQTTIQVDGSEKKIVSIKDFLGSENKRTGALGNLKNSYSYNKDNNELSHFATTCSDKSRPLNDLVSQKTTQLSDITSRFNSAIEALNRFIQKYDSVMQRLLDDTSGKD. Y. pestis F1V(L1) (F1-GGSG-LcrV)(SEQ ID NO: 19)MKKISSVIAIALFGTIATANAADLTASTTATATLVEPARITLTYKEGAPITIMDNGNIDTELLVGTLTLGGYKTGTTSTSVNFTDAAGDPMYLTFTSQDGNNHQFTTKVIGKDSRDFDISPKVNGENLVGDDVVLATGSQDFFVRSIGSKGGKLAAGKYTDAVTVTVSNQGGSGMIRAYEQNPQHFIEDLEKVRVEQLTGHGSSVLEELVQLVKDKNIDISIKYDPRKDSEVFANRVITDDIELLKKILAYFLPEDAILKGGHYDNQLQNGIKRVKEFLESSPNTQWELRAFMAVMHFSLTADRIDDDILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDKSINLMDKNLYGYTDEEIFKASAEYKILEKMPQTTIQVDGSEKKIVSIKDFLGSENKRTGALGNLKNSYSYNKDNNELSHFATTCSDKSRPLNDLVSQKTTQLSDITSRFNSAIEALNRFIQKYDSVMQRLLDDTSGKE. Y. pestis VF1YscF(L3) (LcrV-[GGGS]3-F1-[GGGS]3-YscF)(SEQ ID NO: 20)MIRAYEQNPQHFIEDLEKVRVEQLTGHGSSVLEELVQLVKDKNIDISIKYDPRKDSEVFANRVITDDIELLKKILAYFLPEDAILKGGHYDNQLQNGIKRVKEFLESSPNTQWELRAFMAVMHFSLTADRIDDDILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDKSINLMDKNLYGYTDEEIFKASAEYKILEKMPQTTIQVDGSEKKIVSIKDFLGSENKRTGALGNLKNSYSYNKDNNELSHFATTCSDKSRPLNDLVSQKTTQLSDITSRFNSAIEALNRFIQKYDSVMQRLLDDTSGKGGGGSGGGGSGGGGSADLTASTTATATLVEPARITLTYKEGAPITIMDNGNIDTELLVGTLTLGGYKTGTTSTSVNFTDAAGDPMYLTFTSQDGNNHQFTTKVIGKDSRDFDISPKVNGENLVGDDVVLATGSQDFFVRSIGSKGGKLAAGKYTDAVTVTVSNQGGGGSGGGGSGGGGSMSNFSGFTKGTDIADLDAVAQTLKKPADDANKAVNDSIAALKDKPDNPALLADLQHSINKWSVIYNINSTIVRSMKDLMQGILQKFP

Construction and Verification of Bivalent rLVS ΔcapB 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

We have constructed the following Francisella-vectored vaccine candidates expressing B. anthracis and Y. pestis antigens (Table 2): 1) rLVS ΔcapB 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 ΔcapB/BaLFPA(L1) and rLVS ΔcapB/BaLFPA(L2); 2) rLVS ΔcapB 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 ΔcapB/katGss-BaLFPA(L1) and rLVS ΔcapB/PepOss-BaLFPA(L1); 3) rLVS ΔcapB expressing the Y. pestis cafI-encoded F1 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 ΔcapB/YpF1V(D) and rLVS ΔcapB/YpF1V(L1); 4) rLVS ΔcapB expressing the Y. pestis F1V(L1) ligated in frame with the C-terminal of F. novicida PepPOss, resulting in rLVS ΔcapB/PepOss-YpF1V(L1); 5) rLVS ΔcapB 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 ΔcapB/YpVF1YscF(L3).

Construction and Verification of Monovalent rLm Booster Vaccines Expressing Ba Fustion Protein LFD1-GGSG-PAD4 and Yp Fusion Protein F1-GGSG-LcrV

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 ΔactA and Lm ΔactA ΔinlB prfA*Lm vectors, resulting in rLm ΔactA/actA-BaLFPA(L1), rLm ΔactA ΔinlB prfA*/actA-BaLFPA(L1), rLm ΔactA/hly-BaLFPA(L1), and rLm ΔactA ΔinlB 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 ΔactA/actA-YpF1V(L1), rLm ΔactA ΔinlB prfA*Lm/actA-YpF1V(L1), rLm ΔactA/hly-YpF1V(L1), and rLm ΔactA ΔinlB 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

rLVS ΔcapB and rLm vaccines express Ba and Yp fusion proteins in broth (FIG. 1 and FIG. 2).

Demonstration that B. anthracis and Y. pestis Fustion Proteins are Expressed by rLVS ΔcapB from Infected Macrophase-Like THP-1 Cells

To examine whether the F. tularensis rLVS ΔcapB platform can express the B. anthracis and Y. pestis fusion proteins from infected macrophage-like cells, we seeded THP-1 cells at 3×105 cells/well on 24-well plates and differentiated them in the presence of PMA for 3 days. Vaccine vector (LVS ΔcapB) and vaccines were grown on Chocolate agar supplemented without or with kanamycin (7.5 μg/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 1000×g 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 μg/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 (FIG. 3). At five hours post infection (FIG. 3A), double protein bands of ˜51-52 kDa (expected size of the Ba fusion protein) were detected from THP-1 cells infected with rLVS ΔcapB/BaLFPA(L1) (lane 4), but not from the uninfected THP-1 cells (lane 2), or THP-1 cells infected with the parental LVS ΔcapB (lane 3), rLVS ΔcapB/YpF1V(D) (lane 5) or the rLVS ΔcapB/YpF1V(L1) (lane 6) strains. The mAb antibody to PA also detected the full length PA protein and its degraded forms (lane 8). Also at five hours post infection (FIG. 3A), a major single protein band of ˜50 kDa (expected size of the Yp fusion protein) was also detected from THP-1 cells infected with rLVS ΔcapB/YpF1V(L1) (lane 5) and the rLVS ΔcapB/YpF1V(L1) (lane 6), similar to the positive control of F1-LcrV monomer protein (lane 9). At 24 h post infection (FIG. 3B), the fusion protein of B. anthracis LFD1-PAD4 (lane 4) was expressed at a level similar to that expressed at 5 hours post infection; the fusion protein of Y. pestis F1-LcrV (lane 5) and Y. pestis F1-GGSG-LcrV (lane 6) were expressed to a level higher than that detected at 5 hours post infection. These results indicated that the F. tularensis LVS ΔcapB platform could be used as a multi-valent vaccine platform for expressing B. anthracis and Y. pestis antigens.

Demonstration that F. tularensis rLVS ΔcapB Expressing Fusion Proteins of B. anthracis and Y. pestis Grow SImilarly to the Parental LVS ΔcapB in Infected THP-1 Cells

To examine the growth kinetics of the rLVS ΔcapB vaccine candidates expressing B. anthracis and Y. pestis antigens, we infected THP-1 cells as described above for FIG. 3. At various times post infection, we lysed the cells; serially diluted the lysates, plated on chocolate agar supplemented with kanamycin, incubated at 37° C. in a CO2 incubator for 3-5 days, and enumerated colonies. As shown in FIG. 4, the rLVS ΔcapB strains expressing B. anthracis or the Y. pestis antigens grew similarly to the parental LVS ΔcapB strain in infected THP-1 cells.

Demonstration that Immunization with rLVS ΔcapB- and/or rLm ΔactA ΔinlB prfA*—Vectored Ba and Yp Vaccines Includes Antigen-Specific Functional Humoral and Strong LF—Specific T-cell Mediated Immune Responses

Mice immunized with rLVS ΔcapB/BaLF-PA (rLVS ΔcapB/BaLFPA[L1]) or prime-boosted with rLVS ΔcapB/BaLF-PA and rLmprfA*/BaLF-PA (rLm ΔactA ΔinlB 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 (FIG. 5a-5d). Importantly, heterologously prime-boosted mice also produced higher titers of serum antibodies that neutralized anthrax toxin (assayed in mouse macrophage cell line J774A.1) than sham-immunized mice and mice vaccinated with only the LVS ΔcapB-vectored vaccine (data not shown). Priming with LVS ΔcapB-vectored Ba vaccine with or without boosting with Lm-vectored Ba vaccines induces Francisella (HI-LVS) and anthrax antigen (LF>PA)-specific Th1- and Th2-type cytokine secretion (FIG. 5e-5h) and LF- and HI-LVS specific multifunctional CD4 T cells (data not shown). In contrast, AVA vaccine immunization yielded little or no cytokine secretion to Ba antigens. Homologous prime-boosting with LVS ΔcapB/YpF1-V (LVS ΔcapB/YpF1V[L1]) [but not rLmprfA */Yp (rLm ΔactA ΔinlB prfA*/hly-YpF1V(L1)] or heterologous prime-boosting with the LVS ΔcapB/YpF1-V (i.n.)+rLmprfA */YpF1-V (i.n. or i.m.) vaccines similarly induce Yp F1/LcrV antigen-specific antibody responses (FIG. 6A) and T-cell mediated immune responses (FIG. 6B).

Demonstration that Immunization with Francisella- and Lm-Vectored B. anthracis Vaccines Induces Antigen specific T-cell Mediated Immune Responses

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 ΔcapB/BaLFPA(L1) intranasally twice, or primed with rLVS ΔcapB/BaLFPA(L1) intranasally and boosted twice with rLmprfA */hly-BaLFPA intramuscularly produced greater amounts of interferon gamma (IFN-γ) than sham-immunized mice, mice immunized only once with rLVS ΔcapB/BaLFPA(L1) intranasally, or mice primed with rLVS ΔcapB/BaLFPA(L1) intranasally and boosted twice with rLmprfA*/hly-BaLFPA intranasally, although the difference did not reach statistical significance (FIG. 7A). In response to LF, spleen cells from mice primed with rLVS ΔcapB/BaLFPA(L1) intranasally and boosted twice with rLmprfA*/hly-BaLFPA intramuscularly produced significantly greater amounts of IFN-γ than mice from all other groups (FIG. 7B); lung cells from heterologously prime-boosted mice, whether the boost was intranasal or intramuscular, also produced the highest amounts of IFN-γ in response to LF (FIG. 7E). In response to HI-LVS, spleen and lung cells from all mice immunized with rLVS ΔcapB/BaLFPA(L1), whether once or twice, or whether heterologously boosted or not, produced significantly greater amounts of IFN-γ than sham-immunized mice or mice immunized with the AVA vaccine (FIGS. 7C and F). Consistently, spleen and lung cells from mice primed with Francisella and boosted with Lm vaccines produced significantly greater frequencies of T cells producing IFN-γ, TNF, IL-2, and IL17, especially in response to LF and HI-LVS (FIGS. 8 & 9). Spleen and lung cells from mice primed with Francisella and boosted with Lm vaccines produced significantly greater frequencies of multifunctional CD4+ T cells expressing IFN-γ, TNF and IL2 in response to in vitro stimulation with PA and LF antigens, especially LF antigen (FIGS. 10 & 11, Panels A & B); lung cells from mice prime-boosted by these vaccines intranasally also produced large numbers of multifunctional CD4+ T cells expressing IFN-γ, TNF, IL2, and IL17 in response to in vitro stimulation with PA and LF antigens (FIG. 11, Panels A & B). Spleen and lung cells from mice primed with Francisella-vectored vaccines produced significantly greater frequencies of multifunctional CD4+ T cells in response to HI-LVS (FIGS. 10 & 11, Panel C). In contrast, in response to stimulation with PMA, spleen and lung cells from all groups produced comparable frequencies of cytokine-producing CD4+ T cells (FIGS. 8, 9, 10 & 11). These results indicate that priming with Francisella-vectored B. anthracis vaccines and boosting with Lm-vectored B. anthracis vaccines induce Francisella and anthrax antigen specific Th1-type cytokine secretion and multifunctional CD4 T cells.

Demonstration that Systemic Homologous Vaccination with rLVS ΔcapB/Ba LFPA(L1) and Both Systemic and i.n. Heterologous Prime-Boost Vaccination with rLVS ΔcapB/BaLFPA(L1) and rLm ΔactA ΔinlB prfA*/hly-BaLFPA Induces Strong Protective Immunity Against Respiratory Challenge wih Ba and protection is Superior to Systemic Homologous Boosting with the AVA Vaccine

Mice were sham-immunized, or immunized subcutaneously twice with AVA (Biothrax, 0.025m1), i.d. or i.n. twice with rLVS ΔcapB/Ba (rLVS ΔcapBIBaLFPA(L1)) or prime-boosted with rLVS ΔcapB/Ba once and rLm/Ba (rLm ΔactA ΔinlB prfA*/hly-BaLFPA) once systemically (i.d./i.m.) or mucosally (i.n./i.n.) (106 of each) at Week 0 and 4, as indicated in FIG. 12, challenged at week 8 with Ba Ames spores (5× LD50), and monitored for survival. Homologous vaccination i.d. and heterologous prime boosting by either route of immunization showed significant protection and heterologous prime-boost vaccination was significantly better than the AVA vaccine (P=0.03 and 0.006 by mucosal and systemic route, respectively).

