Novel safe potent single vector platform vaccines against melioidosis and glanders

Abstract

Embodiments of the invention include single vector platform vaccine compositions for preventing the disease Melioidosis caused by Burkholderia pseudomallei in humans and animals. Embodiments of the invention include single vector platform vaccine compositions for preventing the disease glanders caused by Burkholderia mallei in humans and animals. Embodiments of the invention include methods of immunizing a susceptible host against Burkholderia pseudomallei and / or Burkholderia mallei using the disclosed compositions.

Description

[0001] NOVEL SAFE POTENT SINGLE VECTOR PLATFORM VACCINES AGAINST MELIOIDOSIS AND GLANDERS CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. Section 119(e) of co- pending and commonly-assigned U.S. Provisional Patent Application No.63 / 552,844, filed February 13, 2024, entitled “NOVEL SAFE POTENT SINGLE VECTOR PLATFORM VACCINES AGAINST MELIOIDOSIS”, the contents of which is incorporated by reference herein. This application is related to U.S. Patent No. 11,224,647, entitled “SAFE POTENT SINGLE PLATFORM VACCINE AGAINST TIER 1 SELECT AGENTS AND OTHER PATHOGENS” the contents of which are incorporated herein by reference. STATEMENT REGARDING FEDERAL FUNDING This invention was made with government support under AI141390 awarded by the National Institutes of Health. The government has certain rights in the invention. CROSS-REFERENCE TO RELATED APPLICATIONS TECHNICAL FIELD The invention relates to single platform homologous and heterologous priming and boosting vaccines for preventing melioidosis and / or glanders. BACKGROUND OF THE INVENTION Burkholderia pseudomallei (Bp) is a Gram-negative bacterium that exists as an environmental saprophyte in soils and freshwater of the tropics. Bp is capable of “accidental” virulence in humans and animal hosts, and is the causative agent of melioidosis, a disease endemic in many tropical areas with an estimated 165,000 cases and 89,000 deaths per year. Bp virulence is in large part due to its intracellular lifestyle; Bp can invade both phagocytic and nonphagocytic cells, replicate in the host cytosol after escaping the vacuole, and directly spread from cell to cell. Infection with Bp occurs by inhalation, ingestion, and through broken skin. It is assumed that most natural disease occurs via percutaneous inoculation with contaminated soil or water but inhalation may be an important route of natural infection as well. In addition to its significant public health burden, Bp possesses a number of properties that have resulted in its categorization as a potential bioweapon and a Tier 1 Select Agent by the Centers for Disease Control (CDC): Bp is easily aerosolized and inhalation of low doses of Bp can cause rapidly fatal pneumonia; Bp is inherently resistant to numerous antibiotics making treatment difficult; symptoms of melioidosis are non-specific, often making it difficult to diagnose; Bp can be readily isolated from the environment and cultured for bioterrorism purposes; and there is no licensed vaccine against melioidosis (7, 8). Thus, a safe and effective vaccine against Bp would be of great benefit to people living in endemic regions, travelers, and military personnel stationed in these areas, and would also reduce the risk from an intentional release in a bioterrorist attack. Glanders disease, caused by the related bacterium Burkholderia mallei, is a highly contagious zoonotic infection that primarily affects livestock including horses, donkeys, and mules. It may also spread to people, usually by touching sick animals or contaminated objects. Glanders presents in acute and chronic forms, with symptoms in humans ranging from fever, chills, and muscle aches to respiratory issues and skin lesions. There is a need for a safe and effective melioidosis vaccines in endemic areas of the world and to protect military personnel and civilians against an intentional bioterrorism attack. In addition, there is a similar need for a safe and effective glanders vaccines to protect livestock and humans. SUMMARY OF THE INVENTION The present disclosure provides a single vector platform vaccine and associated methods for preventing, reducing the possibility of or treating melioidosis and / or glanders in humans and animals. Embodiments of the invention include vaccines that are easier and cheaper to manufacture than both virus-vectored vaccines, which must be grown in tissue culture cells and then purified, and protein-in-adjuvant vaccines, where the protein needs to be purified. Aspects of the invention disclosed herein are discussed in Tullius et al., “LVS ΔcapB-vectored multiantigenic melioidosis vaccines protect against lethal respiratory Burkholderia pseudomallei challenge in highly sensitive BALB / c mice” ASM Journals, mBio, Vol. 15, No. 4, 21 March 2024 (hereinafter “Tullius et al.”), the contents of which are incorporated by reference. As discussed herein, we have discovered that certain constellations of different Burkholderia pseudomallei and / or Burkholderia mallei proteins perform surprisingly well when utilized in combination in the immunogenic compositions disclosed herein (i.e., can elicit significant protection against Burkholderia pseudomallei and / or Burkholderia mallei infection). In particular, in the studies that led to the invention disclosed herein, we discovered that certain constellations of immunogenic epitopes presented on selected Burkholderia pseudomallei and / or Burkholderia mallei proteins can significantly enhance the protective efficacy of vaccines against Burkholderia pseudomallei and / or Burkholderia mallei (e.g., in the LVS ΔcapB fusion protein platforms disclosed herein). This surprising observation illustrates the potential therapeutic efficacy of the specific vaccine compositions disclosed herein. The invention disclosed herein has a number of embodiments that are based upon the discovery that in fusion proteins produced by the vaccine platform disclosed herein, certain combinations of Burkholderia pseudomallei and / or Burkholderia mallei polypeptide epitopes can be selected to optimize expression of the fusion protein (and facilitate an immune response). In this context, one such embodiment of the invention is a composition of matter comprising at least one immunogenic fusion protein comprising antigenic epitopes present in at least one Burkholderia pseudomallei and / or Burkholderia mallei protein selected from HCP-1 and HCP-2; in combination with antigenic epitopes present in at least one Burkholderia pseudomallei and / or Burkholderia mallei protein selected from HCP-6 and LolC, wherein the composition comprises live attenuated LVS ΔcapB expressing the at least one fusion protein. In illustrative embodiments of the invention, an immunogenic fusion protein comprising HCP-1 and / or HCP-2 in combination with HCP-6 and / or LolC is surprisingly expressed at levels that are at least 50% greater than expression levels of control immunogenic fusion proteins comprising HCP-1 and / or HCP-2 in the absence of HCP-6 and / or LolC. In some embodiments of the invention, the immunogenic fusion protein comprises antigenic epitopes present in HCP6, HCP1 and HCP2. In other embodiments of the invention, the immunogenic fusion protein comprises antigenic epitopes present in HCP6, HCP1 and LolC. Embodiments of the invention can further include other antigenic epitopes found in Burkholderia pseudomallei and / or Burkholderia mallei proteins. In certain embodiments of the invention, the Listeria monocytogenes vaccine platform expresses at least one Burkholderia pseudomallei and / or Burkholderia mallei protein from a native Burkholderia pseudomallei and / or Burkholderia mallei polynucleotide sequence and / or does not express a functional InlB protein; and / or does not express a functional actA protein; and / or expresses prfA protein having a G155S substitution mutation. In some embodiments of the invention, the Listeria monocytogenes vaccine platform expresses at least two fusion proteins. Embodiments of the invention include methods of generating an immune response to a Burkholderia pseudomallei and / or Burkholderia mallei in a mammal comprising administering to the mammal a vaccine composition disclosed herein such that an immune response to Burkholderia pseudomallei and / or Burkholderia mallei is generated. Typically, the mammal is immunized with the composition intranasally, subcutaneously, intradermally, intramuscularly or orally. Embodiments of the invention include use of a vaccine composition disclosed herein as a vaccine to generate an immune response to Burkholderia pseudomallei and / or Burkholderia mallei in a mammal. A related embodiment is a method of immunizing a susceptible host against an infection from Burkholderia pseudomallei and / or Burkholderia mallei. The method comprises administering to the host a prime vaccine composition disclosed herein (and optionally a homologous and / or heterologous booster vaccine). The vaccine comprises an attenuated recombinant live vaccine strain lacking a polynucleotide encoding CapB (LVS ΔcapB), wherein the LVS ΔcapB expresses a fusion protein having at least two antigens that are present on at least two proteins expressed by Burkholderia pseudomallei and / or Burkholderia mallei. Each vaccine is administered to the host in an amount sufficient to induce an immune response in the host to Burkholderia pseudomallei and / or Burkholderia mallei. In certain implementations, the host is administered a single dose of the prime vaccine and one or more doses of a homologous or heterologous booster vaccine. One major advantage of the vaccine compositions disclosed herein is the capacity to manufacture the vaccine cheaply and quickly. Live, attenuated bacterial vaccines, such as the LVS ΔcapB – vectored vaccine against melioidosis and / or glanders disclosed herein are much less expensive to manufacture, as they can be grown readily in inexpensive broth and require no purification. Vaccine cost is of critical importance in developing countries. Another major advantage of our vaccine embodiments of the invention is that the vector is a more attenuated derivative of a vaccine already safely administered to people. Hence it is anticipated to be extremely safe. Another likely advantage of our vaccine is that as a live attenuated vaccine, it is much more likely to induce long-lasting protection than a protein / adjuvant vaccine, DNA / RNA vaccine, or non-replicating virus-vectored vaccine. 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 Figure 1. Expression of B. pseudomallei Hcp6 by rLVS ΔcapB strains. Cleared cell lysates from rLVS ΔcapB strains expressing B. pseudomallei Hcp6 were analyzed by SDS-PAGE (A) and Western blotting (B) using anti-FLAG monoclonal antibody (the amount of lysate loaded per lane is equivalent to the amount of cells from 10 µL of overnight culture). The version of the gene (native or coLm) and the presence or absence of a FLAG tag (N-terminal or C-terminal) is indicated above the lanes. Red arrows indicate the positions of the recombinant proteins. M, molecular mass markers in kDa. Figure 2. Expression of B. pseudomallei LolC by rLVS ΔcapB strains. Cleared cell lysates from rLVS ΔcapB strains expressing B. pseudomallei LolC were analyzed by SDS-PAGE (A) and Western blotting (B) using anti-FLAG monoclonal antibody (the amount of lysate loaded per lane is equivalent to the amount of cells from 25 µL of overnight culture). The version of the gene (coLm or coLVS) and the presence or absence of a FLAG tag (N-terminal or C-terminal) is indicated above the lanes. The plasmid used to construct the two LolC (coLm) clones with an N-terminal FLAG tag was discovered to be defective after construction of the strains, thus explaining the lack of expression of LolC for these two clones. Red arrows indicate the positions of the recombinant proteins. M, molecular mass markers in kDa. Figure 3. Expression of B. pseudomallei Hcp1 by rLVS ΔcapB strains. Cleared cell lysates from rLVS ΔcapB strains expressing B. pseudomallei Hcp1 were analyzed by SDS-PAGE (A) and Western blotting using anti-Hcp1 antibody (B) or anti-FLAG monoclonal antibody (C) (the amount of lysate loaded per lane is equivalent to the amount of cells from 10 µL of overnight culture). The version of the gene (native, coLm, or coLVS) and the presence or absence of a FLAG tag (N-terminal or C- terminal) is indicated above the lanes. M, molecular mass markers in kDa. Figure 4. Expression of B. pseudomallei Hcp2 by rLVS ΔcapB strains. Cleared cell lysates from rLVS ΔcapB strains expressing B. pseudomallei Hcp2 were analyzed by SDS-PAGE (A) and Western blotting (B) using anti-FLAG monoclonal antibody (the amount of lysate loaded per lane is equivalent to the amount of cells from 25 µL of overnight culture). The version of the gene (native, coLm, or coLVS) and the presence of a FLAG tag (N-terminal or C-terminal) is indicated above the lanes. M, molecular mass markers in kDa. Figure 5. Expression of two-antigen fusion proteins (Hcp6-Hcp1, Hcp6-Hcp2, LolC-Hcp1, and LolC-Hcp2) by rLVS ΔcapB strains. Cleared cell lysates from rLVS ΔcapB strains expressing B. pseudomallei two-antigen fusion proteins with a C- terminal FLAG tag were analyzed by SDS-PAGE (A) and Western blotting (B) using anti-FLAG monoclonal antibody (the amount of lysate loaded per lane is equivalent to the amount of cells from 20 µL of overnight culture). The version of the gene (native, coLm, or coLVS) is indicated above the lanes. Red arrows indicate the positions of the recombinant two-antigen fusion proteins. Strains expressing untagged LolC (blue arrow) and untagged Hcp6 (green arrow) were included as controls. M, molecular mass markers in kDa. Figure 6. rLVS ΔcapB vaccines expressing Bp antigens are protective against IN challenge with virulent B. pseudomallei 1026b in highly sensitive BALB / c mice. BALB / c mice (n = 8 / group) were immunized by the ID route three times, four weeks apart (Weeks 0, 4, and 8) with 1 x 106CFU of LVS ΔcapB (Parental Vector control), Bp82 (Positive Control: a moderately attenuated single deletional mutant strain of B. pseudomallei), or rLVS ΔcapB strains expressing fusion proteins of Bp antigens (Hcp6- Hcp1, Hcp6-Hcp2, LolC-Hcp1, and LolC-Hcp2). Mice sham-immunized with PBS served as a negative control. At Week 12, mice were challenged by the IN route with 2,225 CFU (5x LD50) B. pseudomallei 1026b and monitored for survival for 6 weeks. Surviving mice were euthanized and the lung, liver, and spleen examined for abscesses and then cultured for B. pseudomallei. Mice with no abscesses and no detectable B. pseudomallei in the three organs were recorded as having sterile immunity. Survival curves were compared using the Log-rank (Mantel-Cox) test (Prism 9.3.1) with Holm- Bonferroni correction of p-values for multiple comparisons. *, P < 0.05; **, P < 0.01; ns, not significant (P ≥ 0.05). Figure 7. Expression of three-antigen fusion proteins (LolC-Hcp6-Hcp1, LolC- Hcp6-Hcp2, Hcp6-Hcp1-LolC, and Hcp6-Hcp2-LolC) by rLVS ΔcapB strains. Cleared cell lysates from rLVS ΔcapB strains expressing B. pseudomallei three-antigen fusion proteins with a C-terminal FLAG tag were analyzed by SDS-PAGE (A and C) and Western blotting (B and D) using anti-FLAG monoclonal antibody (the amount of lysate loaded per lane is equivalent to the amount of cells from 25 µL of overnight culture). The third antigen (LolC) was fused to either the N-terminus or the C-terminus of Hcp6-(GSSG (SEQ ID NO: 1))2-Hcp1 or Hcp6-(GSSG (SEQ ID NO: 1))2-Hcp2, using one of four linkers (indicated above the gels): A) GSAGSAAGSGEF (SEQ ID NO: 3); B) A(EAAAK (SEQ ID NO: 2))3A; C) (AP)10; D) direct linkage (single A residue). Red arrows indicate the positions of the recombinant three-antigen fusion proteins when apparent. M, molecular, molecular mass markers in kDa. Figure 8. Expression of Hcp6-(GSSG (SEQ ID NO: 1))2-Hcp1-Hcp2 by rLVS ΔcapB. Cleared cell lysate from an rLVS ΔcapB strain expressing the three-antigen fusion protein Hcp6-(GSSG (SEQ ID NO: 1))2-Hcp1-Hcp2 with a C-terminal FLAG tag was analyzed by SDS-PAGE (A) and Western blotting (B) using anti-FLAG monoclonal antibody (the amount of lysate loaded per lane is equivalent to the amount of cells from 25 µL of overnight culture). Red arrow indicates the position of the recombinant three-antigen fusion protein. M, molecular mass markers in kDa. Figure 9. Expression of four-antigen fusion proteins by rLVS ΔcapB. Cleared cell lysate from rLVS ΔcapB strains expressing four-antigen fusion proteins with a C- terminal FLAG tag were analyzed by SDS-PAGE (A) and Western blotting (B and C) using anti-FLAG monoclonal antibody (the amount of lysate loaded per lane is equivalent to the amount of cells from 100 µL of overnight culture). The blot in C is the same blot as in B, but the brightness and contrast adjusted in order to visualize the very poorly expressed four-antigen fusion proteins (blue arrows). To construct the four- antigen fusion proteins, LolC-(GSSG (SEQ ID NO: 1))2-Hcp2 was joined to the C- terminus of Hcp6-(GSSG (SEQ ID NO: 1))2-Hcp1 using one of four linkers (indicated above the gel): A) GSAGSAAGSGEF (SEQ ID NO: 3); B) A(EAAAK (SEQ ID NO: 2))3A; C) (AP)10; D) direct linkage (single A residue). Red arrows indicate the position of recombinant fusion proteins when apparent. M, molecular mass markers in kDa. Figure 10. Expression of four-antigen fusion proteins by rLVS ΔcapB. Cleared cell lysate from rLVS ΔcapB strains expressing four-antigen fusion proteins with a C- terminal FLAG tag were analyzed by SDS-PAGE (A) and Western blotting (B) using anti-FLAG monoclonal antibody (the amount of lysate loaded per lane is equivalent to the amount of cells from 25 µL of overnight culture). To construct the four-antigen fusion proteins, LolC was joined to either the N-terminus or C-terminus of Hcp6- (GSSG (SEQ ID NO: 1))2-Hcp1-Hcp2 using one of four linkers (indicated above the gels): A) GSAGSAAGSGEF (SEQ ID NO: 3); B) A(EAAAK (SEQ ID NO: 2))3A; C) (AP)10; D) direct linkage (single A residue). M, molecular mass markers in kDa. Figure 11. rLVS ΔcapB vaccines expressing three Bp antigens are comparable to or better than vaccines expressing two Bp antigens and immunization doses of ≥ 2x106CFU provide optimal protection. BALB / c mice (n = 8 / group, except for the group immunized with the Hcp6-Hcp2 vaccine at 2 x 106CFU, which had only 4 mice) were immunized by the ID route three times, four weeks apart (Weeks 0, 4, and 8) with 2 x 106CFU of LVS ΔcapB (Parental Vector control), 1 x 106CFU Bp82 (Positive Control), or 1, 2, 4, or 8 x 106CFU rLVS ΔcapB vaccines expressing fusion proteins of Bp antigens (Hcp6-Hcp1-Hcp2, Hcp6-Hcp1-LolC, Hcp6-Hcp1, Hcp6-Hcp2). Mice sham-immunized with PBS served as a negative control. At Week 12, mice were challenged by the IN route with 1,800 CFU (4x LD50) B. pseudomallei 1026b and monitored for survival for 6 weeks. The Negative Control is shown on all three graphs, but the Positive Control and Parental Vector are only displayed on the leftmost graph to facilitate visualization of the dose-response groups. Surviving mice were euthanized and the lung, liver, and spleen were examined for abscesses and then cultured for B. pseudomallei. Mice with no abscesses and no detectable B. pseudomallei in the three organs were recorded as having sterile immunity. Survival curves were compared using the Log-rank (Mantel-Cox) test (Prism 9.3.1) with Holm-Bonferroni correction of p- values for multiple comparisons. *, P < 0.05; **, P < 0.01; ns, not significant (P ≥ 0.05). Although the group immunized with the Hcp6-Hcp2 vaccine at 2 x 106CFU had good protection, it was not significant vs. sham due to having only 4 mice in the vaccine group. Figure 12. rLVS ΔcapB vaccines expressing three Bp antigens administered by the IN route are highly protective against a high challenge dose of B. pseudomallei 1026b. BALB / c mice (n = 7 or 8 / group) were immunized by the IN route three times, four weeks apart (Weeks 0, 4, and 8) with 2 x 106CFU of LVS ΔcapB (Parental Vector control) or rLVS ΔcapB strains expressing fusion proteins of Bp antigens (Hcp6-Hcp1- Hcp2, Hcp6-Hcp1-LolC). The positive control strain, Bp82, was administered at 1 x 106CFU by the ID route, as in the first two animal efficacy experiments (Fig. 6 and Fig.11). Mice sham immunized with PBS served as a negative control. At Week 12, mice were challenged by the IN route with 2,700 CFU (6x LD50) B. pseudomallei 1026b and monitored for survival for 6 weeks. The Negative Control is shown on all three graphs, but the Positive Control is only displayed on the leftmost graph. Surviving mice were euthanized and the lung, liver, and spleen were examined for abscesses and then cultured for B. pseudomallei. Mice with no abscesses and no detectable B. pseudomallei in the three organs were recorded as having sterile immunity. Survival curves were compared using the Log-rank (Mantel-Cox) test (Prism 9.3.1) with Holm- Bonferroni correction of p-values for multiple comparisons. *, P < 0.05; ns, not significant (P ≥ 0.05). Figure 13. IN immunization with rLVS ΔcapB vaccines and the LVS ΔcapB vector is superior to ID immunization with Bp82. The data from Experiment 3 (Fig. 12) was re-analyzed, combining groups for the same vaccine that received 1, 2, or 3 doses. Survival curves were compared using the Log-rank (Mantel-Cox) test (Prism 9.3.1) with Holm-Šídák correction of p-values for multiple comparisons. *, P < 0.05; **, P < 0.01. Figure 14. Serum IgG titers to LVS and Bp antigens. Immunized mice from Experiment 2 (Fig. 11) were bled one week prior to challenge and serum IgG titers were determined by ELISA. (A) Serum IgG titers for five antigens (HI LVS ΔcapB, Hcp6, Hcp1, LolC, and HI Bp82). Circles represent the values for individual mice and the mean ± SEM is shown with bars. Only four groups were tested for antibody to LolC. For each antigen, the mean serum IgG titers were compared to a control group (Sham, LVS ΔcapB, or Bp82 as indicated in the graphs) using ordinary One-Way ANOVA with Dunnett's multiple comparisons test (Prism 9.3.1). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (B) Dose response of antibody titers and Log CFU of the rLVS ΔcapB / Bp Hcp6-Hcp1 vaccine which was given at 1, 2, 4, and 8 x 106CFU. The data for the three different antigens (HI LVS ΔcapB, Hcp6, and Hcp1) were fit to a straight line using non-linear regression (Prism 9.3.1) and the R2value is shown. Shaded areas represent the 95% confidence interval. The dashed lines in (A) and (B) indicate the lower limit of detection (200-fold initial serum dilution). Figure 15. Frequency of cytokine-producing CD4+ T cells (as a percentage of total CD3+ T cells) in response to HI LVS ΔcapB. Mice were sham immunized (A) or immunized with LVS ΔcapB (B), rLVS ΔcapB / Bp Hcp6-Hcp1-Hcp2 (C), or rLVS ΔcapB / Bp Hcp6-Hcp1-LolC (D) three times at weeks 0, 4, and 8. One week after the last immunization, splenocytes and lung cells were isolated, stimulated in vitro with HI LVS ΔcapB for 6 h, and analyzed by multiparameter flow cytometry. Background numbers of cells producing cytokines without antigen stimulation were subtracted. The results from two independent experiments are shown. Figure 16. Expression of Bp antigens by rLVS ΔcapB strains. Cleared cell lysates from rLVS ΔcapB strains expressing Bp Hcp6 (A), LolC (B), Hcp1 (C), or Hcp2 (D) were analyzed by SDS-PAGE (upper image) and Western blotting (lower image or images) using anti-FLAG monoclonal antibody or anti-Hcp1 antibody, as indicated. The amount of lysate loaded per lane is equivalent to the amount of cells from 10 µL of overnight culture (A and C) or 25 µL of overnight culture (B and D). The version of the gene [native, codon-optimized for L. monocytogenes (coLm), or codon-optimized for LVS (coLVS) and the presence or absence of a FLAG tag [N-terminal (N) or C- terminal (C)] is indicated above the lanes. Red arrows indicate the positions of the recombinant proteins in SDS-PAGE images (A and B). For LolC (B), the plasmid used to construct the two LolC (coLm) clones with an N-terminal FLAG tag was discovered to be defective after construction of the strains, thus explaining the lack of expression of LolC for these two clones. M, molecular mass markers in kDa. Lane numbers are indicated underneath the lower image for A, B, C, and D. Figure 17. Expression of Hcp6 by rLVS ΔcapB strains. Boiled cell pellets from rLVS ΔcapB strains expressing Bp Hcp6 were analyzed by SDS-PAGE. Four different clones for each construct are shown. The version of the gene [codon-optimized for L. monocytogenes (coLm) or codon-optimized for LVS (coLVS)] is indicated above the lanes. Red arrows indicate the positions of the recombinant proteins. M, molecular mass markers in kDa. Lane numbers are indicated underneath the image. Figure 18. Expression of two-antigen and three-antigen fusion proteins by rLVS ΔcapB strains. Cleared cell lysates from rLVS ΔcapB strains expressing Bp fusion proteins with a C-terminal FLAG tag were analyzed by SDS-PAGE (upper images in A-C; left image in D) and Western blotting using anti-FLAG monoclonal antibody (lower image in A-C; right image in D). A. Two-antigen fusion proteins (LolC-Hcp1, LolC-Hcp2, Hcp6-Hcp1, and Hcp6-Hcp2): The version of the gene (native, coLm, or coLVS) is indicated above the lanes. Strains expressing untagged LolC (yellow arrow) and untagged Hcp6 (green arrow) were included as controls. B and C. Three-antigen fusion proteins (LolC-Hcp6-Hcp1, LolC-Hcp6-Hcp2, Hcp6- Hcp1-LolC, and Hcp6-Hcp2-LolC): The third antigen (LolC) was fused to either the N-terminus (B) or the C-terminus (C) of Hcp6-(GSSG (SEQ ID NO: 1))2-Hcp1 or Hcp6-(GSSG (SEQ ID NO: 1))2-Hcp2, using one of four linkers (indicated above the gels): (1) GSAGSAAGSGEF (SEQ ID NO: 3); (2) A(EAAAK (SEQ ID NO: 2))3A; (3) (AP)10; (4) direct linkage (single alanine residue). D. Expression of Hcp6-Hcp1-Hcp2 by rLVS ΔcapB. Cleared cell lysate from an rLVS ΔcapB strain expressing the three- antigen fusion protein Hcp6-(GSSG (SEQ ID NO: 1))2-Hcp1-Hcp2 with a C-terminal FLAG tag (lane 3) was analyzed by SDS-PAGE (left image) and Western blotting (right image) using anti-FLAG monoclonal antibody. Lane 1: rLVS ΔcapB strain with an empty plasmid, Lane 2: rLVS ΔcapB strain with an alternate version of Hcp6-(GSSG (SEQ ID NO: 1))2-Hcp1-Hcp2. The amount of lysate loaded per lane is equivalent to the amount of cells from 20 or 25 µL of overnight culture. Red arrows indicate the positions of the recombinant three-antigen fusion proteins when apparent. M, molecular mass markers in kDa. Lane numbers are indicated underneath the lower image for A, B, and C. Figure 19. Efficacy Experiment 1: rLVS ΔcapB vaccines expressing two Bp antigens are protective against IN challenge with virulent Bp 1026b in highly sensitive