Demonstration that Systemic Heterologous Prime-Boost Vaccination with rLVS ΔcapB/YpF1V and rLm ΔactA ΔinlB prfA*/hly-YpF1V Protects Against Respiratory Challenge with Yp (CO92)

Mice were sham-immunized, or immunized subcutaneously with EV76 (106), i.d. or i.n. twice with rLVS ΔcapB/Yp (rLVS ΔcapB/YpF1V[L1]) or prime-boosted with rLVS ΔcapB/Yp once and rLm/Yp (ΔactA ΔinlB prfA*/hly-YpF1V) once systemically (i.d./i.m.) or mucosally (i.n./i.n.) (106 of each) at Week 0 and 4, as indicated in FIG. 13, challenged 5 weeks later with Yp (CO92, 10×LD50), and monitored for survival. Homologous or heterologous prime-boosting by the systemic route (intradermally/intramuscularly), with only one boost, showed significant protection (P=0.02/0.01), albeit less than EV76.

Demonstration that Homologous Prime-Boost Vaccination with rLVS ΔcapB/BaLFPA(L1) or with rLm ΔactA ΔinlB prfA*/hly-BaLFPA(L1) and Heterologous Prime-Boost Vaccination with rLVS ΔcapB/BaLFPA(L1)—rLm ΔactA ΔinlB prfA*hly-BaLFP A(L1) via Systemic and Mucosal Routes Induce Antigen-Specific Functional Humoral Immune Responses

As described and indicated in the legend to FIG. 28, mice were sham-immunized, or immunized subcutaneously (s.c.) with AVA three times at weeks 0, 4, and 8 (Biothrax, 0.025m1), i.d. or i.n. with rLVS ΔcapB/BaLFPA three times at weeks 0, 4, and 8 (rLVS ΔcapB/BaLFPA[L1]) or rLm/BaLFPA (rLm ΔactA ΔinlB prfA*/hly-BaLFPA), or primed i.n. or i.d. with rLVS ΔcapB/BaLFPA once at week 0 and boosted i.n. or i.m. with rLm/BaLFPA (106 of each) once at week 0 or twice at weeks 4 and 8, bled at week 11, challenged at week 12 and monitored for 3 weeks. Sera were analyzed for antigen specific antibody. Similar to mice immunized with AVA, mice homologously primed-boosted (immunized three times) with rLVS ΔcapB/BaLFPA (rLVS ΔcapB/BaLFPA[L1]) and heterologously primed-boosted with rLVS ΔcapB/BaLFPA-rLm/BaLFPA (rLm ΔactA ΔinlB prfA*/hly-BaLFPA) produced significantly greater amounts of Ba PA antigen—specific serum IgG antibody than sham-immunized mice, dominated by subtype IgG2a (FIG. 28a). Mice homologously primed-boosted (immunized three times) with rLm/BaLFPA (rLm ΔactA ΔinlB prfA*/hly-BaLFPA) showed increased IgG and IgG2a when immunized by either the i.n. or i.m. routes and increased IgG1 when immunized by the i.m. route, but the differences were not statistically significant. In contrast to mice immunized with AVA, mice homologously primed-boosted with rLVS ΔcapB/BaLFPA or rLm/BaLFPA, and mice heterologously primed-boosted with rLVS ΔcapB/BaLFPA—rLm/BaLFPA produced significantly greater amounts of Ba LF antigen—specific serum IgG antibody than sham-immunized mice, balanced between subtypes IgG1 and IgG2a (FIG. 28b).

Demonstration that Both Systemic and Mucosal Homologous Prime-Boost Vaccination with rLVS ΔcapB/BaLFPA or rLm ΔactA ΔinlB prfA*/hly-BaLFPA and Heterologous Prime-Boost Vaccination with rLVS ΔcapB/BaLFPA—rLm ΔactA ΔinlB 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

Mice were immunized as described above and as indicated in FIG. 29, challenged at week 12 with Ba Ames spores (371,000 CFU), and monitored for 21 days for survival. Mice homologously primed-boosted i.d. with rLVS ΔcapB/BaLFPA or i.m. or i.n. with rLm ΔactA ΔinlB prfA*/hly-BaLFPA and heterologously primed with rLVS ΔcapB/BaLFPA and boosted with rLm ΔactA ΔinlB prfA*/hly-BaLFPA by either route of immunization showed significantly increased survival compared with sham immunized mice. In contrast, survival of mice immunized with the AVA vaccine was not significantly different from that of sham-immunized mice (P=0.3) (FIG. 29).

Demonstration that Systemic and Mucosal Homologous Prime-Boost Vaccination with rLVS ΔcapB/YpF1V and Systemic Heterologous Prime-Boost Vaccination with rLVS ΔcapB/YpF1V—rLm ΔactA ΔinlB prfA*/hly-YpF1V Induce Antigen-Specific Humoral Immune Responses

Mice were sham-immunized, or immunized subcutaneously (s.c.) with EV76 (106) once, i.n. or i.d. with rLVS ΔcapB/YpF1V or rLm ΔactA ΔinlB prfA*/hly-YpF1V three times at weeks 0, 4, and 8, or primed i.d. with rLVS ΔcapB/YpF1V once at week 0 and boosted i.m. with rLm ΔactA ΔinlB prfA*/hly-YpF1V (106 of each) once at week 4 or twice at weeks 4 and 8, as indicated in FIG. 30, bled at week 11 and sera assayed for antigen-specific antibody. Among all the vaccine regimens tested, systemic (i.d.) homologous prime-boost vaccination with rLVS ΔcapB/YpF1V induced significantly elevated serum antibody to F1 antigen in comparison with Sham-immunized mice, balanced between subtypes IgG1 and IgG2a (FIG. 30A), similar to mice vaccinated with EV76. In response to LcrV antigen, homologous prime-boost vaccination i.n. and i.d with rLVS ΔcapB/YpF1V but not i.m. with rLm ΔactA ΔinlB prfA*/hly-YpF1V, and systemic heterologous prime-boost vaccination induced significantly elevated LcrV-specific IgG antibody in comparison with Sham-immunized mice, balanced between subtypes IgG1 and IgG2a (FIG. 30B).

Demonstration that Systemic Homologous Prime-Boost Vaccination with rLVS ΔcapB/YpF1V Induces Strong Protective Immunity against Yp CO92 Respiratory Challenge

Mice were immunized as described above and in the legend to FIG. 30, challenged at week 12 with Yp CO92 (1,800 CFU), and monitored for 21 days for survival. Mice immunized three times i.d. with rLVS ΔcapB/YpF1V survived significantly longer than sham-immunized mice (P=0.03); mice primed-boosted with rLVS ΔcapB/YpF1V—rLm ΔactA ΔinlB prfA*/hly-YpF1V also survived longer than sham-immunized mice, although the difference was not statistically significant (FIG. 31).

Example 2Construction and Characterization of LVS ΔcapB 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

A. Recombinant LVS ΔcapB Overexpressing the Fusion Protein of IglA, IglB, and IglC

Rationale and Construction of LVS ΔcapB Overexpressing the Fusion Protein of IglA, IglB, and IglC as a Vaccine Candidate against F. tularensis

Previously, we have constructed recombinant LVS ΔcapB (rLVS ΔcapB) 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 ΔcapBlgro-iglA, rLVS ΔcapBlgro-iglB, rLVS ΔcapBlgro-iglC and rLVS ΔcapB/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 ΔcapBlgro-iglA and rLVS ΔcapBlgro-iglC survive longer than sham-immunized mice and mice immunized with the parental rLVS ΔcapB vaccine against lethal aerosol challenge with virulent F. tularensis Schu S4 strain (27). To improve the potency of the rLVS ΔcapB 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 ΔcapB 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 ΔcapB 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 (FIG. 14A).

The translated amino acid sequences of IglABC(D), IglABC(GGSG), and IglABC(2GGSG) are listed below:

A. IglABC(D)(SEQ ID NO: 21)MLVVGDLSKGRSVDAKKEFADREVRRVNNGVDRVLEEMNISFDFEAPNFVSKDRSNLKVNYRIESVKDFRPDAVAKKVPEIRALLEMKEILASFAKDIENNQLPLEMARYPFRNVSIEVETIPGKPGWYSCKINVIPHIQFEGMNITMTIDIRLEPELFGINNNTGNCRLFIDSLTIAGEKLDKNIVAIDGGEDVIKADSATAAASVIRLSITPGSINPTISITLGVLIKSNVRTKIEEKVSSILQASATDMKIKLGNSNKKQEYKTDEAWGIMIDLSNLELYPIB. IglABC(GGSG)(SEQ ID NO: 22)MLVVGDLSKGRSVDAKKEFADREVRRVNNGVDRVLEEMNISFDFEAPNFVSKDRSNLKVNYRIESVKDFRPDAVAKKVPEIRALLEMKEILASFAKDIENNQLGGSGPLEMARYPFRNVSIEVETIPGKPGWYSCKINVIPHIQFEGMNITMTIDIRLEPELFGINNNGGSGTGNCRLFIDSLTIAGEKDKNIVAIDGGEDVIKADSATAAASVIRLSITPGSINPTISITLGVLIKSNVRTKIEEKVSSILQASATDMKIKLGNSNKKQEYKTDEAWGIMIDLSNLELYPIC. IglABC(2GGSG)(SEQ ID NO: 23)MLVVGDLSKGRSVDAKKEFADREVRRVNNGVDRVLEEMNISFDFEAPNFVSKDRSNLKVNYRIESVKDFRPDAVAKKVPEIRALLEMKEILASFAKDIENNQLGGSGGGSGPLEMARYPFRNVSIEVETIPGKPGWYSCKINVIPHIQFEGMNITMTIDIRLEPELFGINNNGGSGGGSGTGNCRLFIDSLTIAGEKLDKNIVAIDGGEDVIKADSATAAASVIRLSITPGSINPTISITLGVLIKSNVRTKIEEKVSSILQASATDMKIKLGNSNKKQEYKTDEAWGIMIDLSNLELYPI

Demonstration that rLVS ΔcapB Overexpresses IglC and IglIABC in Broth and in Infected Human Macrophage-like THP-1 Cells

To examine the protein expression by rLVS ΔcapB, 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 ΔcapB/bfr-iglA, rLVS ΔcapB/bfr-iglB, and rLVS ΔcapB/bfr-iglC was 1.5-1.6-fold higher than that of the parental LVS ΔcapB (FIG. 14B, 14C). IglABC fusion proteins, whether the three protein residues were directly fused or linked by flexible linkers, were expressed by each rLVS ΔcapB/bfr-iglABC and detected by antibody to IglA or IglC; fusion protein expression was higher when the protein residues were linked by flexible linkers than when fused directly, and the IglABC fusion protein with two linkers had a higher expression level than the one with one linker. To examine further the protein expression by rLVS ΔcapB, we infected PMA-differentiated THP-1 cells with rLVS ΔcapB overexpressing IglA, IglB, IglC, or IglABC under the control of the bfr promoter (rLVS ΔcapB/bfr-iglA, rLVS ΔcapB/bfr-iglB, rLVS ΔcapB/bfr-iglC, and rLVS ΔcapB/bfr-iglABC) for 72 hours, lysed the cells, and analyzed the cell lysates for F. tularensis protein expression by Western blotting using polyclonal antibodies specific to IglA, IglB, or IglC. Our results showed that IglC expression by rLVS ΔcapB/pbfr-iglC was approximately 1.7-fold higher than the parental LVS ΔcapB; IglA and IglB expression by rLVS ΔcapB/pbfr-iglA and rLVS ΔcapB/pbfr-iglB, respectively, were equivalent to LVS ΔcapB; and the IglABC fusion protein, whether the three protein residues were directly fused or linked by flexible linkers, was expressed by rLVS ΔcapB/bfr-iglABC (data not shown) (FIG. 14B). rLVS ΔcapB/bfr-iglABC induced greater IglC- and HI-LVS-specific humoral and cell-mediated immune responses than rLVS ΔcapB expressing IglABC linked directly or by GGSGGGSG (SEQ ID NO: 63) (data not shown). We chose the rLVS ΔcapB/bfr-iglABC for further analysis.

Demonstration that rLVS ΔcapB Overexpressing IglABC (rLVS ΔcapB/bfr-iglABC) Grows Similarly to the Parental LVS ΔcapB in Broth Culture and Intracellularly in Infected Human Macrophage-like THP-1 Cells

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 ΔcapB 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 FIG. 15, rLVS ΔcapB strains overexpressing IglA, IglB, or IglC under the control of the groE, bfr or omp promoter grew similarly to LVS and LVS ΔcapB in TSBC (FIG. 15A-C) and CDM (FIG. 15D-F). Moreover, these vaccines grew similarly in PMA-differentiated THP-1 cells (FIG. 15G-I). These results indicate that the shuttle plasmids carried by the rLVS ΔcapB vaccines did not affect their growth kinetics. The shuttle plasmids were stable under non-selective conditions in infected THP-1 cells (FIG. 15).