BALB / c mice. A. Experimental Schedule. B. Kaplan-Meier survival curves. BALB / c mice (n = 8 / group) were immunized by the ID route three times, four weeks apart (Weeks 0, 4, and 8) with 1 x 106CFU of LVS ΔcapB (Parental Vector control), Bp82 (Positive Control: a moderately attenuated single deletional mutant strain of Bp), or rLVS ΔcapB strains expressing fusion proteins of Bp antigens (Hcp6-Hcp1, Hcp6- Hcp2, LolC-Hcp1, and LolC-Hcp2). Mice sham-immunized with PBS served as a negative control. At Week 12, mice were challenged by the IN route with 2,225 CFU (5x LD50) Bp 1026b and monitored for survival for 6 weeks. Surviving mice were euthanized and the lung, liver, and spleen examined for abscesses and then cultured for Bp. Groups were compared using pairwise Log-rank tests with Holm-Bonferroni correction of p-values for multiple comparisons. Significance vs. sham: *, P < 0.05; **, P < 0.01; ns, not significant (P ≥ 0.05). Figure 20. Expression of four-antigen fusion proteins by rLVS ΔcapB. Cleared cell lysate from rLVS ΔcapB strains expressing four-antigen fusion proteins with a C- terminal FLAG tag were analyzed by SDS-PAGE (A) and Western blotting (B and C) using anti-FLAG monoclonal antibody (the amount of lysate loaded per lane is equivalent to the amount of cells from 100 µL of overnight culture). The blot in C is the same blot as in B, but the brightness and contrast adjusted in order to visualize the very poorly expressed four-antigen fusion proteins (yellow arrows). To construct the four-antigen fusion proteins, we joined LolC-(GSSG (SEQ ID NO: 1))2-Hcp2 to the C- terminus of Hcp6-(GSSG (SEQ ID NO: 1))2-Hcp1 using one of four linkers (indicated above the gel): (1) GSAGSAAGSGEF (SEQ ID NO: 3); (2) A(EAAAK)3A; (3) (AP)10; (4) direct linkage (single alanine residue). Red arrows indicate the position of recombinant fusion proteins when apparent. M, molecular mass markers in kDa. Lane numbers are indicated underneath the images. Figure 21. rLVS ΔcapB vaccines expressing three Bp antigens and the LVS ΔcapB parental vector provide protection against inhalational B. mallei China 7 challenge. A. Experimental Schedule. B, C, D, E, and F. Kaplan-Meier survival curves. BALB / c mice (n = 12 / group) were sham-immunized with PBS (Negative Control) or immunized three times, four weeks apart (Weeks 0, 4, and 8) with 2 x 106CFU of LVS ΔcapB (Parental Vector control) by the intradermal (ID) or intranasal (IN) route, 1 x 106CFU Bp82 (Positive Control) by the ID route, or 2 x 106CFU rLVS ΔcapB vaccines expressing fusion proteins of Bp antigens (Hcp6-Hcp1-Hcp2 or Hcp6- Hcp1-LolC) by the ID or IN route. At Week 14, mice were challenged by the IN route with 190 CFU (12.7x LD50) B. mallei China 7 and monitored for survival for 6 weeks. Each graph includes the Negative Control (Group A, PBS) and Positive Control (Group B, Bp82) for comparison. B. LVS ΔcapB and rLVS ΔcapB vaccines administered by the IN route. C. LVS ΔcapB and rLVS ΔcapB vaccines administered by the ID route. D. LVS ΔcapB and rLVS ΔcapB vaccines administered by the IN route (combined). E. LVS ΔcapB and rLVS ΔcapB vaccines administered by the ID route (combined). F. All six LVS ΔcapB and rLVS ΔcapB vaccines administered by the IN and ID route (combined). 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. 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. 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. Patent No. 8,481,024, titled “VACCINES AGAINST TULAREMIA”, U.S. Patent No. 8,206,700, titled “METHODS AND COMPOSITIONS FOR TREATING TULAREMIA”, and U.S. Patent No.11,224,647, entitled “SAFE POTENT SINGLE PLATFORM VACCINE AGAINST TIER 1 SELECT AGENTS AND OTHER PATHOGENS”. Aspects of the invention disclosed herein are discussed in Tullius et al., “LVS ΔcapB-vectored multiantigenic melioidosis vaccines protect against lethal respiratory Burkholderia pseudomallei challenge in highly sensitive BALB / c mice” ASM Journals, mBio, Vol. 15, No. 4, 21 March 2024 (hereinafter “Tullius et al.”), the contents of which are incorporated by reference. The present disclosure provides a single vector platform vaccine and associated methods for preventing, reducing the possibility of or treating melioidosis and / or glanders in humans and animals. 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 which comprises a heterologous promoter that expresses a fusion protein comprising a selected constellation of multiple antigenic polypeptide epitopes present in Burkholderia pseudomallei and / or Burkholderia mallei. Another exemplary embodiment of the invention is a method of immunizing a susceptible host against an infection from Burkholderia pseudomallei and / or Burkholderia mallei, comprising administering to the host a vaccine disclosed herein, for example one comprising an attenuated recombinant live vaccine strain lacking a polynucleotide encoding CapB (LVS ΔcapB), wherein the LVS ΔcapB expresses a polypeptide having the constellation of Burkholderia pseudomallei and / or Burkholderia mallei antigens disclosed herein. Embodiments of the invention include a single vector platform vaccine for preventing the disease melioidosis caused by Burkholderia pseudomallei in humans and animals. The invention utilizes our previously described vector LVS ΔcapB, a live attenuated capB mutant of Live Vaccine Strain (LVS), itself attenuated by serial passage in the 20th century from Francisella tularensis, subsp. holarctica; LVS has two major attenuating deletions and several minor mutations. In an exemplary working example of this embodiment of the immunogenic composition, a heterologous promoter (e.g. a bfr promoter and / or an omp promoter) expresses a fusion protein comprising at least 8 contiguous amino acids of Hcp1; and / or at least 8 contiguous amino acids of Hcp2; in combination with at least 8 contiguous amino acids of Hcp6 and / or at least 8 contiguous amino acids of at least 8 contiguous amino acids of LolC. Embodiments of the invention include a single vector platform vaccine for preventing the disease glanders caused by Burkholderia mallei in humans and animals. The invention utilizes our previously described vector LVS ΔcapB, a live attenuated capB mutant of Live Vaccine Strain (LVS), itself attenuated by serial passage in the 20th century from Francisella tularensis, subsp. holarctica; LVS has two major attenuating deletions and several minor mutations. In an exemplary working example of this embodiment of the immunogenic composition, a heterologous promoter (e.g. a bfr promoter and / or an omp promoter) expresses a fusion protein comprising at least 8 contiguous amino acids of Hcp1; and / or at least 8 contiguous amino acids of Hcp2; in combination with at least 8 contiguous amino acids of Hcp6 and / or at least 8 contiguous amino acids of at least 8 contiguous amino acids of LolC. As discussed in Example 2 below, we obtained experimental results evaluating the vector and the vector expressing one of two combinations of 3 antigens (the same combinations of antigens used against B. pseudomallei) for efficacy in protecting against intranasal challenge with Burkholderia mallei, the agent of glanders. The vaccines protected well. In these studies, we saw cross-protection against B. mallei by the vector as we also saw for B. pseudomallei, presumably because of shared antigens Surprisingly, the vector alone showed efficacy in addition to the vector expressing one or the other of the two 3-antigen combinations. In this context, embodiments of the invention directed towards glanders include vectors alone (i.e., not expressing one or the other of the two 3-antigen combinations) and their use. Embodiments of the invention are useful for preventing melioidosis and / or glanders, infections caused by Burkholderia pseudomallei (Bp) and Burkholderia mallei respectively. One such embodiment is a vaccine composition for expressing the constellation of Burkholderia pseudomallei and / or Burkholderia mallei epitopes disclosed herein. Typically, the 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. Previous human trials have demonstrated reasonable safety of the double- deletional parent vector (LVS). The even more attenuated but still highly immunogenic triple-deletional platform vector (LVS ∆capB) derived from the parent is >10,000 fold less virulent in a mouse model (as measured by intranasal LD50; all animals survived highest dose tested). Because the vaccine is based upon a bacterial vector, it can be inexpensively manufactured in broth culture – no purification is necessary as in the case of viral-vectored vaccine. This vaccine is the latest in a series of vaccines developed using the LVS ΔcapB Vector platform. This provides several additional advantages. For example, a single vector 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. 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. Furthermore, a single vector platform vaccine also has the advantage that different vaccines comprising the same vector but expressing different antigens can be safely and effectively administered at the same time. That is, individual LVS ΔcapB vaccines expressing Burkholderia pseudomallei (Bp) antigens and / or Burkholderia mallei antigens, Francisella tularensis subsp. tularensis (Ft) antigens, Bacillus anthracis (Ba) antigens, Yersinia pestis (Yp) antigens, SARS-CoV- 2 antigens, and the antigens of other pathogens, can be administered together. 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 Burkholderia pseudomallei and / or Burkholderia mallei polypeptide. In one such embodiment, the method comprises administering a LVS immunogenic composition disclosed herein in a primary vaccination; and optionally 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. To construct illustrative rLVS ΔcapB / Bp vaccines, we selected four different antigens (Hcp1, Hcp2, Hcp6, and LolC) for expression in the LVS ΔcapB vector platform. As detailed below, it was not obvious how to achieve good expression of multiple antigens in a single vaccine strain prior to our studies. In our studies we observed a large variation in expression level for individual antigens. We first tried expressing each individual antigen using a strong promoter and ribosome-binding site (RBS). We could detect high levels of two of the antigens - Hcp6 (Fig.16A, top image, Lanes 1-6 and 9-11) and LolC (Fig.16B, top image, Lanes 1-4, 8-9, and 11-14) - using a total protein stain following SDS-PAGE of cell lysates from the recombinant vaccine strains, but surprisingly, Hcp1 (Figure 16C, top image, Lanes 1-12) and Hcp2 (Figure 16D, top image, Lanes 2-7, 9-14) were undetectable by this method. In our studies we observed that codon optimization does not always work as expected. To optimize expression levels, we tested 2 or 3 versions of each gene: 1) the native gene, 2) a version of the gene codon optimized for LVS (coLVS), and 3) a version of the gene codon optimized for a different attenuated vaccine vector (Listeria monocytogenes) (coLm). We found for Hcp6, rather unexpectedly, that expression was significantly worse using the version of the gene codon optimized for LVS (Figure 17, Lanes 5-8), compared with both the native gene and the gene codon optimized for L. monocytogenes (Figure 17, Lanes 1-4). LolC, produced the opposite result, where the gene codon optimized for LVS had better expression than the gene codon optimized for L. monocytogenes. For Hcp1 and Hcp2, the two antigens with poor expression, surprisingly, the native gene was expressed as well as, or in some cases better than, the two codon optimized versions of the genes (Figure 16C, middle and bottom images, Lanes 1-12; Figure 16D, bottom image, Lanes 2-7 and 9-14). For example, for Hcp1, the untagged native gene (Figure 16C, middle image, Lanes 1 and 2), had greater expression than the codon-optimized genes (Figure 16C, middle image, Lanes 3-6); and for Hcp2, expression of the C-terminal tagged native gene (Figure 16D, lower image, Lanes 9 and 10) was modestly greater than the expression of the L. monocytogenes codon optimized gene (Figure 16D, lower image, Lanes 11 and 12) but substantially greater than the LVS codon optimized gene (Figure 16D, lower image, Lanes 13 and 14). In our studies we discovered an improved expression of Hcp1 and Hcp2 by expressing them as fusion proteins with Hcp6 or LolC. Because codon optimization of the hcp1 and hcp2 genes did not improve the poor expression of Hcp1 and Hcp2, as described above, we sought other methods to improve their expression. We discovered that joining these two antigens to either Hcp6 or LolC (the two antigens with good expression) as the lead proteins to create two-antigen fusion proteins allowed for much greater expression of Hcp1 and Hcp2 such that they were now apparent on SDS-PAGE gels (Figure 18A, top image, Lanes 1-6, 10-15). Thus, in all cases, the fusion proteins were now visible using a total protein stain following SDS-PAGE of cell lysates, whereas previously Hcp1 and Hcp2 were undetectable by this method when expressed by themselves. We also discovered that fusion proteins with Hcp6 as the lead protein had greater expression levels compared with fusion proteins with LolC as the lead protein (Figure 18A, top and bottom images, Lanes 10-15 vs. Lanes 1-6). In our studies we discovered that mice immunized with vaccines expressing Hcp6-Hcp1 and Hcp6-Hcp2 have better survival than mice immunized with vaccines expressing LolC-Hcp1 and LolC-Hcp2. Mice immunized with two recombinant vaccines, those expressing Hcp6-Hcp1 and Hcp6-Hcp2, had greater survival than mice immunized with positive control vaccine Bp82 (88% vs.75%), while mice immunized with two other vaccines, expressing LolC-Hcp1 and LolC-Hcp2, had lower survival (38% vs. 75%) (Figure 19). Bp82, the positive control vaccine, is a live attenuated Burkholderia pseudomallei strain with a single major attenuating deletion and hence unsuitable for clinical use. The construction of vaccines expressing more than two antigens is particularly challenging. As a first attempt to construct vaccines expressing more than two antigens, we took the simple approach of linking two of our two-antigen fusion proteins (Hcp6- Hcp1 and LolC-Hcp2), which were expressed well (see above), to create a four-antigen fusion protein (Hcp6-Hcp1-LolC-Hcp2), joined by various peptide linkers. Expression of the four-antigen fusion proteins was just barely detectable using a highly sensitive Western blotting method (Figure 20, Lanes 3-6, 10-13). Because we obtained such poor expression using a simple approach, we took a more systematic approach to first construct vaccines expressing three-antigen fusion proteins. We did this by linking LolC to either the N-terminus or C-terminus of Hcp6-Hcp1 or Hcp6-Hcp2 [two of the most highly expressed fusion proteins and most protective vaccine constructs (Figures 18 and 19)]. For each combination of antigens, the C-terminal portion was linked to the N-terminal portion by one of three peptide linkers or linked directly (only a single alanine inserted between the two). We transformed the 16 plasmids into LVS ΔcapB, and analyzed individual clones for expression of the three-antigen fusion proteins by SDS-PAGE and Western blotting (Figure 18B, 18C). Surprisingly, seven of the eight constructs with LolC as the lead protein were expressed well (Figure 3B, Lanes 2-8), but LolC linked directly to Hcp6-Hcp2 had poor expression (Figure 18B, Right section, Lane 9 with Linker 4). All four constructs comprising Hcp6-Hcp1-LolC were expressed well and the type of linkage between Hcp6-Hcp1 and LolC seemed to have minimal influence on expression level (Figure 18C, Left section, Linkers 1-4). Also, surprisingly, in the case of Hcp6-Hcp2-LolC, all four constructs were expressed poorly (Figure 18C, Right section, Linkers 1-4). Thus, multiple experimental approaches were required to identify ones that achieved good expression of multi-antigen fusion proteins in the LVS ΔcapB vector platform. EXAMPLES EXAMPLE 1: RECOMBINANT FRANCISELLA TULARENSIS LVS ΔCAPB - VECTORED VACCINE EXPRESSING BURKHOLDERIA PSEUDOMALLEI PROTEINS PROTECTS AGAINST LETHAL RESPIRATORY CHALLENGE IN THE HIGHLY SENSITIVE BALB / C MOUSE MODEL OF MELIOIDOSIS Results Construction of Bp vaccines expressing a single Bp antigen based on the LVS ΔcapB platform Our lab has previously constructed vaccines against tularemia, anthrax, plague, and SARS-CoV-2 by expressing antigens from the pathogens responsible for those diseases in the LVS ΔcapB vector, a highly attenuated bacterium1-7. In the current study, to construct vaccines against melioidosis, we selected four promising antigens from B. pseudomallei for expression in LVS ΔcapB, Hcp1, Hcp2, Hcp6, and LolC. Hcp1 (BPSS1498), Hcp2 (BPSS0518), and Hcp6 (BPSL3105) are surface-associated components from three of the six Type VI secretion systems (T6SSs) present in B. pseudomallei8. LolC (BPSL2277) is a membrane protein involved in lipoprotein sorting in gram-negative bacteria9. All four of these proteins have been shown to provide some level of protective efficacy against B. pseudomallei challenge in mice when administered as recombinant protein in adjuvant8-10. To facilitate construction and characterization of rLVS ΔcapB vaccines expressing B. pseudomallei antigens, we adapted the E. coli-Francisella shuttle expression vector pFNL / pbfr-SD-iglA5. To make it compatible with Electra cloning11, we removed the four SapI sites in the vector backbone using site directed mutagenesis and then replaced iglA with a sacB cassette flanked by SapI site with Electra compatible overhangs. We first constructed three Electra compatible DAUGHTER plasmids that allow for ORFs from Electra compatible MOTHER plasmids to be cloned downstream of the strong Francisella bacterioferritin promoter with a ribosomal binding site. The three versions of the expression plasmid allow cloning of ORFs without an additional tag [pFNL-bfr-D1 (sacB)]; with an N-terminal fusion of a dual 3xFLAG-His8 tag [pFNL-bfr-D2[N3F-8H] (sacB)]; or with a C- terminal fusion of a dual His8-3x FLAG tag [pFNL-bfr-D3[C8H-3F] (sacB)]. We then transferred ORFs coding for the four B. pseudomallei antigens from Electra compatible MOTHER plasmids to the three Electra compatible DAUGHTER plasmids, electroporated the plasmids into LVS ΔcapB, and analyzed cleared cell lysates from individual clones for expression of the heterologous proteins. To improve our chances of obtaining good expression, we tested two or three versions of each gene: 1) the native gene amplified from B. pseudomallei K96243 gDNA, 2) a synthetic gene codon optimized for LVS (coLVS), and 3) a synthetic gene codon optimized for Listeria monocytogenes (coLm). The coLm gene was intended for a separate project using attenuated L. monocytogenes as a vaccine vector, but as it was available, we tested it in LVS ΔcapB as well. We obtained strong expression of Hcp6 by LVS ΔcapB, with or without a fusion tag, with bands visible on an SDS-PAGE gel when stained for total protein (Fig. 1A). The coLm gene appeared to be expressed somewhat better than the native gene. Expression of the C-terminally tagged protein appeared slightly reduced compared with the untagged protein, while the N-terminally tagged protein was even further reduced (for both native and coLm versions of the hcp6 gene). Surprisingly, expression of the gene codon optimized specifically for LVS (coLVS) was lower than expression of the gene codon optimized specifically for L. monocytogenes (coLm), and so we decided to use the coLm version of hcp6 for further constructs. As with Hcp6, we obtained good expression of LolC in LVS ΔcapB, with bands visible on an SDS-PAGE gel when stained for total protein, although in this case the coLVS gene was superior to the coLm gene (Fig.2A). Based on total protein, the C-terminally tagged LolC was expressed at a similar level to the untagged LolC, and the N-terminally tagged protein appeared to have somewhat reduced expression. However, the N-terminally tagged LolC produced stronger bands than the C-terminally tagged LolC by Western blotting (Fig.2B). Hcp1 had seemingly less expression than Hcp6 and LolC, with no bands visible on an SDS- PAGE gel stained for total protein (Fig. 3A). The stain we used for detection of total protein becomes fluorescent after reacting with tryptophan residues. That Hcp1 only has a single tryptophan, whereas Hcp6 has four and LolC has three, may account for its apparent reduced expression compared with these other two proteins. Based on Western blotting using anti-Hcp1 and anti-FLAG antibodies, the untagged native hcp1 gene seemed to have the best expression, followed by the coLVS gene, and then the coLm gene, which had the least expression (Fig. 3B, 3C). Finally, Hcp2 (containing three tryptophan residues) also had lower expression than Hcp6 and LolC, with no bands visible on an SDS-PAGE gel stained for total protein (Fig. 4A). However, the FLAG tagged proteins were easily detected by Western blotting (Fig. 4B). There is seemingly greater expression for C-terminal Hcp2 fusions compared with N-terminal fusions, although this may reflect different affinities of the anti-FLAG antibody for N- terminal and C-terminal fusions and not just the expression level. Expression of N- terminally tagged Hcp2 appears similar for native, coLm, and coLVS genes, but expression of C-terminally tagged Hcp2 appears somewhat better for the native construct compared with the coLm and coLVS constructs. Construction of Bp vaccines expressing two Bp antigens To increase the antigen repertoire of the rLVS ΔcapB vaccines, we next sought to express fusion proteins consisting of two Bp antigens joined by a flexible linker. We constructed pFNL expression plasmids in which Hcp6 or LolC (the two best expressed proteins) were the lead protein fused via a GSSGGSSG (SEQ ID NO: 4) flexible peptide linker to a C-terminal Hcp1 or Hcp2 protein, electroporated the plasmids into rLVS ΔcapB, and analyzed expression by SDS-PAGE and Western blotting (Fig. 5). Expression of the two-antigen fusion proteins was maintained at a relatively high level, similar to the individual Hcp6 and LolC proteins (Fig.1A and 2A), with bands visible on an SDS-PAGE stained for total protein (Fig.5A). Expression levels appeared to be unaffected by which gene (hcp1 or hcp2) or which version of the gene (native, coLVS, or coLm) was at the C-terminus, suggesting that the lead gene (hcp6 or lolC) has the greater influence on expression level. Efficacy of LVS ΔcapB platform vaccines expressing combinations of two B. pseudomallei antigens in the highly sensitive BALB / c mouse model of pneumonic melioidosis To assess the protective efficacy of the two-antigen vaccines described above, we immunized BALB / c mice, a strain of mice highly sensitive to B. pseudomallei infection, by the intradermal (ID) route using a homologous boosting regimen (1 x 106CFU administered at Weeks 0, 4, and 8). Three control groups were included: a negative control group (sham immunized), a positive control group (immunized with Bp82, a moderately attenuated single deletional mutant strain of B. pseudomallei that is too toxic for human use but which has been demonstrated to induce good protection12), and the parental vector (LVS ΔcapB). At Week 12, the mice were challenged by the intranasal (IN) route with a lethal dose of B. pseudomallei 1026b (2,225 CFU, 5x LD50) and monitored for survival for 6 weeks (Fig. 6). Sham-immunized mice and mice immunized with LVS ΔcapB succumbed rapidly to infection with B. pseudomallei. Survival of all other groups was statistically significantly better than for sham- immunized mice, except for the rLVS ΔcapB / Bp LolC-Hcp1 group. Mice immunized with two recombinant vaccines, those expressing Hcp6-Hcp1 and Hcp6-Hcp2, had greater survival than mice immunized with Bp82 (88% vs. 75%), while mice immunized with two other vaccines, expressing LolC-Hcp1 and LolC-Hcp2, had lower survival (38%). At the end of the 6-week period for monitoring survival, the surviving mice were euthanized and the lung, liver, and spleen were examined for abscesses and then cultured for B. pseudomallei. Mice with no abscesses and no detectable B. pseudomallei in the three organs were recorded as having sterile immunity. We obtained moderate to high levels of sterile immunity with rLVS ΔcapB / Bp vaccines, comparable, and in some cases superior, to that achieved with Bp82 (Fig.6). Construction of vaccines expressing three or four Bp antigens As we obtained good protection with all four rLVS ΔcapB vaccines expressing two antigen fusion proteins in Experiment 1 (Hcp6-Hcp1, Hcp6-Hcp2, LolC-Hcp1, and LolC-Hcp2, Fig. 6), but not 100% protection, we sought to determine if we could improve upon our results by adding a third antigen. Therefore, we constructed vaccines expressing three-antigen fusion proteins by linking LolC to either the N-terminus or C- terminus of Hcp6-Hcp1 or Hcp6-Hcp2 (two of the best constructs from Experiment 1). For each combination of antigens, the C-terminal portion was linked to the N-terminal portion by one of three peptide linkers or linked directly (only a single alanine inserted between the two). We transformed the 16 plasmids into rLVS ΔcapB, and analyzed individual clones for expression of the three-antigen fusion proteins by SDS-PAGE and Western blotting (Fig. 7). Seven of the eight constructs with LolC as the lead protein were expressed well, but LolC linked directly to Hcp6-Hcp2 had poor expression. All four constructs comprising Hcp6-Hcp1-LolC were expressed well and the type of linkage between Hcp6-Hcp1 and LolC seemed to have minimal influence on expression level. In the case of Hcp6-Hcp2-LolC, all four constructs were expressed poorly. We selected one of the best expressing constructs, Hcp6-Hcp1-GSAGSAAGSGEF (SEQ ID NO: 3)-LolC, to pursue further. We also constructed an rLVS ΔcapB vaccine expressing Hcp6-Hcp1-Hcp2 with Hcp2 linked directly to Hcp6-Hcp1, which had good expression (Fig.8). To construct vaccines with all four antigens, we joined two of the two-antigen constructs that were expressed well (Hcp6-Hcp1 and LolC-Hcp2, Fig. 5) with various linkers. As before, the Hcp6-Hcp1 fusion protein was expressed very well, as were derivatives of Hcp6-Hcp1 with peptide linkers on the C-terminus (but lacking LolC- Hcp2) (Fig. 9). Also, as previously observed, LolC-Hcp2 was expressed well enough to be visualized with a total protein stain. However, the four-antigen fusion proteins (Hcp6-Hcp1-LolC-Hcp2), although present, were barely detectable by Western blotting (Fig. 9C) and so this strategy was abandoned. We then tried a second approach to construct vaccines with all four antigens, by linking Hcp6-Hcp1-Hcp2 with LolC fused to either the N-terminus or the C-terminus. As for the three-antigen constructs, the C- terminal portion was linked to the N-terminal portion by one of three peptide linkers or directly. We