Demonstration that a) rLVS ΔcapB Overexpressing IglA, IglB, IglC are Safe in Mice; b) rLVS ΔcapB Overexpressing IglA, IglB, IglC Disseminate and are Cleared Similarly to the Parental rLVS ΔcapB Strain in Mice; and c) the Shuttle Plasmid for Overexpressing IglA, IglB, or IglC is Stable in Mice

To evaluate the safety, dissemination and clearance of rLVS ΔcapB/iglA, iglB, iglC, and iglABC, we infected BALB/c mice i.n. or i.d. with LVS, LVS ΔcapB, or rLVS ΔcapB/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 FIG. 16, after i.n. vaccination, rLVS ΔcapB/bfr-iglA, iglB, iglC, and iglABC peaked in the spleen (FIG. 16A), liver (FIG. 16B), and lung (FIG. 16C) at Day 4 post-vaccination and were largely cleared by most mice at day 14 post-vaccination, similar to parental LVS ΔcapB in all three organs. LVS grew to much higher levels (1-2 logs higher) at Day 4 and/or Day 7 and maintained higher levels through Day 14, at which point its level was >1 log higher than the other vaccines in all three organs. After i.d. vaccination (FIG. 16D-H), rLVS ΔcapB/pbfr-iglA, iglB, iglC, and iglABC peaked at Day 4 post-vaccination in the spleen and liver and had minimal growth in the lung, and these vaccines were cleared from spleen, liver, and lung of all mice at Day 14 post-vaccination. In the local skin, the rLVS ΔcapB strains were detected on Day 1 and Day 4 post-vaccination and cleared by all mice at Day 7 post-vaccination. In the inguinal lymph nodes, the rLVS ΔcapB strains were detected at 1, 4, and 7 days post-vaccination and cleared (Limit of Detection) at Day 14 post-vaccination, similarly to the parental LVS ΔcapB. LVS grew to higher levels in all these sites, peaking at Day 4 (spleen, liver, skin, and lymph nodes) or Day 7 (lung) and was not cleared from the spleen and lymph nodes by Day 14. Both i.n. and i.d. vaccination with rLVS ΔcapB strains did not induce any signs of illness, indicating that these vaccines were as safe as the LVS ΔcapB parental strain. In contrast, ˜25% of mice immunized i.n. with 200 CFU LVS died and mice immunized i.d. with 106 CFU LVS showed ruffled fur in some of our experiments, evidence of toxicity of LVS by both the i.n. and i.d. route in BALB/c mice.

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 ΔcapB Overexpressing IglABC are well Tolerated by Mice after Intranasal Vaccination

To test the safety of rLVS ΔcapB/bfr-iglABC at relatively high doses, we immunized BALB/c mice intranasally, 4 per group, with six different doses of rLVS ΔcapB/bfr-iglABC, ranging from 1×107 to 4×108 CFU/mouse. Mice immunized with 1-2.5×107 CFU of rLVS ΔcapB/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 ΔcapB/bfr-iglABC 5-20×107 CFU lost 6-12% of their weight at 3-4 days post immunization (FIG. 17) and showed signs of moderate illness (ruffled fur and inactive). However, all the mice immunized with rLVS ΔcapB/bfr-iglABC recovered by 7 days post immunization and none of them died. These results suggest that intranasal immunization with as many as 4×108 CFU rLVS ΔcapB/bfr-iglABC is reasonably safe.

Demonstration that Immunization with rLVS ΔcapB Overexpressing FPI T6SS Proteins Induces Antigen-Specific Cytokine Production and Th1-Type Antibody Responses

To examine T cell immune responses generated by the rLVS ΔcapB 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 ΔcapB/iglC or iglABC secreted greater amounts of IL-17A (FIG. 18A) and IFN-γ (FIG. 18B) into the culture supernatant than sham- or LVS ΔcapB-immunized mice, although the differences did not reach statistical significance. In response to in vitro stimulation with HI-LVS, splenocytes from mice immunized with rLVS ΔcapB/iglC or iglABC also secreted greater amounts of IL-17A into the culture supernatant than sham- or LVS ΔcapB-immunized mice, comparable to splenocytes from LVS-immunized mice. Consistently, splenocytes from these mice generated significantly greater frequencies of Th1-type CD4+ T cells expressing IFN-γ (FIG. 19A), or IFN-γ+TNF (FIG. 19B), TNF+IL-2, or IFN-γ+TNF+IL-2 in response to in vitro stimulation with IglC, IglC peptide or HI-LVS than splenocytes from LVS ΔcapB-immunized mice (data not shown). Splenocytes from mice immunized with rLVS ΔcapB/bfr-iglABC showed the highest frequencies of Th1-type CD4+ T cells expressing IFN-γ (FIG. 19A) or IFN-γ+TNF (FIG. 19B) in response to in vitro stimulation with IglC and HI-LVS. However, splenocytes from LVS ΔcapB- and rLVS ΔcapB-immunized mice had lower frequencies of CD8+ IFN-γ+ T cells in response to HI-LVS (FIG. 19C) than splenocytes from LVS-immunized mice. With respect to humoral immune responses, all vaccine candidates induced HI-LVS-specific IgG2a and IgG1 antibodies (differences not statistically significant) (FIG. 18C). Thus, overall, the rLVS ΔcapB vaccines overexpressing IglC or the fusion protein of IglABC had enhanced T-cell mediated immune responses.

Demonstration that Immunization with rLVS ΔcapB/bfr-iglABC Induces Improved Protective Immunity Against Respiratory Challenge with F. tularensis Schu S4

To evaluate rLVS ΔcapB/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 ΔcapB, or rLVS ΔcapB, challenged them 7 weeks later i.n. with a high lethal dose ofF. tularensis Schu S4 (16 CFU, equivalent to 5×LD50), and observed the mice closely for signs of illness and death. Mice immunized with rLVS ΔcapB/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 ΔcapB or rLVS ΔcapB/bfr-iglA, iglB, or iglC; (mean survival time 6.6-8.0 days; difference not statistically significant (FIG. 20A).

To verify further the efficacy of rLVS ΔcapB 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 LD50, respectively. Consistently, after i.n. challenge with 31 CFU of Schu S4, mice immunized with rLVS ΔcapB/bfr-iglA , iglB, iglC, or iglABC survived longer than sham-immunized mice (P<0.0001) and generally longer than LVS ΔcapB-immunized mice (differences not significant). In this experiment, mice immunized with rLVS ΔcapB/bfr-iglA were especially well protected—mean survival time 11.6 days vs. 4 days for sham-immunized mice and 7.1 days for LVS ΔcapB-immunized mice—but not as well protected as LVS-immunized mice (mean survival time 15.8 days), but this difference was not statistically significant (FIG. 20B, upper section). After i.n. challenge with 310 CFU of Schu S4, although all mice died by day 6 post-challenge, mice immunized with rLVS ΔcapB/bfr-iglABC survived significantly longer than all other mice including LVS-immunized mice (FIG. 20B, lower section). Mice immunized with rLVS ΔcapB/bfr-iglA or iglB survived significantly longer than sham- and LVS ΔcapB-immunized mice, comparable to LVS-immunized mice.

To explore further the efficacy of rLVS ΔcapB/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 FIG. 21, mice immunized with rLVS ΔcapB/bfr-iglABC survived significantly longer than sham-immunized mice, comparable to LVS-immunized mice (FIG. 21A). Mice immunized with rLVS ΔcapB/bfr-iglABC that survived challenge recovered from temporary weight loss such that their weights were similar to mice immunized with LVS by 2 weeks post-challenge (FIG. 21B).

Demonstration that Immediate Pre- or Post-Exposure Prophylaxis with rLVS ΔcapB/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

To evaluate the efficacy of rLVS ΔcapB/bfr-iglABC as a pre- or post-exposure vaccine, we immunized mice with PBS (Sham), 100 CFU LVS, or 1×106 or 5×106 CFU rLVS ΔcapB/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 (FIG. 22A, upper section). In contrast, mice immunized with 1×106 CFU of rLVS ΔcapB/bfr-iglABC at −2 days were not sick until day 6 post-challenge and survived significantly longer (mean survival time 10.5 days) than both sham- and LVS-immunized mice (mean survival time 5 days). Mice immunized with 1×106 CFU or 5×106 CFU (FIG. 22A, lower section) of rLVS ΔcapB/bfr-iglABC on the day of challenge (Day 0) also survived significantly longer (mean survival times 5.8 and 6.4 days, respectively) than both sham-immunized mice and mice immunized with LVS at Day 0. When immunized at 1 or 2 days post-challenge, mice immunized with rLVS ΔcapB/bfr-iglABC had mean survival times comparable to sham- and LVS-immunized mice (FIG. 22A, lower section).

To explore the efficacy of a higher dose of rLVS ΔcapB/bfr-iglABC as immediate pre- and post-exposure prophylaxis against Schu S4 challenge, we repeated the experiment described above with an immunizing dose of 5×107 CFU (tested as safe, FIG. 17). The extent of protection was directly dependent upon the time relative to challenge, with mice immunized sooner surviving longer. Mice immunized with 5×107 CFU rLVS ΔcapB/bfr-iglABC at −2, −1, or even 1 day post-challenge with Schu S4 survived significantly longer than sham-immunized mice (FIG. 22B, middle and right panel).

To explore the mechanism of protection provided by immediate pre-exposure prophylaxis with rLVS ΔcapB/bfr-iglABC, we immunized mice in groups of 3 i.n. with PBS (Sham), 102 CFU LVS, or 1×106 or 5×107 CFU rLVS ΔcapB/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 1×106 or 5×107 rLVS ΔcapB/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 (FIG. 23A-23D); many of the cytokines in rLVS ΔcapB/bfr-iglABC-immunized mice were orders of magnitude higher than in sham- and LVS-immunized mice. In most but not all cases, mice immunized with the higher dose of rLVS ΔcapB/bfr-iglABC had higher cytokine/chemokine levels than mice immunized with the lower dose of this vaccine. In contrast, there were no significant differences in cytokine/chemokine levels between LVS- and sham-immunized mice in the lungs or sera (FIG. 23A-23D). These results show that mice immunized with rLVS ΔcapB/bfr-iglABC, which could be administered safely at very high doses, rapidly develop a strong innate immune response in the lung and blood, whereas mice immunized with LVS, which could be administered safely at only very low doses, do not. Taken together with the above results on the efficacy of immediate pre-exposure prophylaxis, where rLVS ΔcapB/bfr-iglABC but not LVS provided near-term protection, these data show that the level of near-term protection correlates with the level of the innate immune response.

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

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 ΔcapB or LVS ΔcapB overexpressing F. tularensis IglA or IglC as the prime vaccine and rLm/iglC as the booster vaccine. We show that the LVS ΔcapB-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).

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 ΔactA or Lm ΔactA ΔinlB 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 ΔactA/hly-iglABC, rLm ΔactA/actA-iglABC, rLm ΔactA ΔinlB prfA*/hly-iglABC, and rLm ΔactA ΔinlB prfA*/actA-iglABC. We have shown that all four rLm vaccine candidates express the F. tularensis IglABC (FIG. 24).

Demonstration that Priming with rLVS ΔcapB/bfr-iglABC and Boosting with rLmprfA*/hly-iglABC Induces Antigen Specific T Cell-Mediated and Humoral Immune Responses

To examine the immune responses induced by priming with rLVS ΔcapB/bfr-iglABC and boosting with rLmprfA*/hly-iglABC (rLm ΔactA ΔinlB prfA*/hly-iglABC), we immunized mice i.d. at week 0 with rLVS ΔcapB/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 ΔcapB/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 ΔcapB/bfr-iglABC and boosted with rLmprfA*/hly-iglABC produced significantly greater frequencies of CD4+ T cells expressing IFN-γ, TNF, or IL-2 in their spleens (FIG. 25A, 25B, 25C) and lungs (FIG. 25D, 25E, 25F) than sham-immunized mice, mice immunized with only rLVS ΔcapB/bfr-iglABC, or mice immunized with only rLm/hly-iglABC. In response to stimulation with HI-LVS, mice primed with rLVS ΔcapB/bfr-iglABC and boosted with rLmprfA*/hly-iglABC produced significantly greater frequencies of CD4+ T cells expressing IFN-γ, TNF, or IL-2 in their spleens and lungs than sham-immunized mice and mice immunized with only the booster vaccine, rLm/hly-iglABC, and comparable to that produced by mice immunized with only the prime vaccine, rLVS ΔcapB/bfr-iglABC (FIG. 25).

After analysis of the 7 possible combinations of CD4+ T cells producing one or more cytokines among IFN-γ, 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-γ, 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 ΔcapB/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 ΔcapB/bfr-iglC—rLm/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 ΔcapB/bfr-iglC—rLm vector (FIG. 26A). The IgG antibody is balanced between IgG1 and IgG2a (FIG. 26B and 26C).

Demonstration that Priming with rLVS ΔcapB/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 ΔcapB/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 ΔcapB/bfr-iglABC (1×106 CFU) at Week 0 and boosted them once at Week 4 or twice at Week 4 and 6 with rLm vaccines (1×106 CFU) expressing IglABC where the genes encoding IglABC are expressed by the LmprfA* vector (Lm ΔactA ΔinlB 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 (1×106 CFU). The mice were challenged intranasally with 29 CFU of F. tularensis Schu S4 (equivalent to 10×LD50) 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 times—both with MST of 9.4 days.
C. Homologous Prime-Boosting with rLVS ΔcapB/bfr-iglABC and Heterologous Prime-Boosting with rLVS ΔcapB/bfr-iglABC as the Prime Vaccine and rLm/iglABC as the Booster Vaccine
Demonstration that Homologous Prime-Boosting with rLVS ΔcapB/bfr-iglABC or Heterologous Prime-Boosting with rLVS ΔcapB/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.