failed to obtain a correct plasmid for one of the eight constructs and did not pursue it further; the correct 7 plasmids were transformed into rLVS ΔcapB, and individual clones were analyzed for expression of the four-antigen fusion proteins by SDS-PAGE and Western blotting (Fig. 10). Although none of the four-antigen fusion proteins were apparent in a total protein stain (Fig.10A), six of the seven proteins were detectable by Western blotting (Fig. 10B). We selected one of the constructs, LolC- (AP)10-Hcp6-Hcp1-Hcp2, to test as a vaccine. Efficacy of LVS ΔcapB platform vaccines expressing combinations of three B. pseudomallei antigens in the BALB / c mouse model of pneumonic melioidosis and dose-response study of two-antigen vaccines To evaluate the efficacy of three-antigen vaccines, we immunized BALB / c mice by the ID route using a homologous boosting regimen (Weeks 0, 4, and 8) as in Experiment 1. We administered the new three-antigen vaccines and the LVS ΔcapB parental vector at 2 x 106CFU. Simultaneously, to determine the optimal dose of vaccine, we selected the two best vaccines from Experiment 1 (expressing Hcp6-Hcp1 and Hcp6-Hcp2) and performed a dose-response study, using 1, 2, 4, or 8 x 106CFU for Hcp6-Hcp1 and 2 or 4 x 106CFU for Hcp6-Hcp2. A negative control group (sham immunized) and a positive control group (immunized with Bp82) were included as in Experiment 1. A vaccine expressing a four-antigen fusion protein, LolC-(AP)10-Hcp6- Hcp1-Hcp2, was also included. At Week 12, mice were challenged by the IN route with a lethal dose of B. pseudomallei 1026b (1,800 CFU, 4x LD50) and monitored for survival for 6 weeks (Fig.11). As in Experiment 1, sham-immunized mice succumbed rapidly to infection with B. pseudomallei. However, in contrast to Experiment 1, mice immunized with LVS ΔcapB, the parental vector, were moderately protected. The difference in results may be due to using a higher vaccination dose (2 x 106vs.1 x 106CFU) and / or a somewhat lower challenge dose in Experiment 2 (1,800 vs.2,225 CFU). All vaccines tested in Experiment 2 produced statistically significant protection compared with sham vaccinated mice, with the exception of the lowest dose of the Hcp6-Hcp1 vaccine. The new three-antigen vaccines expressing Hcp6-Hcp1-Hcp2 or Hcp6-Hcp1-LolC provided good protection (88% and 75% survival, respectively), comparable or superior to the two-antigen vaccines expressing Hcp6-Hcp1 or Hcp6- Hcp2, although the differences were not statistically significant. We obtained moderate levels of sterile immunity for the three-antigen vaccines, comparable to that achieved with Bp82. Based on these results, we decided to move forward with the three-antigen vaccines reasoning that a greater antigen repertoire may be beneficial in outbred populations. Immunization with the four-antigen vaccine (expressing LolC-Hcp6- Hcp1-Hcp2) resulted in 50% survival, somewhat less than efficacy of the three-antigen vaccines. In the dose-response study, the lowest dose (1 x 106CFU) had the lowest survival of any of the rLVS ΔcapB vaccine groups. Since there was no apparent advantage to doses greater than 2 x 106CFU, and potentially lesser efficacy at 1 x 106CFU, we decided to use 2 x 106CFU as the dose in subsequent experiments. Efficacy of three-antigen vaccines delivered intranasally in the BALB / c mouse model of pneumonic melioidosis and efficacy of 1 vs.2 vs.3 immunizations In our third experiment, we tested intranasal (IN) delivery of our two three- antigen vaccines (expressing Hcp6-Hcp1-Hcp2 and Hcp6-Hcp1-LolC) compared with the LVS ΔcapB parental vector, to determine if this might further improve protective efficacy. We immunized BALB / c mice with 2 x 106CFU by the IN route using a homologous boosting regimen (Weeks 0, 4, and 8). Additionally, to determine the optimal number of doses of vaccine, we immunized other groups of mice with only 1 dose (Week 8) or 2 doses (Weeks 4 and 8) of vaccine instead of the usual 3 doses while maintaining a constant immunization-challenge interval. A negative control group (sham immunized) and a positive control group (immunized with 1 x 106CFU Bp82 by the ID route) were included as in Experiments 1 and 2. At Week 12, mice were challenged by the IN route with a lethal dose of B. pseudomallei 1026b (2,700 CFU, 6x LD50) and monitored for survival for 6 weeks (Fig.12). As in previous experiments, sham immunized mice succumbed rapidly to infection with B. pseudomallei. Bp82, while still protective, had the worst survival of the three experiments (25% vs. 75% and 100%), presumably due to Experiment 3 having the highest challenge dose of the three experiments (2,700 CFU vs. 2,225 and 1,800 CFU). Despite the high challenge dose, the six groups immunized by the IN route with the two three-antigen vaccines (1, 2, or 3 doses) had very high survival (75- 100%). The LVS ΔcapB parental vector was also highly protective. Although 1 dose of LVS ΔcapB was not as effective as 2 or 3 doses (63% vs. 88% and 100%), the differences in the survival curves were not statistically significant. Despite the high efficacy of the three-antigen rLVS ΔcapB vaccines and the LVS ΔcapB parental vector, most groups had lower sterile immunity than observed in the two previous experiments, as did Bp82. This likely reflects the higher challenge dose in Experiment 3. Although all nine IN vaccine groups had better protection than the Bp82 positive control (62.5% to 100% survival vs. 25% survival), individually differences between the survival curves were not statistically significant. Combining groups for the same vaccine that received 1, 2, or 3 doses demonstrates the superiority of IN vaccination with the LVS ΔcapB vaccine platform over ID immunization with Bp82 (Fig.13). Serum IgG response to LVS ΔcapB platform vaccines expressing two or more B. pseudomallei antigens To evaluate the immune response of mice immunized by the ID route, we bled mice from Experiment 2 (Fig.11) one week prior to challenge and analyzed serum IgG titers to LVS and Bp antigens (HI LVS ΔcapB, Hcp6, Hcp1, LolC, and HI Bp82, Fig. 14). All groups immunized with the LVS ΔcapB parental strain or an rLVS ΔcapB vaccine, produced strong antibody titers to HI LVS ΔcapB, whereas sham-immunized and Bp82 immunized mice did not. Mice immunized with rLVS ΔcapB / Bp Hcp6-Hcp1 and rLVS ΔcapB / Bp Hcp6-Hcp2 had anti-Hcp6 antibody titers significantly greater than LVS ΔcapB immunized mice at doses of 4 and 8 x 106CFU; lower doses of vaccine also generated elevated titers, but these differences were not statistically significant. Despite the presence of Hcp6 in the three-antigen and four-antigen vaccines, these vaccines did not produce a response to Hcp6 significantly different from LVS ΔcapB. This may be explained in part by the immunization dose (2 x 106CFU) used for these vaccines. Mice immunized with rLVS ΔcapB / Bp Hcp6-Hcp1 had anti-Hcp1 antibody titers significantly greater than LVS ΔcapB immunized mice at all four doses (1, 2, 4, and 8 x 106CFU) and the response to Hcp1 was more pronounced than the response to Hcp6 for this same vaccine. The three-antigen vaccine, rLVS ΔcapB / Bp Hcp6-Hcp1- LolC, produced a moderate response to Hcp1 when given at 2 x 106CFU, with a greater, and more consistent, response to LolC. The four-antigen vaccine, rLVS ΔcapB / Bp LolC-Hcp6-Hcp1-Hcp2, which had fairly low expression of the fusion protein (Fig.10) produced weak responses to Hcp6 and LolC, which were not significantly different from LVS ΔcapB. Mice immunized with Bp82 produced high titers of IgG to HI Bp82, significantly greater than all other groups. Mice immunized with LVS ΔcapB or rLVS ΔcapB vaccines produced antibody to HI Bp82 that was greater than sham-immunized mice, indicating some cross-reactivity between LVS and Bp. The dose-response study with rLVS ΔcapB / Bp Hcp6-Hcp1 demonstrated that serum IgG titers to Hcp6 and Hcp1 both increased with increasing doses of vaccine, whereas serum IgG titers to HI LVS ΔcapB were already maximal at the lowest dose of vaccine tested (Fig.14B). T cell mediated response to LVS ΔcapB platform vaccines expressing three B. pseudomallei antigens To further assess the response of mice immunized by the ID route, we performed two independent experiments in which mice were immunized three times, as in the protective efficacy studies. Each experiment consisted of four groups (sham- immunized, LVS ΔcapB, LVS ΔcapB / Bp Hcp6-Hcp1-Hcp2, and LVS ΔcapB / Bp Hcp6-Hcp1-LolC). One week after the last immunization, spleen and lung cells were stimulated in vitro with LVS antigens (HI LVS ΔcapB) and Bp antigens (rHcp1, rHcp6, and rLolC, Hcp2 peptide pool, and HI Bp82) and then analyzed by multiparameter flow cytometry. Surprisingly, we did not detect any vaccine specific increased T cell responses to any of the Bp antigens. However, spleen and lung cells from mice immunized with the parental LVS ΔcapB, as well as the two three-antigen rLVS ΔcapB / Bp vaccines, produced IFNγ, TNFα, and IL-2 in response to stimulation with HI LVS ΔcapB (Fig.15). Materials and Methods Bacterial strains and media Escherichia coli was grown on Luria-Bertani or YT agar and Luria-Bertani broth at 37°C. Ampicillin (100 µg / mL) and kanamycin (30 or 50 µg / mL) were included as appropriate. Bp82 was grown on Luria-Bertani (Lennox) agar plates containing 0.6 mM Adenine at 37°C. LVS ΔcapB and rLVS ΔcapB strains were grown on Chocolate Agar plates (CA; Difco GC Medium Base + 1% (w / v) Bovine Hemoglobin + 1% (v / v) IsoVitaleX enrichment) and modified Medium T broth13,14at 37°C [we substituted N- Z-Amine A (enzymatic digest of casein) for the Casamino Acids (acid hydrolysate of casein) component of the formula]. Kanamycin (7.5 µg / mL) was included for rLVS ΔcapB strains maintaining pFNL plasmids. Construction of Electra compatible E. coli-Francisella shuttle plasmids To facilitate cloning and expression analysis of B. pseudomallei antigens (individually or as fusion proteins), we first constructed three Electra compatible DAUGHTER plasmids. Electra cloning11is similar to fragment exchange (FX) cloning15and uses SapI, a type IIS restriction enzyme which produces 3 bp overhangs, together with T4 DNA ligase in a single-tube reaction. To generate Electra compatible DAUGHTER plasmids, we first removed the four SapI restriction sites from the E. coli-Francisella shuttle expression vector pFNL / pbfr-SD-iglA5, using the QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent; Santa Clara, CA). We used PrimerX software16to design four mutagenesis primers and, as all four SapI restriction sites occurred within ORFs, we ensured that the mutations did not affect the protein sequence from the mutated ORF’s translation. Next, we replaced the iglA coding sequence with three different Electra compatible sacB cassettes, to generate three Electra compatible DAUGHTER plasmids, pFNL-bfr-D1 (sacB), pFNL-bfr-D2[N3F-8H] (sacB), and pFNL-bfr- D3[C8H-3F] (sacB). Using Electra cloning, ORFs from Electra compatible MOTHER plasmids can be readily cloned in frame into the three different DAUGHTER vectors, with expression of the ORF driven from the strong Francisella bacterioferritin promoter. Cloning into the D1 vector results in no fusion with the ORF, cloning into the D2 vector results in an N-terminal fusion of a dual 3xFLAG-His8 tag (MDYKDHDGDYKDHDIDYKDDDDKHHHHHHHHGGGS (SEQ ID NO: 5)) with the ORF, and cloning into the D3 vector results in a C-terminal fusion of the ORF with a dual His8-3x FLAG tag (GGSHHHHHHHHDYKDHDGDYKDHDIDYKDDDDK (SEQ ID NO: 6)). ORFs excised from Electra MOTHER plasmids with SapI have a 5’ overhang of ATG on the coding strand (coding for Met) and an ACC overhang on the complementary strand (compatible with the GGT overhang of the vector, coding for Gly). Thus, ORFs cloned into the D1 or D2 DAUGHTER vectors, in most cases, will have an extra Gly residue on the C-terminus of the protein, unless the native sequence ends in a Gly residue. The presence of the sacB cassette in these vectors (which is replaced by the desired ORF in the cloning reaction), permitted us to use undigested DAUGHTER vectors in Electra reactions instead of gel-purified, linearized vector. Selection of transformants on agar plates with kanamycin and 5% sucrose was highly successful in selecting for recombinant DAUGHTER plasmids as transformants that take up unmodified DAUGHTER plasmids are killed in the presence of sucrose due to expression of the sacB gene. We used In Vivo Assembly (IVA)17to construct several additional Electra compatible DAUGHTER plasmids derived from the original three plasmids. We first created a new series of Electra plasmids, in which the unnecessary ampicillin resistance gene is removed, by amplifying the original Electra plasmids using primers pFNL- delAmp-F and pFNL-delAmp-R and transforming the PCR products into E. coli to generate pFNLdA-bfr-D1 (sacB), pFNLdA-bfr-D2[N3F-8H] (sacB), and pFNLdA-bfr- D3[C8H-3F] (sacB). We also used IVA to construct additional derivatives of these new pFNLdA DAUGHTER plasmids with modified SapI overhangs to allow for cloning of ORFs from MOTHER plasmids which have different SapI overhangs (see further details below). Derivatives with a modified SapI overhang at the 5’ end of the cloning site (GCA or TCT, instead of ATG) have an ATG codon immediately preceding the SapI overhang for translation of the cloned ORF. Recombinant plasmids were confirmed to be correct by restriction analysis and DNA sequencing.

[0002] Electra cloning to construct expression plasmids with a single B. pseudomallei antigen ORF We purchased two Electra MOTHER plasmids from DNA2.0 (now ATUM; Newark, CA) containing an ORF encoding the B. pseudomallei Hcp6 antigen (hcp6, BPSL3105), with one version codon optimized for Francisella tularensis subsp. holarctica LVS (http: / / www.kazusa.or.jp / codon / cgi- bin / showcodon.cgi?species=376619&aa=1&style=GCG), and one version codon optimized for Listeria monocytogenes (http: / / www.kazusa.or.jp / codon / cgi- cloned into and MOTHER plasmids, respectively. For construction of additional Electra MOTHER plasmids containing ORFs for other B. pseudomallei antigens (hcp1 [BPSS1498], hcp2 [BPSS0518], and lolC [BPSL2277]), we first generated plasmid pM264-sacB, a derivative of pM264 with a sacB cassette inserted in between the SapI restriction sites. This allowed us to use undigested pM264-sacB, rather than gel-purified, linearized vector, in Electra reactions to clone PCR products and synthetic DNA fragments. Transformation of Electra reactions into E. coli and plating on agar plates with ampicillin and 5% sucrose was highly successful in selecting for recombinant MOTHER plasmids, as transformants that take up residual pM264-sacB are killed in the presence of sucrose due to expression of the sacB gene. For hcp2, hcp6, and lolC, we used GeneDesigner 2.0 software (DNA2.0 / ATUM) to generate coding sequences codon optimized for LVS and L. monocytogenes, purchased Electra compatible synthetic DNA fragments for these codon optimized genes from SGI-DNA (San Diego, CA), and cloned them into the pM264-sacB. In the case of lolC, we only cloned the sequence encoding the periplasmic region (amino acid residues 51 to 273)9. For hcp1, hcp2, and hcp6, we also amplified the native genes from B. pseudomallei K96243 gDNA (gift from Christopher T. French) and cloned the PCR products into pM264-sacB. We were unsuccessful amplifying the native B. pseudomallei lolC. For a typical cloning reaction, we would mix a MOTHER plasmid containing a B. pseudomallei antigen ORF, a pFNL DAUGHTER plasmid, and Electra Cloning reagents (DNA2.0 / ATUM) containing buffer, SapI, and T4 DNA ligase in a final volume of 5 µL and incubate at room temperature for 1 h. We would then use 1 µL of the cloning reaction to transform 10 µL of competent E. coli and select for recombinant clones on YT plates containing kanamycin and 5% sucrose. Recombinant plasmids were confirmed to be correct by restriction analysis and DNA sequencing. Electra cloning to construct expression plasmids with multiple B. pseudomallei antigens joined as a fusion protein For our first set of fusion constructs, we joined hcp6 or lolC to either hcp1 or hcp2 with a sequence coding for a flexible peptide linker (GSSG-GSSG (SEQ ID NO: 4)) in between the two ORFs. To accomplish this, we first amplified hcp6 and lolC by PCR, using a primer for the pM264 vector (pM264-FP) and a primer specific for the gene (hcp6_coLm-L-R, lolC_coLm-L-R, or lolC_coLVS-L-R) which included the sequence for the peptide linker in the primer tail. The PCR products were reamplified in a second PCR using primers pM264-FP and Linker-3gca-R, which allowed the resulting PCR products to be cloned by Gibson Assembly18into the SapI cloning sites of the pM264 MOTHER vector, replacing the 3’GGT SapI overhang with a GCA overhang. We then amplified hcp1 and hcp2 by PCR, using a primer specific for the gene (hcp1_nat-5gca-F, hcp1_coLm-5gca-F, hcp1_coLVS-5gca-F, hcp2_nat-5gca-F, hcp2_coLm-5gca-F, or hcp2_coLVS-5gca-F) and a primer specific for the pM264 vector (pM264-RP). The gene specific primer included 30-33 nt of homology to the pM264 vector allowing the PCR product to be cloned by Gibson Assembly into the pM264 SapI cloning sites, replacing the 5’ATG SapI overhang with a GCA overhang. Fusions of two ORFs (hcp6 plus hcp1 or hcp2; and lolC plus hcp1 or hcp2) were done by combining pM264 MOTHER plasmids with compatible GCA overhangs together with a pFNL DAUGHTER plasmid using Electra cloning to generate the desired expression plasmid. For example: pFNL-bfr-D3[C8H-3F] (sacB) + pM264(3'gca)-Bp hcp6 (coLm)-(GSSG (SEQ ID NO: 1))2 + pM264(5'gca)-Bp (coLVS) ^ To facilitate construction of subsequent fusion proteins, we made additional modifications to the Electra cloning system to simplify the in frame joining of two or three ORFs. Using Gibson Assembly or IVA, we constructed five modified pM264- sacB MOTHER plasmids which vary in the SapI overhangs, with the 5’ ATG overhang on the coding strand (coding for Met) and / or the GGT overhang for the 3’ end of the ORF (coding for Gly) being replaced by GCA (Ala) or TCT (Ser). We then cloned ORFs into the modified pM264-sacB plasmids. Finally, we performed Electra reactions combining two or three modified MOTHER plasmids (each with an ORF) and a DAUGHTER plasmid to construct expression plasmids with fusion protein genes. Fusions of two ORFs were done by combining pM264 MOTHER plasmids with compatible GCA or TCT overhangs (see GCA example above). Likewise, fusions of three ORFs were done by combining three MOTHER plasmids with compatible GCA and TCT overhangs together with a pFNL / pFNLdA DAUGHTER. For example: pFNLdA-bfr-D1 (sacB) + pM264(3'gca)-ORF1 + pM264(5'gca, 3'tct)-ORF2 + pM264(5'tct)-ORF3 ^ To enable joining two ORFs with a peptide linker, we constructed three separate linkers, a flexible linker (GSAGSAAGSGEF (SEQ ID NO: 3))19and two rigid linkers [A(EAAAK (SEQ ID NO: 2))3A and (AP)10]20,21, by annealing complementary oligonucleotides with the appropriate 3 bp overhangs for cloning into pM264(5'gca, 3'tct)-sacB. We also constructed a series of modified pFNLdA plasmids using IVA that allows for direct cloning of inserts from modified pM264-sacB MOTHER plasmids (i.e. the modified insert can be cloned by itself, without fusion to one or two other inserts), such that individual components of the desired fusion protein can be tested. For example: pFNLdA-bfr-D31(5'gca)[C8H-3F]-sacB + pM264(5'gca)-ORF2 ^ pFNLdA-bfr-ORF2-C8H-3F For expression of four-antigen fusion proteins, we first attempted to combine two of the two-antigen constructs that were expressed well in LVS ΔcapB, Hcp6- (GSSG (SEQ ID NO: 1))2-Hcp1 and LolC-(GSSG (SEQ ID NO: 1))2-Hcp2. To achieve this, we amplified the Hcp6-(GSSG (SEQ ID NO: 1))2-Hcp1 ORF from the pFNL-bfr-Hcp6 (coLm)-(GSSG (SEQ ID NO: 1))2-Hcp1 (coLVS)-C8H-3F expression plasmid using primers hcp6_coLm-(ATG)-F and hcp1_coLVS-(GCA)-R and cloned the PCR product into pM264(3’gca)-sacB, generating pM264(3'gca)-Hcp6 (coLm)- (GSSG (SEQ ID NO: 1))2-Hcp1 (coLVS). Likewise, we amplified the Hcp6-(GSSG (SEQ ID NO: 1))2-Hcp1 ORF using primers hcp6_coLm-(ATG)-F and hcp1_coLVS- (TCT)-R and cloned the PCR product into pM264(3’tct)-sacB, generating pM264(3'tct)-Hcp6 (coLm)-(GSSG (SEQ ID NO: 1))2-Hcp1 (coLVS). The final required MOTHER plasmid, pM264(5'tct)-LolC (coLVS)-(GSSG (SEQ ID NO: 1))2- Hcp2 (coLVS) was constructed by cloning a PCR product of the LolC-(GSSG (SEQ ID NO: 1))2-Hcp2 ORF [using primers lolC_coLVS-(TCT)-F and hcp2_coLVS- (GGT)-R] into pM264(5’tct)-sacB. We then performed Electra reactions combining two MOTHER plasmids (each with a two-antigen ORF) and a DAUGHTER plasmid, with or without one of the three MOTHER plasmids containing a peptide linker ORF, to construct expression plasmids with four-antigen fusion protein genes as detailed above. As these constructs were very poorly expressed by LVS ΔcapB, we next turned to expressing three-antigen fusion proteins. For expression of three-antigen fusion proteins, we took a systematic approach, linking LolC to the either the N-terminus or C-terminus of the most highly expressed two-antigen ORFs [Hcp6-(GSSG (SEQ ID NO: 1))2-Hcp1 and Hcp6-(GSSG (SEQ ID NO: 1))2-Hcp2]. For each combination of antigens, the C-terminal portion was linked to the N-terminal portion by one of three peptide linkers or directly (only a single alanine inserted between the two), for a total of 16 three-antigen fusion protein variations, using the same cloning approach as detailed above for the four-antigen constructs. Construction and characterization of rLVS ΔcapB / Bp strains Expression plasmids were introduced into LVS ΔcapB by electroporation or chemical transformation. To prepare electrocompetent cells, LVS ΔcapB was grown overnight in Medium T at 37°C with shaking, subcultured in Medium T to an OD600 of 0.1 or 0.2, and grown for an additional 4 or 5 h, reaching an OD600 of 0.7 to 1.1. The bacteria were harvested by centrifugation at 4°C, washed two or three times with ice-cold sucrose-glycerol wash buffer [SGWB; 10% (v / v) glycerol, 500 mM sucrose; pH ~ 7]22, resuspended in a final volume of SGWB equivalent to 1 / 50thto 1 / 10thof the original culture volume, and aliquots stored at -80°C until needed. For electroporation, 1-2 µL of plasmid DNA was mixed with 30-50 µL of electrocompetent cells on ice and transferred to a pre-chilled electroporation cuvette (0.1 cm, BioRad). After the pulse was applied (1.8 kv, 25 µF, 200 Ω), electroporated bacteria were resuspended in 0.5 mL Medium T, incubated at 37°C for 3-5 h, and then plated on CA containing 7.5 µg / mL kanamycin. Typically, individual clones were picked for analysis after 3 to 5 days of incubation at 37°C. To prepare chemically competent cells, we grew LVS ΔcapB overnight in Medium T at 37°C with shaking, subcultured in Medium T to an OD600 of 0.1, and cultured for an additional 4 h, reaching an OD600 of 0.5. Competent cells were prepared using the method Inoue et al. described for E. coli23, except that we grew LVS ΔcapB at 37°C instead of a reduced temperature. Aliquots of competent cells were stored at -80°C until needed. For transformation of LVS ΔcapB, we modified a typical protocol we use for small scale E. coli transformations. We mixed 1 µL of plasmid DNA with 20 µL of competent cells in a PCR tube on ice; incubated on ice for 30 minutes; applied a 42°C heat shock for 45 seconds in a thermal cycler machine and cooled to 4°C; incubated on ice for 2 minutes; added recovery medium (130 µL Medium T) and mixed gently; incubated at 37°C for 3 h (no shaking); and plated the entire transformation on CA containing 7.5 µg / mL kanamycin. Typically, individual clones were picked for analysis after 3 to 5 days of incubation at 37°C. Individual clones were picked and suspended into 2 mL Medium T broth containing 