To further evaluate homologous prime-boosting with rLVS ΔcapB/hfr-iglABC or heterologous prime-boosting with rLVS ΔcapB/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), 104 CFU LVS (Group B), 106 CFU LVS ΔcapB vector (Group H), or 106 rLVS ΔcapB/bfr-iglABC (Group C); immunized them i.d. or i.n. twice at weeks 0 and 4 with 106 rLVS ΔcapB/bfr-iglABC (Groups D & E, resp.); primed them i.d. once at week 0 with rLVS ΔcapB/bfr-iglABC (Groups F & G) or LVS ΔcapB (Groups I & J) and boosted them intramuscularly (i.m.) twice at weeks 4 and 6 with rLmprfA* Vector (Lm ΔactA ΔinlB prfA*) (Groups F & I) or rLmprfA*/hly-iglABC (Groups G & J); or immunized them simultaneously i.d. with rLVS ΔcapB/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 FIG. 27, mice immunized i.d. once or twice with rLVS ΔcapB/bfr-iglABC and mice immunized i.n. twice with rLVS ΔcapB/bfr-iglABC (Groups C, D, and E, respectively) survived significantly longer than sham-immunized mice (P=0.03, P=0.0001, and P=0.0003, respectively) and survived significantly longer than mice immunized once with the LVS ΔcapB vector (P=0.006, P=0.0001, and P=0.0005, respectively); the survival of mice immunized i.d. or i.n. twice with rLVS ΔcapB/bfr-iglABC (Groups D and E) was equivalent to mice immunized with LVS (Group B) (Panel a). Mice primed with rLVS ΔcapB/bfr-iglABC or LVS ΔcapB vector and boosted with LmprfA* vector or rLmprfA*/hly-iglABC (Groups F, G, I and J) survived significantly longer than sham-immunized mice (P=0.0001, P=0.0001, P=0.0001, and P=0.0001, respectively); the survival of mice primed once with rLVS ΔcapB/bfr-iglABC and boosted twice with rLmprfA*/hly-iglABC (Groups G) was not statistically different from LVS-immunized mice. Mice immunized simultaneously with rLVS ΔcapB/bfr-iglABC and rLmprfA*/hly-iglABC (Groups K) survived longer than sham-immunized mice and their survival was not statistically different from LVS-immunized mice (Panel b).

Aspects of Working Embodiments with Antigenic Polypeptide Epitopes Present in a Francisella tularensis IglA, IglB, and IglC Epitopoes

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).

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 [FIG. 4]. IglA is localized to the cytoplasm of Ft and its expression/stability is dependent on IglB. In particular, residues 33 to 132 of IglA are necessary for efficient binding to IglB, for the stability of IglB and for the intramacrophage growth of LVS. The T-cell epitopes of IglB were recently found to be within residues 454-514, especially residues 471-514. Bioinformatic analysis identifies the FPI including IglA, IglB, and VgrG, IcmF/pdpB, and DotU as a putative Type VI secretion system (T6SS) cluster as found in pathogens like Pseudomonas aeruginosa, Aeruginosa hydrophila, Vibrio cholerae, enteroaggregative E. coli, S. typhimurium, and others. The IglA and IglB homologues VipA and VipB in V. cholerae are shown to form tubular structures that are remodeled by ClpV into small complexes, a proposed essential step in T6SS formation. We show that IglA/IglB heterodimers assemble to form Francisella T6SS sheaths. Ft VgrG, significantly smaller than any known VgrG and lacking an active C-terminal domain, is secreted into Ft culture supernatant and the cytosol of infected macrophages. In view of these discoveries, one focus will be on tularemia vaccines expressing these highly immunogenic secreted FPI-encoded Ft proteins.

Fustion Protein Promoters

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 ΔcapB strains expressing Ft-Ag downstream of the bfr promoter.

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 ΔcapB 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 (FIG. 32)

Route of Immunization (e.g., Intradermal [I.D.] vs. Intranasal [I.N.])

We have tested systemic routes (intradermal [i.d.] or intramuscular [i.m.]) vs. mucosal route of immunization (i.n.).

B. anthracis: As shown in FIGS. 12 & 29, homologous prime-boost vaccination with rLVS ΔcapB/Ba and heterologous prime-boost vaccination with rLVS ΔcapB/Ba—rLm/Ba vaccines by either systemic (intradermal) or mucosal (intranasal) route induces strong protection against challenge with Ba Ames spores.

Y. pestis: As shown in FIGS. 13 & 31, heterologous prime-boost vaccination with rLVS ΔcapB/Yp—rLm/Yp vaccines by the systemic (intradermal/intramuscular) route is superior to mucosal (intranasal) route against challenge with virulent Y. pestis CO92 challenge. Three doses of systemic (intradermal) homologous prime-boost vaccination with rLVS ΔcapB/Yp is also superior to the same vaccines administered by the intranasal route (FIG. 31).

F. tularensis: Mucosal route (intranasal) is generally superior to systemic (intradermal) route. However, two doses of rLVS ΔcapB/bfr-iglABC is comparable to LVS at the safe dose by the intradermal route (FIG. 27).

TABLES

TABLE 1Tier 1 Select AgentsFrancisella tularensis (Tularemia)Bacillus anthracis (Anthrax)Yersinia pestis (Plague)Burkholderia pseudomallei (melioidosis)Burkholderia mallei (glanders)Category B PathogensCoxiella burnetii (Q Fever)Brucella species (Brucellosis)Chlamydia psittaci (Psittacosis)Rickettsia prowazekii (Typhus)Listeria monocytogenes (Listeriosis)Campylobacter jejuni (Gastroenteritis)Yersinia enterocolitica (Yersiniosis)Additional PathogensChlamydia pneumoniaChlamydia trachomatisMycoplasma pneumoniaLegionella pneumophilaStaphylococcus aureusStreptococcus pneumoniae

TABLE 2Plasmids, recombinant F. tularensis subsp. holarctica, Listeriamonocytogenes for B. anthracis and Y. pestis vaccinesPlasmidsNameDescriptionReferencepFNLTP6 groE-E. coli-Francisella shuttle vector with gfp driven(13)gfpby Francisella groE promoter; Ampr KanrpBC11pFNLTP6-derived E. coli-Francisella shuttleThisPomp-Ypcaf1-D-vector for expressing the fusion protein of Y. pestisstudylcrVF1 (encoded by caf1) and LcrV (encodedby lcrV) linked directly and downstream of F. tularensisomp promoterpBC12/pFNLTP6-derived E. coli-Francisella shuttleThisPomp-Ypcaf1-vector for expressing the fusion protein of Y. pestisstudyGGSG-lcrVF1 (encoded by caf1) and LcrV (encodedby lcrV) separated by a GGSG linker (SEQ IDNO: 62) and downstream of F. tularensis omppromoterpSG39/pFNLTP6-derived E. coli-Francisella shuttleThisPomp-pepOss-vector for expressing the fusion protein of Y. pestisstudyYpcaf1-GGSG-F1 (encoded by caf1) and LcrV (encodedlcrVby lcrV) separated by a GGSG linker (SEQ IDNO: 62) and downstream of F. tularensis omppromoter and PepO signal peptidepBC14/pFNLTP6-derived E. coli-Francisella shuttleThisPbfr-BalefD1-vector for expressing B. anthracis lethal factorstudyGGSG-pagAD4(encoded by lef) domain 1 and Protective antigen(encoded by pagA) domain 4 separated by aGGSG linker (SEQ ID NO: 62) and downstreamof F. tularensis bfr promoterpSG37/pFNLTP6-derived E. coli-Francisella shuttleThisPomp-pepOss-vector for expressing the fusion protein of B. anthracisstudyBalef1D1-GGSG-lethal factor (encoded by lef) domain 1pagAD4and Protective antigen (encoded by pagA) domain4 with a GGSG linker (SEQ ID NO: 62) anddownstream of F. tularensis omp promoter andPepO signal peptidepSG38/pFNLTP6-derived E. coli-Francisella shuttleThisPomp-katGssvector for expressing the fusion protein of B. anthracisstudyBalef1D1-GGSG-lethal factor (encoded by lef) domain 1pagAD4and Protective antigen (encoded by pagA) domain4 with a GGSG linker (SEQ ID NO: 62) anddownstream of F. tularensis omp promoter andKatG signal peptidepSG28/pPL2-derived conjugation vector for integration(14) &ActAN100-Ypcaf1-of the antigen expression cassette ActAN100-ThisGGSG-lcrVYpcaf1-GGSG-lcrV into the tRNAarg locus of thestudyrLm chromosomepSG29/pPL2-derived conjugation vector pPL2-derived(14) &hly-Ypcaf1-GGSG-conjugation vector for integration of the antigenThislcrVexpression cassette hly-Ypcaf1-GGSG-lcrV intostudythe tRNAarg locus of the rLm chromosomepSG40/pPL2-derived conjugation vector for integration(14) &ActAN100-Balef1-of the antigen expression cassette ActAN100-ThisGGSG-pagA4Balef1-GGSG-pa4 into the tRNAarg locus of thestudyrLm chromosomepSG41/pPL2-derived conjugation vector for integration(14) &hly-Balef1-GGSG-of the antigen expression cassette hly-Balef1-ThispagA4GGSG-pa4 into the tRNAarg locus of the rLmstudychromosomepQJ137/pFNLTP6-derived E. coli-Francisella shuttleThisPhfr-BalefD1-vector for optimized expression in Ft LVS of B. anthracisstudy(GSSG)2-pagAD4lethal factor (encoded by lef) domain 1and Protective antigen (encoded by pagA)extended domain 4, separated by a GSSGGSSGlinker (SEQ ID NO: 65) and downstream of F. tularensisbfr promoterpQJ138/pFNLTP6-derived E. coli-Francisella shuttleThisPbfr-YplcrV-vector for optimized expression in Ft LVS of thestudy(GGGS)3-caf1-fusion protein of Y. pestis LcrV (encoded by(GGGS)3-YscFlcrV), F1 (encoded by caf1), and YscF (encodedby yscF), separated by GGGSGGGSGGGS linker(SEQ ID NO: 66) and downstream of F. tularensisbfr promoterStrainDescriptionReferenceAttenuated recombinant F. tularensis subsp. holarcticaLVSLive vaccine strainCDCLVS ΔcapBUnmarked LVS with deletion of capB(10)rLVS ΔcapB/LVS ΔcapB carrying pBC11/Pomp-Ypcaf1-D-ThisYpF1V(D)lcrVstudyrLVSLVS ΔcapB carrying pBC12/Pomp-Ypcaf1-ThisΔcapB/YpF1V(L1)GGSG-lcrVstudyrLVS ΔcapB/LVS ΔcapB carrying pBC14/Pbfr-BalefD1-ThisBaLFPA(L1)GGSG-pagAD4studyrLVS ΔcapB/LVS ΔcapB carrying pSG39/Pomp-pepOss:ThispepOss-Ypcaf1-GGSG-lcrVstudyYpF1V(L1)rLVS ΔcapB/LVS ΔcapB carrying pSG38/Pomp-katGss-ThiskatGss-Balef1D1-GGSG-pagAD4studyBaLFPA(L1)rLVS ΔcapB/LVS ΔcapB carrying pSG37/Pomp-pepOss-ThispepOss-Balef1D1-GGSG-pagAD4studyBaLFPA(L1)rLVS ΔcapB/LVS ΔcapB carrying pQJ137/Pbfr-BalefD1-ThisBaLFPA(L2)(GSSG)2-pagAD4 optimized for expression in FtstudyLVSrLVS ΔcapB/LVS ΔcapB carrying pQJ138/Pbfr-YplcrV-ThisYpVF1YscF(L3)(GGGS)3-caf1-(GGGS)3-YscF optimized forstudyexpression in Ft LVSAttenuated recombinant Listeria monocytogenesLm ΔactA ΔinlBLm ΔactA ΔinlB ΔuvrAB prfA*(G155S)[7]prfA*rLm ΔactA/actA-rLm ΔactA integrated with the actAN100-Balef1-ThisBaLFPA(L1)GGSG-pa4 antigen expression cassette at thestudytRNAarg locusrLm ΔactA ΔinlBrLm ΔactA ΔinlB ΔuvrAB prfA*(G155S)ThisprfA*/actA-integrated with the actA-Balef1-GGSG-pagA4studyBaLFPAantigen expression cassette at the tRNAarg locusrLm ΔactA/rLm ΔactA integrated with the hly-Balef1-GGSG-Thishly-BaLF-PApagA4 antigen expression cassette at the tRNAargstudylocusrLm ΔactA ΔinlBrLm ΔactA ΔinlB ΔuvrAB prfA*(G155S)ThisprfA*/hly-integrated with the hly-Balef1-GGSG-pa4 antigenstudyBaLFPAexpression cassette at the tRNAarg locusrLm ΔactA/actA-rLm ΔactA integrated with the actAN100-Ypcaf1-ThisYpF1VGGSG-lcrV antigen expression cassette at thestudytRNAarg locusrLm ΔactA ΔinlBrLm ΔactA ΔinlB ΔuvrAB prfA*(G155S)ThisprfA*/actA-integrated with the actAN100-Ypcaf1-GGSG-lcrVstudyYpF1Vantigen expression cassette at the tRNAarg locusrLm ΔactA/hly-rLm ΔactA integrated with the hly-Ypcaf1-GGSG-ThisYpF1VlcrV antigen expression cassette at the tRNAargstudylocusrLm ΔactA ΔinlBrLm ΔactA ΔinlB ΔuvrAB prfA*(G155S)ThisprfA*/hly-YpF1Vintegrated with the hly-Ypcaf1-GGSG-lcrVstudyantigen expression cassette at the tRNA arg locus