7.5 µg / mL kanamycin and grown overnight at 37°C with shaking. To analyze recombinant protein expression, we prepared cell lysates from 1 mL of overnight culture by pelleting the cells by centrifugation; freezing the cell pellet at - 30°C for at least 1 h; resuspending the thawed cell pellet in 0.1 mL of B-PER (Complete Bacterial Protein Extraction Reagent, Thermo Scientific) containing 5 mM EDTA and a protease inhibitor cocktail (HALT Protease Inhibitor Single Use Cocktail, Thermo Scientific); and incubating at room temperature for 10-20 minutes, at which point the bacterial suspensions had cleared. After centrifuging to clear any remaining cellular debris, we mixed 20 µL of supernatant with 20 µL of 2x SDS-PAGE sample buffer and boiled for 5 minutes. Boiled lysates were analyzed on Any kD™ Mini-PROTEAN® TGX Stain-Free™ Protein Gels (BioRad) and total protein visualized by UV light and / or by Coomassie Blue staining. Recombinant proteins with a 3x FLAG tag were detected by Western blotting using HRP conjugated anti-FLAG monoclonal antibody (100,000-fold dilution, Sigma) and Clarity Western ECL Substrate (BioRad). Hcp1 was detected by Western blotting using rat anti-Hcp1 antibody (5,000-fold dilution, generously provided by Christopher T. French) and goat anti-rat HRP (10,000-fold dilution, Invitrogen). Preparation of Vaccine Stocks Bp82 was grown overnight at 37°C on Luria-Bertani (Lennox) agar plates containing 0.6 mM adenine. Bacterial colonies were scraped from the agar plates into PBS containing 15% (v / v) glycerol and clumps dispersed by vortexing and pipetting. Large aggregates were allowed to settle for 10 minutes and then the upper portion of the suspension (avoiding any pellet) was aliquoted and stored at -80°C until needed. LVS ΔcapB and rLVS ΔcapB vaccines expressing Bp antigens were grown from a frozen stock overnight at 37°C with shaking in Medium T broth (+ 7.5 µg / mL kanamycin for strains with a pFNL plasmid), subcultured in Medium T to an OD600 of 0.001 to 0.003, and grown for 18-20 h, reaching an OD600 of 0.6 to 3.8 (equivalent to 8.4 to 10.4 generations of growth, doubling time: 1.7 to 2.1 h, median = 1.9 h, N = 29). The bacteria were harvested by centrifugation at 4°C, washed twice with PBS, resuspended in a final volume of PBS-20% (v / v) glycerol equivalent to 1 / 10thof the original culture volume, and aliquots stored at -80°C until needed. We checked the vaccine stocks for stability of the plasmid and antigen expression as follows. Dilutions of a thawed vaccine stock were first plated on CA to determine the post-freeze titer. Twenty individual clones were then patched onto CA plates with and without kanamycin to determine stability of the pFNL plasmid. To validate that the correct plasmids were present in the rLVS ΔcapB vaccines, plasmid DNA was isolated from thawed vaccine stocks using the Zyppy Plasmid Miniprep Kit (Zymo Research; Irvine, CA) and confirmed to be correct by restriction analysis, as well as by DNA sequencing of the expression cassette and / or PCR of the expression cassette followed by DNA sequencing of the PCR product. Finally, vaccine stocks were used to inoculate Medium T broth containing 7.5 µg / mL kanamycin, grown overnight, and lysates prepared to check recombinant protein expression (as described above).

[0003] Mice Protection Studies at CSU: Six to 8-week-old female BALB / c mice were purchased from Charles River or Envigo, held 4 per cage, and provided food and water ad libitum. Mice were acclimated for one week prior to initiating the experiments. Immunology Studies at UCLA: Six to 8-week-old BALB / c mice (half male and half female) were purchased from The Jackson Laboratory, held 4 per cage, and provided food and water ad libitum. Mice were acclimated for one week prior to initiating the experiments. Animal research was conducted within the ethical guidelines outlined under the U.S. Public Health Service policy for the care and use of laboratory animals following protocols approved by the animal research committees of UCLA and CSU. Protective Efficacy and Immunology Studies For rLVS ΔcapB / Bp vaccines, we immunized mice by either the ID route at the base of the tail (1, 2, 4, or 8 x 106CFU) or the IN route (2 x 106CFU) using a homologous boosting regimen administered at Weeks 0, 4, and 8. In the case of IN vaccination, additional groups of mice received only 1 dose (Week 8) or 2 doses (Weeks 4 and 8) of vaccine instead of the usual 3 doses (constant immunization-challenge interval). Mice immunized by the IN route were anesthetized with ketamine / xylazine and administered 20 µL of vaccine divided between both nostrils. Each protective efficacy experiment included three control groups: a negative control group (sham- immunized), a positive control group (immunized with 1 x 106CFU Bp82 by the ID route), and the parental vector (LVS ΔcapB). In protective efficacy studies, blood was collected one week prior to challenge to prepare serum, which was stored at -80°C until analysis for antibody to Bp and LVS antigens. Four, six, or twelve weeks after the last vaccine dose, mice were challenged by the IN route with a lethal dose of B. pseudomallei 1026b (1,430 to 2,680 CFU) and monitored for survival for 6 weeks. Mice were euthanized when they reached humane endpoints. At the end of the 6-week period for monitoring survival, the surviving mice were euthanized, and the lung, liver, and spleen were examined for abscesses and then cultured for B. pseudomallei. Mice with no abscesses and no detectable B. pseudomallei in the three organs were recorded as having sterile immunity. For immunology studies, one week after the last vaccine dose, mice were euthanized and the spleen and lungs were removed to assess immune responses. Expression and purification of recombinant Bp antigens We used the Expresso Rhamnose SUMO Cloning and Expression System (Lucigen) to obtain purified recombinant Bp antigens for immunology assays. ORFs for hcp1, hcp2, hcp6, and lolC (codon optimized for LVS) were amplified from pM264 / pM268 plasmids and cloned into the pRham N-His SUMO Kan expression vector. The coding regions were amplified (starting with the second codon) with tails recommended by Lucigen for cloning into their linearized vector (tails have 18 nt of homology to the vector to allow for IVA) such that the B. pseudomallei genes will be fused downstream of a His6-SUMO tag under the control of the L-rhamnose-inducible rhaPBAD promoter. The second codon for both hcp1 and hcp6 codes for leucine, which is inefficiently cleaved by SUMO protease. Therefore, we inserted codons for Gly-Ser in front of the Leu codon for these two genes to facilitate cleavage of the SUMO tag. Purified PCR products and linearized pRham N-His SUMO Kan expression vector were mixed together, transformed into competent E. coli (E. cloni 10G, Lucigen), and clones selected on YT containing 30 µg / mL kanamycin. We confirmed that the recombinant plasmids were correct by restriction analysis and DNA sequencing and confirmed that we could obtain high level, inducible expression of the N-His-SUMO tagged recombinant proteins before proceeding with purification. To induce fusion protein expression, we used an auto-induction protocol, inoculating 10 mL of an overnight culture into 1 L LB with 0.05% glucose, 0.05% rhamnose, and 30 µg / mL kanamycin in a 2.8 L Fernbach flask, and incubating at 28°C for 24 h with shaking (reaching an OD600 of ~5). The bacteria were harvested by centrifugation at 4°C, washed first with 50 mL ice-cold 2x TE pH 8.0 followed by 25 mL ice-cold ddH2O, and the washed cell pellets stored at -80°C until needed. Recombinant proteins were extracted by resuspending thawed cell pellets in 50 mL B- PER Complete Bacterial Protein Extraction Reagent (Thermo Scientific) and incubating at room temperature for 60 minutes. The extract was then clarified by centrifugation at 10,000 g for 60 minutes at 4°C and the supernatant filtered (0.2 µm). Filtered extract was mixed with 2 mL of HisPur Cobalt Superflow Agarose resin (Thermo Scientific) and rocked gently at 4°C overnight for binding of the His-SUMO tagged recombinant protein to the resin. After extensive washing, recombinant protein was eluted with 150 mM imidazole in 50 mM NaH2PO4-300 mM NaCl buffer (pH 7.4). We performed concentration and buffer exchange of the 150 mM imidazole elution fractions using an Amicon Ultra-15 centrifugal filter device (10,000 MWCO) (Millipore) into 20 mM Tris-150 mM NaCl-10% (v / v) glycerol pH 8.0, added DTT to a final concentration of 2 mM, and digested with SUMO Express Protease (Lucigen) overnight at 4°C at a concentration of ~1 U per 2 mg of His-SUMO tagged recombinant protein. The digests (<0.5 mL) were diluted to 20 mL PBS and passed through a HisPur Cobalt column for subtractive chromatography to remove the cleaved His6-SUMO fragment, the SUMO Express Protease (His tagged), and E. coli protein contaminants that bound to the column during the original binding step. The flow through was concentrated and buffer exchanged into PBS using an Amicon Ultra-15 centrifugal filter device (10,000 MWCO), and then filtered to sterilize with a 0.2 µm Spin-X column (Corning). We measured protein concentration of the purified proteins using the protein’s extinction coefficient at 280 nm (calculated using the ProtParam tool, https: / / web.expasy.org / protparam / )24and assessed purity by SDS-PAGE. Aliquots Other Antigens A peptide pool for Hcp2 (41 individual 15 amino acid peptides with an 11 amino acid overlap between adjacent peptides, >70% purity) was purchased from JPT Peptide Technologies (Berlin, Germany). To prepare heat-inactivated Bp82 and LVS ΔcapB, bacteria were scraped from agar plates, washed with PBS, incubated at 80°C for 30 minutes or 1 h (respectively) to kill the bacteria, and stored in aliquots at -80°C. Isolation of splenocytes and lung cells One week after the last vaccine dose, mice were euthanized and the spleen and lungs removed to assess immune responses. Single-cell suspensions of splenocytes were prepared by gently pressing the cells out of the spleen sac; lysing red blood cells with PharmLyse (BD Pharmingen); washing the cells; and filtering through a 70-μm nylon cell strainer (Falcon). Single-cell suspensions of lung cells were prepared by cutting the lung into small pieces with a scalpel; incubating 37°C for 1 h with shaking in 10 