TABLE 3Plasmids, recombinant F. tularensis subsp. holarctica, Listeriamonocytogenes for F. tularensis vaccinesPlasmidsNameDescriptionReferencepFNLTP6 groE-gfpE. coli-Francisella shuttle vector[2]with gfp driven by FrancisellagroE promoter; Ampr KanrpFNL/bfr-iglAE. coli-Francisella shuttle vector[34]for over-expressing IglAdownstream of bfr promoterpFNL/bfr-iglBE. coli-Francisella shuttle vector[34]for over-expressing IglBdownstream of bfr promoterpFNL/bfr-iglCE. coli-Francisella shuttle vector[34]for over-expressing IglCdownstream of bfr promoterpFNL/bfr-iglABC(D)E. coli-Francisella shuttle vector[34]for over-expressing IglABCdownstream of bfr promoterpFNL/bfr-iglABC(GGSG)E. coli-Francisella shuttle vector[34]for over-expressing IglABC with aGGSG linker (SEQ ID NO: 62) anddownstream of bfr promoterpFNL/bfr-iglABC(GGSG2)E. coli-Francisella shuttle vector[34]for over-expressing IglABC withGGSG2 linker (SEQ ID NO: 63)and downstream of bfr promoterpFNL/omp-iglAE. coli-Francisella shuttle vector[34]for over-expressing IglAdownstream of omp promoterpFNL/omp-iglBE. coli-Francisella shuttle vector[34]for over-expressing IglBdownstream of omp promoterpFNL/omp-iglCE. coli-Francisella shuttle vector[34]for over-expressing IgICdownstream of omp promoterpFNL/omp-iglABC(D)E. coli-Francisella shuttle vector[34]for over-expressing IglABCdownstream of omp promoterpFNL/omp-iglABC(GGSG)E. coli-Francisella shuttle vector[34]for over-expressing IglABC with aGGSG linker (SEQ ID NO: 62) anddownstream of omp promoterpFNL/omp-iglABC(GGSG2)E. coli-Francisella shuttle vector[34]for over-expressing IglABC withGGSG2 linker (SEQ ID NO: 63)and downstream of omppromoterF. tularensis subsp. holarcticaStrainDescriptionReferenceLVSLive vaccine strainCDCLVS ΔcapBUnmarked LVS with deletion of[10]capBrLVS ΔcapB/gro-iglALVS ΔcapB carrying pFNL/gro-[27]iglArLVS ΔcapB/gro-iglCLVS ΔcapB carrying pFNL/gro-[27]iglCrLVS ΔcapB/gro-iglABCLVS ΔcapB carrying pFNL/gro-[34]iglABCrLVS ΔcapB/bfr-iglALVS ΔcapB carrying pFNL/bfr-iglA[34]rLVS ΔcapB/bfr-iglBLVS ΔcapB carrying pFNL/bfr-iglB[34]rLVS ΔcapB/bfr-iglCLVS ΔcapB carrying pFNL/bfr-iglC[34]rLVS ΔcapB/bfr-iglABC(D)LVS ΔcapB carrying pFNL/bfr-[34]iglABC(D)rLVS ΔcapB/bfr-iglABCLVS ΔcapB carrying pFNL/bfr-[34]iglABC(GGSG)rLVS ΔcapB/bfr-LVS ΔcapB carrying pFNL/bfr-iglABC(GGSG2)[34]iglABC(GGSG2)rLVS ΔcapB/omp-iglALVS ΔcapB carrying pFNL/omp-iglA[34]rLVS ΔcapB/omp-LVS ΔcapB carrying pFNL/omp-iglABC(D)[34]iglABC(D)rLVS ΔcapB/omp-LVS ΔcapB carrying pFNL/omp-iglABC(GGSG)[34]iglABC(GGSG)rLVS ΔcapB/omp-LVS ΔcapB carrying pFNL/omp-iglABC(GGSG2)[34]iglABC(GGSG2)Attenuated recombinant Listeria monocytogenesrLm/iglCLm ΔactA integrated with the hly-iglC antigen[9]expression cassette at the tRNAarg locusrLm ΔactA/hly-Lm ΔactA integrated with the hly-iglABC antigen[34]iglABCexpression cassette at the tRNAarg locusrLm ΔactA/actA-Lm ΔactA integrated with the actA-iglABC antigen[34]iglABCexpression cassette at the tRNAarg locusrLm ΔactA ΔinlBLm ΔactA ΔinlB ΔuvrAB prfA*(G155S) integrated[34]prfA*/hly-iglABCwith the hly-iglABC antigen expression cassetteat the tRNAarg locusrLm ΔactA ΔinlBLm ΔactA ΔinlB ΔuvrAB prfA*(G155S) integrated[34]prfA*/actA-iglABCwith the actA-iglABC antigen expression cassetteat the tRNAarg locusPrimersDescriptionSequence (5′-3′)*[34]SnaB_iglA33_Fagatgctacgtaatgctagttgttggcgatttatca (SEQ ID NO:[34]25)iglA132_Rattattttcaatgtccttagcaa (SEQ ID NO: 26)[34]iglA_Mfe_iglB446_Fgacattgaaaataatcaattgcctttagaaatggcgagatatcctttc[34](SEQ ID NO: 27)iglB506_Rgttattatttgtaccgaataattc (SEQ ID NO: 28)[34]iglB_Age_iglC29_Fggtacaaataataacaccggtaattgtagattatttattgattcttta[34](SEQ ID NO: 29)BamH_iglC149_Ragatgcggatccctattatattggatataactctaaattaga (SEQ[34]ID NO: 30)MfeI_GGSG_iglB446_Faagtaacaattgggtggttctggtcctttagaaatggcgagatatcctt[34]tc (SEQ ID NO: 31)AgeI_GGSG_iglB506_Raagtaaaccggttccagaaccaccgttattatttgtaccgaataattct[34]gg (SEQ ID NO: 32)MfeI_GGSG2_iglB446_FAagtaacaattgggtggttctggtggtggttctggtcctttagaaatgg[34]cgagatatcctttc (SEQ ID NO: 33)AgeI_GGSG2_iglB506_RAagtaaaccggttccagaaccaccaccagaaccaccgttattatttgta[34]ccgaataattctgg (SEQ ID NO: 34)*Restriction sites underlined in the primers.

TABLE 4Plasmid stability in vivo (I)Percentage of Kanamycin resistance(# of KanR/# of total clones) on days post infection1a)4b)7c)14d)LVS 0 (0/15) 0 (0/15) 0 (0/15) 0 (0/24)LVS ΔcapB0 (0/2) 0 (0/15)0 (0/10) 0 (0/15)LVS100 (46/46)100 (30/30)100 (30/30)100 (36/36)ΔcapB/bfr-iglALVS100 (40/40)100 (30/30) 93 (26/28)71 (5/7) ΔcapB/bfr-iglBLVS100 (48/48)100 (30/30)100 (30/30)100 (26/26)ΔcapB/bfr-iglCa)Day 1 post infection: Colonies were patched from chocolate agars plated with lung lysates of a randomly chosen single animal.b)Day 4 post infection: colonies were from patched from chocolate agars plated with lung lysates of a randomly chosen single animals.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.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 5Plasmid stability in vivo (II)Percentage of plasmid persistence[# of PCR (+) clones/# of Total clones tested]14714LVSLVS ΔcapBLVS ΔcapB/bfr-iglA100 (8/8)100 (8/8)100 (8/8)100 (4/4)LVS ΔcapB/bfr-iglB100 (8/8)100 (8/8)87.5 (7/8) 100 (4/4)LVS ΔcapB/bfr-iglC100 (8/8)100 (8/8)100 (8/8)100 (3/3)

TABLE 6Efficacy of Heterologous Prime-Boost Vaccine Expressing IgIABCMean SurvivalVaccineVaccine (weekChallenge (weekTime (Days)(week 0)4)Boost (week 6)10)(weeks 10-13)PBSPBSID29 CFU SchuS44.0rLVS29 CFU SchuS47.9ΔcapB/bfr-iglABCrLVSrLm prfA*rLm prfA*29 CFU SchuS46.4ΔcapB/bfr-(vector)(vector)iglABCrLVSrLm prfA*/hly-29 CFU SchuS47.6ΔcapB/bfr-iglABCiglABCrLVSrLm prfA*/hly-rLm prfA*/hly-29 CFU SchuS49.4ΔcapB/bfr-iglABCiglABCiglABCrLVSrLm29 CFU SchuS47.1ΔcapB/bfr-prfA*/ActA-iglABCiglABCrLVSrLmrLm prfA*/ActA-29 CFU SchuS49.4ΔcapB/bfr-prfA*/ActA-iglABCiglABCiglABC

REFERENCES

Note: This application references a number of different publications as indicated throughout the specification by reference numbers enclosed in parenthesis, e.g., (x). A list of these different publications ordered according to these reference numbers can be found below.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. Publications cited herein are cited for their disclosure prior to the filing date of the present application. Nothing here is to be construed as an admission that the inventors are not entitled to antedate the publications by virtue of an earlier priority date or prior date of invention. Further, the actual publication dates may be different from those shown and require independent verification.

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SEQUENCESB. anthracis polypeptide sequencesa. Anthrax protective antigen precursorAAA22637.1 protective antigen precursor [Bacillusanthracis]MKKRKVLIPLMALSTILVSSTGNLEVIQAEVKQENRLLNESESSSQGLLGYYFSDLNFQAPMVVTSSTTGDLSIPSSELENIPSENQYFQSAIWSGFIKVKKSDEYTFATSADNHVTMWVDDQEVINKASNSNKIRLEKGRLYQIKIQYQRENPTEKGLDFKLYWTDSQNKKEVISSDNLQLPELKQKSSNSRKKRSTSAGPTVPDRDNDGIPDSLEVEGYTVDVKNKRTFLSPWISNIHEKKGLTKYKSSPEKWSTASDPYSDFEKVTGRIDKNVSPEARHPLVAAYPIVHVDMENIILSKNEDQSTQNTDSETRTISKNTSTSRTHTSEVHGNAEVHASFFDIGGSVSAGESNSNSSTVAIDHSLSLAGERTWAETMGLNTADTARLNANIRYVNTGTAPIYNVLPTTSLVLGKNQTLATIKAKENQLSQILAPNNYYPSKNLAPIALNAQDDFSSTPITMNYNQFLELEKTKQLRLDTDQVYGNIATYNFENGRVRVDTGSNWSEVLPQIQETTARIIENGKDLNLVERRIAAVNPSDPLETTKPDMILKEALKIAFGFNEPNGNLQYQGKDITEFDFNFDQQTSQNIKNQLAELNATNIYTVLDKIKLNAKMNILIRDKRFHYDRNNIAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYAVIKENTIINPSENGDISTNGIKKILIFSKKGYEIG (SEQID NO: 6)b. Anthrax toxin lethal factor precursor; lef, plasmid pXO1,Protein ID: NP_052803.1NP_052803.1 pXO1-107 (plasmid) [Bacillusanthracis]MNIKKEFIKVISMSCLVTAITLSGPVFIPLVQGAGGHGDVGMHVKEKEKNKDENKRKDEERNKTQEEHLKEIMKHIVKIEVKGEEAVKKEAAEKLLEKVPSDVLEMYKAIGGKIYIVDGDITKHISLEALSEDKKKIKDIYGKDALLHEHYVYAKEGYEPVLVIQSSEDYVENTEKALNVYYEIGKILSRDILSKINQPYQKFLDVLNTIKNASDSDGQDLLFINQLKEHPIDFSVEFLEQNSNEVQEVFAKAFAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEINLSLEELKDQRMLSRYEKWEKIKQHYQHWSDSLSEEGRGLLKKLQIPIEPKKDDIIHSLSQEEKELLKRIQIDSSDFLSTEEKEFLKKLQIDIRDSLSEEEKELLNRIQVDSSNPLSEKEKEFLKKLKLDIQPYDINQRLQDTGGLIDSPSINLDVRKQYKRDIQNIDALLHQSIGSTLYNKIYLYENMNINNLTATLGADLVDSIDNIKINRGIFNEFKKNFKYSISSNYMIVDINERPALDNERLKWRIQLSPDTRAGYLENGKLILQRNIGLEIKDVQIIKQSEKEYIRIDAKVVPKSKIDTKIQEAQLNINQEWNKALGLPKYTKLITENVHNRYASNIVESAYLILNEWKNNIQSDLIKKVINYLVDGNGRFVFIDITLPNIAEQYTHQDEIYEQVHSKGLYVPESRSILLHGPSKGVELRNDSEGFIHEFGHAVDDYAGYLLDKNQSDLVINSKKFIDIFKEEGSNLTSYGRTNEAEFFAEAFRLMHSTDHAERLKVQKNAPKTFQFINDQIKFIINS (SEQ ID NO: 5)c. AhpC, Alkyl hydroperoxide reductase subunit C [Bacillusanthracisstr. Ames]NP_842892.1 alkyl hydroperoxide reductase subunit C [Bacillusanthracis str. Ames]MLLIGTEVKPFKANAYHNGEFIQVIDESLKGKWSVVCFYPADFIFVCPTELEDLQNQYAILKELGVEVYSVSIDTHFTHKAWHDSSETIGKIEYIMIGDPIRTITTNFNVLMEEEGLAARGIFIIDPDGVIQSMEINADGIGRDASILVNKIKAAQYVRNNPGEVCPAKWQEGSATLKPSLDLVGKI (SEQ ID NO: 7)Yersiniapestis polypeptide sequencesa. F1 capsule antigenNP_395430.1 F1 capsule antigen (plasmid) [Yersiniapestis CO92]MKKISSVIAIALFGTIATANAADLTASTTATATLVEPARITLTYKEGAPITIMDNGNIDTELLVGILTLGGYKTGITSTSVNFTDAAGDPMYLIFTSQDGNNHQFTTKVIGKDSRDFDISPKVNGENLVGDDVVLATGSQDFFVRSIGSKGGKLAAGKYTDAVIVIVSNQ (SEQ ID NO: 8)b. V antigen (LcrV) proteinNP_395165.1 secreted effector protein (plasmid) [YersiniapestisCO92]MIRAYEQNPQHFIEDLEKVRVEQLIGHGSSVLEELVQLVKDKNIDISIKYDPRKDSEVFANRVITDDIELLKKILAYFLPEDAILKGGHYDNQLQNGIKRVKEFLESSPNTQWELRAFMAVMHFSLTADRIDDDILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDKSINLMDKNLYGYTDEEIFKASAEYKILEKMPQTTIQVDGSEKKIVSIKDFLGSENKRTGALGNLKNSYSYNKDNNELSHFATTCSDKSRPLNDLVSQKTTQLSDITSRFNSAIEALNRFIQKYDSVMQRLLDDTSGK (SEQ ID NO: 9)c. YscFNP_395189.1 needle complex major subunit (plasmid) [YersiniapestisCO92]MSNFSGETKGTDIADLDAVAQTLKKPADDANKAVNDSIAALKDKPDNPALLADLQHSINKWSVIYNINSTIVRSMKDLMQGILQKFP (SEQ ID NO: 10)d. YopEAJJ86307.1 outer membrane virulence protein yopE (plasmid) [Yersiniapestis CO92]MKISSFISTSLPLPTSVSGSSSVGEMSGRSVSQQTSDQYANNLAGRTESPQGSSLASRIIERLSSVAHSVIGFIQRMFSEGSHKPVVTPAPTPAQMPSPTSFSDSIKQLAAETLPKYMQQLNSLDAEMLQKNHDQFATGSGPLRGSITQCQGLMQFCGGELQAEASAILNTPVCGIPFSQWGTIGGAASAYVASGVDLTQAANEIKGLAQQMQKLLSLM (SEQ ID NO: 11)e. Ybt/pesticin receptor Psn [Yersiniapestis]AAC69592.1 Ybt/pesticin receptor Psn [Yersiniapestis]MKMTRLYPLALGGLLLPAIANAQTSQQDESTLVVTASKQSSRSASANNVSSTVVSAPELSDAGVTASDKLPRVLPGLNIENSGNMLFSTISLRGVSSAQDFYNPAVTLYVDGVPQLSTNTIQALTDVQSVELLRGPQGTLYGKSAQGGIINIVTQQPDSTPRGYIEGGVSSRDSYRSKFNLSGPIQDGLLYGSVTLLRQVDDGDMINPATGSDDLGGTRASIGNVKLRLAPDDQPWEMGFAASRECTRATQDAYVGWNDIKGRKLSISDGSPDPYMRRCTDSQTLSGKYTTDDWVFNLISAWQQQHYSRTFPSGSLIVNMPQRWNQDVQELRAATLGDARTVDMVFGLYRQNTREKLNSAYDMPTMPYLSSTGYTTAETLAAYSDLTWHLTDRFDIGGGVRFSHDKSSTQYHGSMLGNPFGDQGKSNDDQVLGQLSAGYMLTDDWRVYTRVAQGYKPSGYNIVPTAGLDAKPFVAEKSINYELGTRYETADVTLQAATFYTHTKDMQLYSGPVRMQTLSNAGKADATGVELEAKWRFAPGWSWDINGNVIRSEFTNDSELYHGNRVPFVPRYGAGSSVNGVIDTRYGALMPRLAVNLVGPHYFDGDNQLRQGTYATLDSSLGWQATERMNISVYVDNLFDRRYRTYGYMNGSSAVAQVNMGRTVGINTRIDFF (SEQ ID NO: 12)F. tularensis polypeptide sequencesa. IglA (intracellular growth locus, subunit A) [Francisellatularensis subsp. tularensis SCHU S4]CAG45992.1 intracellular growth locus, subunit A [Francisellatularensis subsp. tularensis SCHU S4]MLIRCCEKKDNKMAKNKIPNSRLMINYETNVDGVLKKKELPYRVLVVGDLSKGRSVDAKKEFADREVRRVNNGVDRVLEEMNISFDFEAPNFVSKDRSNLKVNYRIESVKDFRPDAVAKKVPEIRALLEMKEILASFAKDIENNRNLKKTIDMIFSDSNELESLKSKIPALTNYTIKDSCDAAESQDLSNQQVDGK (SEQ ID NO:2)b. IglB (intracellular growth locus, subunit B) [Francisellatularensis subsp. tularensis SCHU S4]CAG45991.1 intracellular growth locus, subunit B [Francisellatularensis subsp. tularensis SCHU S4]MVSREDFIMTINKLSLIDELLNNFGGSTEVDSVLKNIDFDVSDDASKVLSLSIDYNARNLMALSLVLANNDNINNYNQKYIQKVITVIDKLIDLQVNSIISNDEFRALEQEWLKVQEVCQEDYDNVEVSILDVKKEELQYDFERNLYDISSSDFFKKVYVSEFDQYGGEPYGAILGLYNFENTINDIIWLIGMGMVAKNSHAPFIASIDKSFFGVKDLSEITHIKSFEALLEHPRYKEWNDFRNLDVAAYIGLTVGDFMLRQPYNPENNPVQYKLMEGFNEFVDYDKNESYLWGPASIHLVKNMMRSYDKTRWFQYIRGVESGGYVKNLVACVYDNKGILETKSPLNVLFADYMELSLANIGLIPFVSEKGISNACFFSVNSAKKVEEFVDGFDSANSRLIANLSYTMCISRISHYIKCVIRDKIGSIVDVESIQKILSDWISEFVTIVYQPIPLEMARYPFRNVSIEVETIPGKPGWYSCKINVIPHIQFEGMNITMTIDIRLEPELFGINNN (SEQ ID NO: 3)c. IglC (intracellular growth locus, subunit C) [Francisellatularensis subsp. tularensis SCHU S4]CAG45990.1 intracellular growth locus, subunit C [Francisellatularensis subsp. tularensis SCHU S4]MIMSEMITRQQVISGETIHVRTDPTACIGSHPNCRLFIDSLTIAGEKLDKNIVAIDGGEDVIKADSATAAASVIRLSITPGSINPTISITLGVLIKSNVRTKIEEKVSSILQASATDMKIKLGNSNKKQEYKTDEAWGIMIDLSNLELYPISAKAFSISIEPTELMGVSKDGMRYHIISIDGLITSQGSLPVCCAASTDKGVAKIGYIAAA (SEQ ID NO: 4)d. VgrG, conserved hypothetical protein [Francisellatularensissubsp. tularensis SCHU S4]CAG45980.1 conserved hypothetical protein [Francisellatularensissubsp. tularensis SCHU S4]MSKADHIFNLEEQGLLIDIKDDSKGCTTKLESSGKITHNATESIESSADKQIIENVKDSKISITEKEILLATKKSSIMLSEDKIVIKIGNSLIILDDSNISLESATINIKSSANINIQASQNIDIKSLNNSIKADVNLNAEGLDVNIKGSVTASIKGSTATMVG (SEQ ID NO: 15)e. capsule biosynthesis protein CapB [Francisellatularensis]MTTLDFWLIVVVFVILCVYLIIENIVHNNSIKSIPIRIHNGTRGKSSVARLIAAGVRAGGYRTVAKTTGTLARYIDVDGSETPVFRIGSNIAEQVKIMFKARRAKADAIVIECMALQPLLQSLCELKLIKATHGVLTNARPDHLDVMGPTERDVAKALAATVPVGAKYFTAEDIHLDFFEYACKDRGELIAATAQDAEKISDEEINKFVYSEFKINVALALKVIDDLGIPREIALKGMWEATPDPGAMTEYNFNIKAEINFANAFAANDPVSTKMLWDKLCAKYSGCDKKVLVVNCRDDREDRSKQMAEAALGWQKQDLIVLIGTGTEVFTSFYKYAKSLNKPMTKVIVCEEMTPIQILEKTVDSNPANSYILVGVGNIKDIGMELVDYCDTSHKKKHNL (SEQ ID NO:1)Francisellatularensis promoter sequencesa. Promoter for Francisellatularensis subsp. holarctica FTL_0617(bacterioferritin, bfr)CP009694.1:1332650-1332938 Francisellatularensis subsp. holarcticaLVS, complete genomeTTGTTACCTCCATTATTTAAAACTCTAATCATTATAATCAACATACTAACTAGTATAATTCATATTAGCAATCAAGTTAGCATACAAAAGAAAATTTAATTCTTTATAAGTTATCAATTTAGTATCTAATTATATATCAAAATATCTAAGAATTCAACCTAGATATTTTAATAAAAATGATATTATGCTATTTTTAGATAAGTTAAATTTACTATTTTTAATAATAATATTTAAGAAAAATAAATGAAGAAAATTAATTTTAATATTGTGATGATGGCAATAGTAACCA (SEQ ID NO: 13)b. Promoter for Francisellatularensis subsp. novicida FTN_1451(omp26, omp)CP009633.1:589316-589474 Francisellatularensis subsp. novicida U112,complete genomeTTTGGGTTGTCACTCATCGTATTTGGTTTATAATTTTAAGCTAATAACCTAATTATAACTAATTAATAGTTTTGTATCTTGAAAAAATAGCTATAAAACTTATTTAAATAACGAAGATTTTTGTGTATAAAATATTTATAACAAAAAAAGGAGACTAAA (SEQ ID NO: 14)Burkholderiapseudomallei polypeptide sequencesHcp-1type VI secretion system [Burkholderiapseudomallei]GenBank: CRY35672.1>CRY35672.1 type VI secretion system [Burkholderiapseudomallei]MLHMHLKFGSPAIKGESADKDHEGWIELKSWDHSIVQPRSATASTAGGHTATRCEHGDMVFTKEIDSSSPLLYQHASGGTTFDEVTIDFLRADGEGQRVKYLEIKLKYVIISSIAPSVHTEGLPVETFSLKYAAVQWKQTQQKIGGNQGGNTQGAWSLTKNDKTYAV (SEQ ID NO: 35)Hcp-2 (T6SS)type VI secretion system [Burkholderiapseudomallei]GenBank: CPN31289.1>CPN31289.1 type VI secretion system [Burkholderiapseudomallei]MSHDIFLKINGIDGEAEDATHKGEIEVLSWSWNVSQQSNMHLGSGGGAGKATIDDLQFEHYIDRASPNLVQYCLLGKHIDEARLVVRKAGGSPLEYIKLTMSDVLVTQVSPAGVAQDESRPRELVRLSFSRLKQEYVVQNPQGGSGGAITATFDIKKNAA (SEQ ID NO: 36)Hcp-3 (T6SS)type VI secretion system [Burkholderiapseudomallei]GenBank: CRY29196.1>CRY29196.1 type VI secretion system [Burkholderiapseudomallei]MAQDIFLKIDGINGESLDDSHKDEIEVLNWNWEIQQESTMHIGSGGGAGKASVKDLIFEHAIDRASPNLMKYALIGKHVDQAVLVMRKAGGNPLEYLKLIMSDVIITRVRPSGSRDDTERSRETVSLSFAKVKQEYVVQNAQGGSGGAVITSFDIKGNKEA (SEQ ID NO: 37)Hcp-4 (T6SS)type VI secretion system [Burkholderiapseudomallei]GenBank: CRY33495.1>CRY33495.1 type VI secretion system [Burkholderiapseudomallei]MANALVDYFLQIDGVEGESTDQQYPGLIQIQSWQWAEENSGRWGFGSGGGAGKVEMKDFEFRMVSNKASPKLFLMCATGEHIQNAKLICRKSGKGQQEFLTISFASGLVSSFRILGNMPISQLGHASGEVDGVLPTDQIRINFAQIEFEYREQRNDGTMGAVIKAGYDLKQNAPI (SEQ ID NO: 38)Hcp-6 (T6SS)type VI secretion system [Burkholderiapseudomallei]GenBank: CRY19699.1>CRY19699.1 type VI secretion system [Burkholderiapseudomallei]MGVAMFMKVDGVTGESADAQHKGWIDIQSFSWGASQPGAMASGSGGNAGKASFNDLVVAAYMDKGATAIIKNCASGKHLPTVEISACKTGGSQIEFMRVILQEVLVISAQIAGVDPGDAADRLMMQYGFQAAKVKKQYWQQNDNGGKGAEVSVGWNIKENTEM (SEQ ID NO: 39)LolC (ATP binding cassette system)lipoprotein-releasing system transmembrane subunit LolC [Burkholderiapseudomallei]NCBI Reference Sequence: WP_050865936.1>WP_050865936.1 lipoprotein-releasing system transmembrane subunitLolC [Burkholderiapseudomallei]MKLPYEWQIGWRYTRAGKRAIGNGFISFIALVSMLGIALGVAALIVVLSVMNGFQKEVRDRMLSVLAHVEIFSPIGSMPDWQLTAKEARLNRSVIGAAPYVDAQALLTRQDAVSGVMLRGVEPSLEPQVSDIGKDMKAGALTALAPGQFGIVLGNALAGNLGVGVGDKVILVAPEGTITPAGMMPRLKQFTVVGIFESGHYEYDSTLAMIDIQDAQALFRLPAPTGVRLRLTDMQKAPQVARELAHTLSGDLYIRDWIQQNKTWFSAVQIEKRMMFIILTLIIAVAAFNLVSSLVMTVINKQADIAILRILGAQPGSIMKIFVVQGVTIGFVGTATGVALGCLIAWSIPWLIPMIEHAFGVQFLPPSVYFISELPSELVAGDVIKIGVIAFALSALATLYPSWRGAKVRPAEALRYE(SEQ ID NO: 40)TypAGTP-binding protein TypA [Burkholderiapseudomallei]GenBank: CRY12842.1>CRY12842.1 GTP-binding protein TypA [Burkholderiapseudomallei]MTRALRNIAIIAHVDHGKITLVDQLLRQSGTFRENQQVAERVMDSNDIEKERGITILAKNCAVEYEGTHINIVDTPGHADFGGEVERVLSMVDSVLLLVDAVEGPMPQTREVIKKALALGLKPIVVINKIDRPGARIDWVINQTFDLFDKLGATEEQLDFPIVYASGLNGYASLDPAARDGDMRPLFEAILQHVPVRPADPDAPLQLQITSLDYSTYVGRIGVGRITRGRIKPGQPVVMRFGPEGDVLNRKINQVLSFQGLERVQVDSAEAGDIVLINGIEDVGIGATICAVEAPEALPMITVDEPTLIMNFLVNSSPLAGREGKEVISRQIRDRLMKELNHNVALRVKDTGDETVFEVSGRGELHLTILVENMRREGYELAVSRPRVVMQEIDGVKHEPYELLTVDLEDEHQGGVMEELGRRKGEMLDMVSDGRGRIRLEYRIPARGLIGFQSEFLILTRGTGLMSHIFDSYAPVKEGSVGERRNGVLISQDDGAAVAYALWKLQDRGRMFVKPGDALYEGMIIGIHSRDNDLVVNPIKGKQLINVRASGTDEAVRLVPPIQMSLEYAVEFIDDDELVEVTPQSIRLRKRHLKEHERRRASREAEAG (SEQ ID NO: 41)BipBBipB [Burkholderiapseudomallei]GenBank:AB026356.1>AB026356.1 BipB [Burkholderiapseudomallei]MSSGVQGGPAAHANAYQTHPLRDAASALGTLSPQAYVDVVSAAQRNFLERMSQLASEQCDAQPAAHDARLDDKPALRAPQERDAPPLGASDTGSRASGAAKLTELLGVLMSVISASSLDELKQRSDIWNQMSKAAQDNLSRLSDAFQRATDEAKAAADAAEQAAAAAKQAGADAKAADAAVDAAQKRYDDAVKQGLPDDRLQSLKAALEQARQQAGDAHGRADALQADATKKLDAASALATQARACEQQVDDAVNQATQQYGASASLRTPQSPRLSGAAELTAVLGKLQELISSGNVKELESKQKLFTEMQAKREAELQKKSDEYQAQVKKAEEMQKTMGCIGKIVGWVITAVSFAAAAFTGGASLALAAVGLALAVGDEISRATTGVSFMDKLMQPVMDAILKPLMEMISSLITKALVACGVDQQKAELAGAILGAVVTGVALVAAAFVGASAVKAVASKVIDAMAGQLTKLMDSAIGKMLVQLIEKFSEKSGLQALGSRTATAMTRMRRAIGVEAKEDGMLLANRFEKAGTVMNVGNQVSQAAGGIVVGVERAKAMGLLADVKEAMYDIKLLGDLLKQAVDAFAEHNRVLAQLMQQMSDAGEMQTSTGKLILRNARAV (SEQ IDNO: 42)BipCBipC [Burkholderia pseudomallei]GenBank: AB026357.1>AB026357.1 BipC [Burkholderiapseudomallei]MSIGVQSSGINISHAELSRLVDAGKSEQGDKAVRDDGRALARADAALAAVVGERVAARRDAVAGSGAQRVELARPKPDAQTRATDRRTVSGLEREHKRLAASQTPRVTGMHDALVQRHVSLDGAKAAHGEGVKRAAGDAPRAAADAPQRFAFADDKAFDAMLALGAAMQKNVQSDLAMQGKLTMLAHDAMMSAAAQDRSIGAAQMTAAIAGGALQATTSLGGAMQQMKSLSTKSMSIEKELKPQAELKQFHAEQALELRGINKPVLSNDEVSHVKIKRDTGETVRHEIDHGGERMSDEHASVLAQEAPARQHRIDMHGMRHEENLVKASRQQMKGDLLQSGGQIGKNQIDGASAQQQGADRAEQKEDENAQQTAMAAASTRDEAAHRSREAAQKAIDAAKSQVANDNAVAAQVAGNLRT(SEQ ID NO: 43)BipDBipD [Burkholderiapseudomallei]GenBank: ABL67521.1>ABL67521.1 BipD [Burkholderiapseudomallei]MNMHVDMGRALTVRDWPALEALAKTMPADAGAREMTDDDLRAAGVDRRVPEQKLGAAIDEFASLRLPDRIDGRFVDGRRANLTVFDDARVAVRGHARAQRNLLERLETELLGGTLDTAGDEGGIQPDPILQGLVDVIGQGKSDIDAYATIVEGLTKYFQSVADVMSKLQDYISAKDDKNMKIDGGKIKALIQQVIDHLPTMQLPKGADIARWRKELGDAVSISDSGVVTINPDKLIKMRDSLPPDGTVWDTARYQAWNTAFSGQKDNIQNDVQTLVEKYSHQNSNFDNLVKMLSGAISTLTDAAKSYLQI (SEQ ID NO: 44)Omp3OmpA family protein [Burkholderiapseudomallei]GenBank: ACN64870.1>ACN64870.1 OmpA family protein [Burkholderiapseudomallei]MNKLSKLAFIAATAVMAASASAQSVPASRQAVNDNWVNGTGEWVWMNGTNELCWRDAFWTPATANAKCDGALVAQAPAPAPVAPVAPAITSQKITYQADTLFDFDKAVLKPAGKQKLDELAAKIQGMNVEVVVATGYTDRIGSDKYNDRLSLRRAQAVKSYLVSKGVPANKVYTEGKGKRNPVTGNTCKQKNRKQLIACLAPDRRVEVEVVGTQEVQKTTVPAQ (SEQ ID NO: 45)Omp7OmpA family lipoprotein [Burkholderiapseudomallei]GenBank: ACN64871.1>ACN64871.1 OmpA family lipoprotein [Burkholderiapseudomallei]MTTRRVTMMSKKLRLAFAMLMIGALAACKSGVKLDEHANQGDAVSTQPNPENVAQVTVDPLNDPNSPLAKRSVYFDFDSYSVQDQYQALLQQHAQYLKSHPQRHILIQGNTDERGTSEYNLALGQKRAEAVRRALSLLGVGDAQMEAVSLGKEKPVALGHDEASWAQNRRADLVYQQ (SEQ ID NO: 46)Omp85putative outer membrane protein [Burkholderiapseudomallei K96243]GenBank: CAH36153.1>CAH36153.1 putative outer membrane protein [Burkholderiapseudomallei K96243]MLFKPHREVPKTVAAAALAAHGLAAHATAPFVVQDIKIEGLQRVEAGSVFAYLPIKQGDTFTDDKASEAIRALYATGFFNDVRIATQGGVVIVQVQERPAIASIDFTGIKEFDKDNLNKALKAVGLSQGRYYDKALVDKAEQELKRQYLTRGFYAAEVSTTVTPVDANRVSILFAVAEGPSAKIRQINFIGNKAFKTSTLRDEMQLSTPNWFSWYTKNDLYSKEKLTGDLENVRSYYLNRGYLEFNIESTQVSISPDKKDMYLTVALHEGEPYTVSSVKLAGNLLDRQAELEKLVKIKPGDRFSAEKLQQTTKAIVDKLGQYGYAFATVNAQPEIDQATHKVGLTLVVDPSRRVYVRRINIVGNTRTRDEVVRREMRQLESSWFDSSRLALSKDRVNRLGYFTDVDVTTVPVEGTNDQVDVNVKVAEKPTGAITLGAGESSTDKVVLSAGISQDNVEGSGTSLAVNVNTAKSYRTLTVTQVDPYFTVDGIKRITDVEYRTYQPLYYSTNSSFRIITAGGNLKEGIPFSETDTVYFGAGFEQNRLDVDSNTPQSYQDYVNEFGRVSNTVPLTIGWSRDARDSALIPSRGYFTQANAEYGVPVGKIQYYKMDVQGQYYYSFARGFILGLNFQAGYGNGIGNPYPIFKNYYAGGIGSVRGYEPSSLGPRDTKTNDPIGGSKMVVGNIELTFPLPGTGYDRTLRVFTFLDGGNVWGNAPGGTSTGANGLRYGYGIGLAWISPIGPLKLSLGFPLQKHEGDQYQKFQFQIGTAF(SEQ ID NO: 47)OmpWouter membrane protein W [Burkholderiapseudomallei]GenBank: A1173329.1>ALJ73329.1 outer membrane protein W [Burkholderiapseudomallei]MGDKHRRRRARRDWRAPVSCWLGATLLACAWSAHAQDSGAARWRDGADGIGFFPGGDAPGFDARAWGPVPGDARRAAASDARNGVAASAGSEATAAAPAADANAAPARKLTEERITLGERVAPVADAARRVRADGDDGIGFADAPGGPPAGGATPSAACDDGACVPDGGDAGRAPRRPPAGATPRFIAGVRYDRMPYELHPIDPERLPDLPEAQGPTLLEQLQGDDSNMIGVGWHYVLSTGRSTPVTTSTAALGIGSFANPGSAVSISNTNTPAFTFTHFFGEHVAAEIVAGIPPELTMRGHGSIGLPFDKIFPGVQGRLPLVDLGNTQSNPLGTTRAWLGSAVFKYYLGKREDRLRPYVGLGLSYTRFTNTNLNPVFAHKLASLGGLLSAGISLGDLQSLLTDSGALDRLLQAGANLILPNGVRATADVKSAWTPVFVVGANYQLTRQLSLSTALSYIPLKAAITVNINDTKGILASNTTTLSANVLLCTMLLNFRF (SEQ ID NO: 48)PotFspermidine/putrescine ABC transporter substrate-binding protein PotF [Burkholderiapseudomallei]GenBank: ANW57327.1>ANW57327.1 spermidine/putrescine ABC transporter substrate-bindingprotein PotF [Burkholderiapseudomallei]MKRIAWLAAVLASLACAAARAAGGNVLNIYNWAEYFAPDTIAGFEKETGIKVRLDVYDSNEALQTKLTTGNSGYDLVFPSNDFLARQIQAGLYRKLDKSRLPNLTNLDPAIVARAAEVDPGNQYSVPYMQGTFGLGLNVAKVKQALGGPLPANTLELIFNPAYAAKLERCGIAFNDAGSEVFPLALRYIGRDPNTTDPRDYEAALDMMKKIRPTIRQFIATPVMNDLATGDVCVVTGYSGAVLVAARRAAEAKNGQQIVYSLPSAGAPFWFDSMAIPKGAAHADHALRFIDYILRPDVVAKISNKVMYPNPNRVATPLVDRRLTANPAIYPDAATMRTLWVKRPMPPQAMRMQTRYWTRFKTGY (SEQ ID NO: 49)OppAoligopeptide ABC transporter periplasmic oligopeptide-binding protein [Burkholderiapseudomallei]GenBank: CRY20041.1>CRY20041.1 oligopeptide ABC transporter periplasmic oligopeptide-binding protein [Burkholderiapseudomallei]MKHTHAFAAVLAALALTIAPSAPAVTVASNVTLADQQDLTRQVPAEVESLDPAHIESWTGNTIGLDLFEGLARIDASGAVVPGVAQAWEHKAPDTWIFKLRRDAKWSNGQPVTAADFVYAWQRLADPKTGSKYTILVEFVKNASAIIAGKQPPGDLGIRAIDPYTIEVKTEVPVSYFPELTAMAPLTPVNKDAVAKFGDAWTRPKNIVSNGPYTLVDWQPNNRIVMAKSDKYWNARNVVIRKVTYLPIENDETALRMYQAGQIDYTYSIPAGGFGQISKQFGKELRPGLQLATYYYYLKNSDPALKDKRVREALAMVLDREILTSKITQAGEVPMYGLMPKGVKGVQRPFTPDWASWPMARRVDYAKNLLKQAGHGDANPLTFTLTYNTNDLHKKVALFAASEWRTKLGVTAKLENVEFKVLMKQRHDGKVQVARDGWFADYNDAMTFFDLIRCGSSQNTVGYCNPKVDSLVAEANQKLDDGARAALLTQAHDLAMNDYPMVPLFQYSADRLVKSYVGGYTLTNYIDMRASQDMYLIKH (SEQ ID NO: 50)BopA (Type III Effector)BopA protein [Burkholderiapseudomallei]GenBank: CRY24966.1>CRY24966.1 BopA protein [Burkholderiapseudomallei]MINVGAFVASARSGARVVVGGDARGPVVSAARLGMKERLFAFLAHVPLLKHCDAVRRYAEQVRMENRRSLEVFVLALSKRYGPEGAKAAFDYGARRDGAPLDQRRVRNMVSIAEHFHGTGDAKPLARQMVERSWECRGLDHPGHASLTIKNQADADAGRHVYEHVSWWPNQRLGSKEHFDRIEPKTLDGYRIDKRSEISSATEQRLREGDAARRKILADGFKYANQDERHDARFFPRAGQKLDKDAEWGLSARKVYFPAIGFNHDRRDTDRPRAFVLFGLNEAAMLRDARTVKEGAKSGELKYRMISKKENCASMALRVLRAGGAEHFVPYTAAWISEDPNHAHAYALAVQARIDALNQRRADVERRCERLRDSASVRQAWRAFSEAGGASASPLAEDAGRGRASAHMRQARLDEHAREVERIGAYFAELSAGRSGKHRDRADAALADAMKRCAPSARDDVAALTRKASVLVETLGRHLDAPPPSDSSALRRLAAHAMIGRIEAFMAAAIAA (SEQ ID NO: 51)BimA (Autotransporter protein)BimA [Burkholderiapseudomallei]GenBank: ACF94992.1>ACF94992.1 BimA [Burkholderiapseudomallei]MHAKASSSHAPDAPKPSSIATTLCRALASLSLGLSMDAEANPPEPPGGTNIPVPPPMPGGGANIPVPPPMPGGGANIPPPPPPPGGIGGATPSPPPLTPVNGNPGASTPTKTGLLKTLNRLSAELQNNPRVTEDVVDNVDAVIRNAVNLAPDANGDFSGRSAMPIEMAANAALRSLKKNPGDAGHAAPAYLPAERIGQLREKVRRTIEALESNRPPKPQPRSTPPQSTPPKPTQHPTAPNPNVPDASTPDASTPDASTPDASTPDASTPSRPAPAPRAGTGAPAASAATRAPAFANRVRKPNPAMPAASSHAIASDFASSNAFAIGDDSTAVGAQATAFSEQSIAIGSRAIAAGARSIAVGTDATAAAPDSVALGSGSIAEREGTVSVGRDGHERQITHVASGTEPTDAVNVTQLRAAMSNANAYTNQRIGDLQQSITDTARDAYSGVAAATALTMIPDVDRDKRVSIGVGGAVYKGHRAVALGGTARINENLKVRAGVAMSAGGNAVGIGMSWQW (SEQ ID NO: 52)BPSL1897hypothetical protein BP [Burkholderiapseudomallei K96243]NCBI Reference Sequence: YP_108497.1>YP_108497.1 hypothetical protein BPSL1897 [BurkholderiapseudomalleiK96243]MTRALGRLRSPRRQRGATAIEFAILFPMFFLILYGIITYGMIFAAQQSLTLAATEGARAALNYQVAQTQSAALGLRAAAACTAANNLTGWLSGATCTTSTNYTCSYDSTMYCIQVTLTYPYAANPLVPAVALFDAVLPTTLTSRATVQINPTNII (SEQ ID NO: 53)BPSL3369acetaldehyde dehydrogenase [Burkholderiapseudomallei K96243]GenBank: CAH37382.1>CAH37382.1 acetaldehyde dehydrogenase [BurkholderiapseudomalleiK96243]MNHADMQHLNIEFPYRKQYGNFIGGEWVAPVGGEYFDNVSPVTGRPFTAIPRSREADIELALDAAHAAKAGWAAKGAAERANVLLRIADRMEANLTRLAVAETIDNGKPLRETTAADVPLAIDHFRYFAGCIRAQEGSIADIGGDMVAYHFHEPLGVVGQIIPWNFPLLMAAWKLAPALAAGNCVVLKPAEQTPASILVFAELIQDLLPPGVLNIVNGFGLEAGKPLASSKRIAKIAFTGETSTGRLIMQYASENLIPVTLELGGKSPNIFFADVMDRDDSYFDKALEGFAMFALNQGEVCTCPSRALVEESIYDRFIERALKRVEAIKQGHPLDSQTMIGAQASAEQLEKILSYIDIGRGEGAQCLTGGERNVLGGELAEGYYVKPTVFRGHNKMRIFQEEIFGPVLAVTTFKTEEEALEIANDTLYGLGAGVWTRDGNRAYRFGRGIQAGRVWTNCYHAYPAHAAFGGYKQSGIGRETHKMMLDHYQQTKNLLVSYSEKPLGFF (SEQ ID NO: 54)BP SL2287HesB family protein [Burkholderiapseudomallei K96243]GenBank: CAH36290.1>CAH36290.1 HesB family protein [Burkholderiapseudomallei K96243]MAITLTEKAAQHVQKYLARRGKGLGLRLGVRTTGCSGLAYKLEYVDELTPEDQMFESHGVKVFVDPKSLAYIDGTELDFAREGLNEGFKFNNPNVKDECGCGESFRV (SEQ ID NO: 55)BPSL2765putative OmpA family lipoprotein [Burkholderiapseudomallei K96243]GenBank: CAH36773.1>CAH36773.1 putative OmpA family lipoprotein [Burkholderiapseudomallei K96243]MMSKKLRLAFAMLMIGALAACKSGVKLDEHANQGDAVSTQPNPENVAQVTVDPLNDPNSPLAKRSVYFDFDSYSVQDQYQALLQQHAQYLKSHPQRHILIQGNTDERGTSEYNLALGQKRAEAVRRALSLLGVGDAQMEAVSLGKEKPVALGHDEASWAQNRRADLVYQQ (SEQ ID NO: 56)VgrG5Rhs element Vgr protein [Burkholderiapseudomallei 1710b]GenBank: ABA53677.1>ABA53677.1 Rhs element Vgr protein [Burkholderiapseudomallei 1710b]MRLIELRSPLLDPDAVALSFVVHENLSQEPSYQLDLLSHDSNLDFDALLGSTLSADIDLGEGDIRTENTHVFGGYDTGQMSGQYTYTLELRSWLSFLAENRNSRIFQDLSVPQIVEQVFQGHQRNGYRFELEGTYEPREYCVQFQETDLNFVKRLLEDEGIYFWVEHEPDRHVVVISDTQRFEDLPLPNDTLEYLPDGEESRAIQGREGVQRLQRTRRIKSNNVALRDFDYHAPSKQLDSDAQVEQQSLGGIPLEYYDYAAGYRDPEQGERLARLRLEAIQADAHALGGEANARALAVGRAFTLVGHPALSRNRRYYVTNSELTFIQDGPDSTSQGRNVAVKFRALADDQPFRPLLVTKRPRVPGIQSATVVGPEMSEVHTDKLGRIRVHFHWDRYKTTEADASCWIRVTQAWAGKGWGVLAMPRVGQEVIVVYVDGDLDRPLATGIVYNGENPTPYDLPKDIRYTGLVTRSIKRAGGIPNASQLTFDDQHGAERVMIHAERDLQQTVERNSSTSIAQDLNLSVKGTSTSVVGISVSFTGISVSYTGLSVSFTGVSARFTGVSTSFTGVSTSFTGVSTSFTGVDTSFTGVSTGFKGVDTSFTGVATSMVGVSTSITGSSNSVTGVSNSMTGISSSWKDVSMSTTGQSESITGVSLSYTGTSNSMTGTSTSVTGTSTSITGTSMSNTGSSTSITGTSMSTTGSSVSTTGSSMSATGSSVGTTGSSVSTTGSKMSVTGFSFSYTGASYEDVGVDLKKLGMQTKN(SEQ ID NO: 57)Burkholderiamallei polypeptide sequencesBMA_A0768 mannitol dehydrogenase family proteinmannitol dehydrogenase family protein [Burkholderiamallei ATCC 23344]GenBank: AAU46944.1>AAU46944.1 mannitol dehydrogenase family protein [Burkholderiamallei ATCC 23344]MPLLSSDHCRALPPEVSRPRYDRRALRTGIVHLGLGAFHRAHQACYTETLVERGDLRWGIAGVELRRRHTVERLAAQDHLYSVTERAGDAARTRVVGAVHRTLFAPQALATLLGLIADPSVSIVSLTVTEKGYYRRPGGGGLDLDDPAIRRDLAQPHAPSTTLGVLAAGIRLRAAHAPLSVLSCDNMPSNGDTLRALLAQYAEQTDGALARRIRCDVAFPNTMVDRIVPAATPESLDWVQSRIGVRDEAAIVCEPFAQWVFEDRFAGARPRWEDAGALVAADVRPYEKMKLRLLNGSHSAIAYAGQLRGRRTVSDAMADPLIDALARGVMTRELLATLDVPAGYDVRAYCASLIERFRNPALAHRTAQIATDGTQKVPLRWLPALAESAAAGVERPFLERSLAMWLHYVEVARDESGRPLVLEDPGAQALAARLHGAPGATDAVRAALGLIASRDAARWPEALTARVGAHLETVRTRGTDALLRPLLDA(SEQ ID NO: 58)BMA_2821ABC Transporter ATP binding proteinglutathione ABC transporter ATP-binding protein [Burkholderiamallei]GenBank: KKM47108.1>KKM47108.1 glutathione ABC transporter ATP-binding protein[Burkholderiamallei]MSASRAAPSLPDARVLAVDGLTVTFRREDAAFVAVRDLSFHVDRGETLAIVGESGSGKSVTSLALMRLVEHGGGAIAGGAIALRRRGGAVLDLARATPSTLRTVRGADVAMIFQEPMTSLNPVETVGDQISEAIALHQHKSAGEARAETLRLLDLVRIPEARRVFARHPHQLSGGMRQRVMIAMALSCRPALLIADEPTTALDVTIQAQILQLIRGLQDEMDMGVIFITHDMGVVAEVADRVLVMYRGEKVEEGACDAIFAAPSHPYTKALLAAVPRLGSMRGIDAPAKFPLLRFDPAAGDALVVAGGDATAASGDAARESVLFVDSDAAAASAASTASTASAASAASAAPTACARPAIDAGAPPLLRVRELVIRFPVKSGVFGRVSQYVHAVERVSFELRAGETLALVGESGCGKSITGRSLLRLVERVSGSIEFEGREIGALKGRELQALRRNIQFIFQDPFASLNPRLTVGFSIMEPLLVHGVASGRQAQARVDWLLERVGLPADAARRYPHEFSGGQRQRIAIARALALNPKVVVADESVSALDVSVQAQIVNLMLDLQRELGVAYLFISHDMAVVERISHRVAVMYLGQIVEIGPRRAVFETPRHPYIKKLMSAVPIADPACRHAPRTLPADELPSPIRALGDEPEVAPLVAVGPAHFVAEHRVGGAY (SEQ ID NO: 59)BMA_0816 maltooligosyl trehalose synthasemaltooligosyl trehalose synthase, putative [Burkholderiamallei ATCC 23344]GenBank: AAU49513 .1>AAU49513.1 maltooligosyl trehalose synthase, putative [Burkholderiamallei ATCC 23344]MKPRATLRLQLHAGFTFDDAAAHVGYFARLGVSHLYLSPITAAEPGSRHGYDVIDYSTVNPELGGEAAFVRLIDALRRRGMGAIVDIVPNHMGVGGSSNRWWNDVLEWGARSRFARHFDIDWHASDPALQRKVLLPCLGRPYGEALAAGDIALRADAAHGRFAIACAGRILPVQIGAYPDILRAANRSDLNALAERFDAPGARPSNHARLDAAHAALRDYAAARGPGALDAVLHGFDPRIARSREMLHRLLEQQHYRLAWWRIATDEINWRRFFDISTLACMRIEDAAVFDDVHALLWRLYAAGLVDGVRIDHVDGLADPRGYCRQLRGRLAALRDGEPYIVVEKILAPDERLPEDWRVDGTTGYDFMNDVSALLHDAAGAAPLAALWADMTGAETTFAREALDGKRRVLARQFAAEHERVARAMHRLARASRDARDFALNPIRRAVAELAIRLPVYRLYPSAGAPQRTDRALLAGAWQAARSAIAPADRAALDYVAATLGLPGVARAVAGLGDPARLAARVGFAQLTAPLAAKGVEDTACYRYGRLLSRNEVGAHADALSLAPGAFHTRNRRRRRTFPGALLATATHDHKRGEDARARLAVLSEAHRAWRAAALDWAAFNAPHHHGAPAAADRIPGPAAEAMLYQTLVGAWPPALAPDDAPGLAALTDRVERWQLKALREAKRDTDWLEPNLGYEAGCAAFLRAIMTPRGPDDFAHRLHRLVARIAPAGIVNSLSQAALRLLSPGVPDLYQGAQTWDHTLVDPDNRADVPFARYAAQRIDAPVAAYLRDWADGRVKHALIGRLLALRAAHPETFAAGAYVPLHVRGTRRGHALAFARRDASTTIVVIATRLAYPLLGDAPARPCVEAACWADTAVGLAPGFAGPWRDMLNDGTLDAPSGMLPLAAALAHLPVAVLIREGGAADTPRRGA (SEQ ID NO: 60)GroELmolecular chaperone GroEL [Burkholderiamallei ATCC 23344]NCBI Reference Sequence: YP _103588.1>YP_103588.1 molecular chaperone GroEL [Burkholderiamallei ATCC23344]MAAKDVVEGDSARAKMVEGVNILANAVKVTLGPKGRNVVLERSEGGPTVTKDGVSVAKEIELKDKLQNMGAQMVKEVASKTSDNAGDGTTTATVLAQSIVREGMKYVASGMNPMDLKRGIDKAVAAAVEELKKISKPCTTNKEIAQVGAISANSDSSIGDRIAEAMDKVGKEGVITVEDGKSLADELDVVEGMQFDRGYLSPYFINNPDKQVAVLENPFVLLHDKKVSNIRDLLPVLEQVAKAGRPLLIIAEDVEGEALATLVVNNIRGILKTVAVKAPGFGDRRKAMLEDIAILTGGQVIAEETGLTLEKATLAELGQAKRIEVGKENTTIIDGAGEAMNIEARVKQIRTQIEEATSDYDREKLQERVAKLAGGVAVIKVGAATEVEMKEKKARVEDALHATRAAVEEGIVPGGGVALIRARTAIASLTGVNADQNAGIKIVLRAMEEPLRQIVTNGGEEASVVVAAVAAGKGNYGYNAATGEYVDMVEAGVVDPTKVTRTALQNAASVAGLLLTTDAAVAELPKEDAPMPGGMPGGMGGMGMGMGMDM(SEQ ID NO: 61)

CONCLUSION

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.

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