mL of digestion solution [300 U / mL Collagenase type II (Worthington) and 0.15 mg / mL DNase I (Worthington) in PBS]; filtering through a 40-μm nylon cell strainer (Falcon); lysing red blood cells with PharmLyse (BD Pharmingen); and washing the cells. Advanced RPMI-1640 (Invitrogen) supplemented with 2% heat-inactivated fetal bovine serum, 2 mM Glutamine dipeptide (glutaGRO Supplement, Corning), 10 mM HEPES buffer, 50 µM β-mercaptoethanol, and penicillin (100 IU / mL)-streptomycin (100 μg / mL) was used as the medium. Flow cytometry analysis Single-cell suspensions of splenocytes (5 x 105viable cells per well) and lung cells (2- 3 x 105viable cells per well) were stimulated with individual antigens or left without antigen for 6 h in U-bottom, 96-well tissue culture plates in 200 µL medium at 37°C in a humidified incubator (95% air, 5% CO2). For antigen stimulation, rHcp1, rHcp6, and rLolC were each used at a final concentration of 10 μg / mL and the Hcp2 peptide pool was used at a final concentration of 1 μg / mL (each peptide, 41 µg / mL for the total pool). Heat-inactivated Bp82 and LVS ΔcapB were used at 5 x 106CFU per well (CFU assayed prior to heat inactivation). Anti-CD28 antibody [Clone 37.51] was included in all wells as a co-stimulant at 2 μg / mL. The protein transport inhibitor brefeldin A (5 µg / mL final concentration) was added to all wells for the final 4 h of incubation. At the end of the 6 h incubation, we performed viability staining using Fixable Viability Dye eFluor 780 (eBioscience); fixed and permeabilized the cells using the Cyto-Fast™ Fix / Perm Buffer Set (BioLegend); blocked Fc Receptors with anti-mouse CD16 / 32 antibody (TruStain FcX™ PLUS, BioLegend); and stained intracellular and surface antigens using fluorescent antibodies for CD3, CD4. CD8, IFNγ, TNFα, IL-2, IL-17A, Perforin, and Granzyme B. A minimum of 16,000 live CD3+ T-cells per spleen sample (Median ~ 29,000) and a minimum of 2,500 live CD3+ T-cells per lung sample (Median ~ 5,000 in the first experiment and ~9,000 in the second experiment) were acquired with a BD LSRII flow cytometer equipped with a high throughput sampler and five lasers (355, 405, 488, 561, and 640 nm). The frequencies of live CD3+ CD4+ and CD3+ CD8+ T-cells expressing IFNγ, IL-2, TNFα, IL-17, Granzyme B, and Perforin were determined using FlowJo software (FlowJo; Ashland, OR). Background numbers of cells producing cytokines without antigen stimulation were subtracted. Serum antibody Frozen serum (obtained from vaccinated mice 1 week prior to challenge) was thawed at 4°C and assayed for antibody to Bp and LVS ΔcapB antigens by ELISA. High Binding EIA plates (Corning) were coated with 100 µL of antigen per well in 0.1 M sodium carbonate buffer pH 9.6 (Protein antigens: 1 µg / mL; Heat-inactivated LVS ΔcapB or Bp82: 5 x 107CFU / mL [CFU prior to heat-inactivation]) for 4 h at room temperature and then blocked with 3% (w / v) BSA in PBS. The plates were then washed with PBS-0.05% TWEEN 20 before adding 100 µL per well of immune serum diluted in PBS-1% (w / v) BSA (we typically prepared a 200-fold dilution, followed by serial 4- fold dilutions out to 204,800-fold dilution of the original serum). Plates were incubated overnight at 4°C with immune sera, washed with PBS-0.05% TWEEN 20, and then the AP conjugated secondary antibody was added [100 µL per well at 1:5,000 dilution in PBS-1% (w / v) BSA, Goat anti-mouse IgG-AP (Sigma)]. After 90 minutes at room temperature, the plates were washed with TBS-0.05% TWEEN-80, developed with the Alkaline Phosphatase Substrate Kit (BioRad) according to the Manufacturer’s instructions, and absorbance at 415 and 750 nm measured with a BioRad iMark microplate reader. We plotted Log (A415 – A750) vs. Log Serum Dilution to visualize the data. We calculated the end point titer as the dilution where the measurement intersects the cutoff, using interpolation between data points, and with the cutoff equal to the Sham Mean + 3 SD, but at least 0.1. In a few cases, sham mice clearly reacted to an antigen, so these mice were excluded for purposes of calculating the end point titer cutoff. However, they were not excluded from the final titer results. Statistics Survival curves were compared using the Log-rank (Mantel-Cox) test (Prism 9.3.1) with Holm-Bonferroni correction of p-values for multiple comparisons. Serum IgG titers were compared to a control group using ordinary One-Way ANOVA with Dunnett's multiple comparisons test (Prism 9.3.1). EXAMPLE 2: STUDIES OF THE EFFICACY OF THE MELIOIDOSIS VACCINES AGAINST GLANDERS Burkholderia pseudomallei, the agent of melioidosis, and Burkholderia mallei, the agent of glanders are closely related pathogens and most strains share conserved immunoprotective antigens, including antigens overexpressed by the melioidosis vaccine - Hcp1, Hcp2, and LolC. To evaluate the efficacy of rLVS ΔcapB vaccines expressing three Bp antigens against virulent B. mallei IN challenge (vaccines previously shown to be protective against virulent B. pseudomallei IN challenge), we immunized BALB / c mice by either the ID route or the IN route using a homologous boosting regimen (Weeks 0, 4, and 8). We administered the three-antigen vaccines and the LVS ΔcapB parental vector at 2 x 106CFU (Fig. 1). A negative control group (sham-immunized) and a positive control group (immunized with Bp82) were included. At Week 14, mice were challenged by the IN route with a lethal dose of B. mallei China 7 (190 CFU, 12.7x LD50) and monitored for survival for 6 weeks. Sham-immunized mice progressively succumbed to infection with B. mallei, with 100% mortality by 6 weeks. The positive control group immunized with Bp82 had 67% survival, significantly different from the sham- immunized group (P=0.004, Holm-Šídák method used to correct for multiple comparisons, see Table 1). The six groups immunized with LVS ΔcapB or the rLVS ΔcapB vaccines had survival at 6 weeks that ranged from 25% to 58%. However, statistical analysis of the Kaplan-Meier survival curves indicated that only one group (Group D, LVS ΔcapB, IN) by itself was significantly different from the sham- immunized group (P=0.027). We then combined the groups for further statistical analysis to determine if additional insights could be obtained. Combining all six groups immunized with LVS ΔcapB or the rLVS ΔcapB vaccines administered either IN or ID yielded protection that was significantly different from the sham-immunized group (P=0.004). Combining just the three groups immunized by the IN route also yielded protection significantly different from the sham-immunized group (P=0.004), while combining just the three groups immunized by the ID route did not yield statistically significant protection compared with the sham-immunized group (P=0.11). Finally, we combined the two groups that were immunized with the LVS ΔcapB parental vector and the four groups that were immunized with one of the rLVS ΔcapB vaccines. There was a statistically significant difference in protection for the combined rLVS ΔcapB vaccines (P=0.015) vs. the Sham group and the difference in protection almost reached the significance cutoff for the combined LVS ΔcapB parental vector groups (P=0.052) vs. the Sham group. Our results show that both the LVS ΔcapB vector platform and the rLVS ΔcapB vaccines expressing three Bp antigens are protective against B. mallei challenge. Protection by the vector alone appears to be comparable to protection by the vector expressing Bp antigens. Protection by IN delivery of the vaccines appears to be greater than protection by ID delivery.

[0004] Table 1. Statistical Analysis of Kaplan-Meier Survival Curves Adjusted P Value Burkholderia pseudomallei polypeptide sequences Hcp-1 type VI secretion system [Burkholderia pseudomallei] GenBank: CRY35672.1 >CRY35672.1 type VI secretion system [Burkholderia pseudomallei] MLHMHLKFGSPAIKGESADKDHEGWIELKSWDHSIVQPRSATASTAGGHTATRCEHGDMVFTKEIDSSS PLLYQHASGGTTFDEVTIDFLRADGEGQRVKYLEIKLKYVIISSIAPSVHTEGLPVETFSLKYAAVQWKQTQQKIGGNQGGNTQGAWSLTKNDKTYAV(SEQ ID NO: 7)Hcp-2 (T6SS) type VI secretion system [Burkholderia pseudomallei] GenBank: CPN31289.1 >CPN31289.1 type VI secretion system [Burkholderia pseudomallei] MSHDIFLKINGIDGEAEDATHKGEIEVLSWSWNVSQQSNMHLGSGGGAGKATIDDLQFEHYIDRASPNL VQYCLLGKHIDEARLVVRKAGGSPLEYIKLTMSDVLVTQVSPAGVAQDESRPRELVRLSFSRLKQEYVV QNPQGGSGGAITATFDIKKNAA (SEQ ID NO: 8) Hcp-3 (T6SS) type VI secretion system [Burkholderia pseudomallei] GenBank: CRY29196.1 >CRY29196.1 type VI secretion system [Burkholderia pseudomallei] MAQDIFLKIDGINGESLDDSHKDEIEVLNWNWEIQQESTMHTGSGGGAGKASVKDLTFEHAIDRASPNL MKYALTGKHVDQAVLVMRKAGGNPLEYLKLTMSDVIITRVRPSGSRDDTERSRETVSLSFAKVKQEYVV QNAQGGSGGAVTTSFDIKGNKEA (SEQ ID NO: 9) Hcp-4 (T6SS) type VI secretion system [Burkholderia pseudomallei] GenBank: CRY33495.1 >CRY33495.1 type VI secretion system [Burkholderia pseudomallei] MANALVDYFLQIDGVEGESTDQQYPGLIQIQSWQWAEENSGRWGFGSGGGAGKVEMKDFEFRMVSNKAS PKLFLMCATGEHIQNAKLICRKSGKGQQEFLTISFASGLVSSFRTLGNMPISQLGHASGEVDGVLPTDQ IRINFAQIEFEYREQRNDGTMGAVIKAGYDLKQNAPI (SEQ ID NO: 10) Hcp-6 (T6SS) type VI secretion system [Burkholderia pseudomallei] GenBank: CRY19699.1 >CRY19699.1 type VI secretion system [Burkholderia pseudomallei] MGVAMFMKVDGVTGESADAQHKGWTDIQSFSWGASQPGAMASGSGGNAGKASFNDLVVAAYMDKGATAI IKNCASGKHLPTVEISACKTGGSQIEFMRVTLQEVLVTSAQIAGVDPGDAADRLMMQYGFQAAKVKKQYWQQNDNGGKGAEVSVGWNIKENTEM(SEQ ID NO: 11)LolC (ATP binding cassette system) lipoprotein-releasing system transmembrane subunit LolC [Burkholderia pseudomallei] NCBI Reference Sequence: WP_050865936.1 >WP_050865936.1 lipoprotein-releasing system transmembrane subunit LolC [Burkholderia pseudomallei] MKLPYEWQIGWRYTRAGKRATGNGFISFIALVSMLGIALGVAALIVVLSVMNGFQKEVRDRMLSVLAHV EIFSPTGSMPDWQLTAKEARLNRSVIGAAPYVDAQALLTRQDAVSGVMLRGVEPSLEPQVSDIGKDMKA GALTALAPGQFGIVLGNALAGNLGVGVGDKVTLVAPEGTITPAGMMPRLKQFTVVGIFESGHYEYDSTL AMIDIQDAQALFRLPAPTGVRLRLTDMQKAPQVARELAHTLSGDLYIRDWTQQNKTWFSAVQIEKRMMF IILTLIIAVAAFNLVSSLVMTVTNKQADIAILRTLGAQPGSIMKIFVVQGVTIGFVGTATGVALGCLIA WSIPWLIPMIEHAFGVQFLPPSVYFISELPSELVAGDVIKIGVIAFALSALATLYPSWRGAKVRPAEAL RYE (SEQ ID NO: 12) TypA GTP-binding protein TypA [Burkholderia pseudomallei] GenBank: CRY12842.1 >CRY12842.1 GTP-binding protein TypA [Burkholderia pseudomallei] MTRALRNIAIIAHVDHGKTTLVDQLLRQSGTFRENQQVAERVMDSNDIEKERGITILAKNCAVEYEGTH INIVDTPGHADFGGEVERVLSMVDSVLLLVDAVEGPMPQTRFVTKKALALGLKPIVVINKIDRPGARID WVINQTFDLFDKLGATEEQLDFPIVYASGLNGYASLDPAARDGDMRPLFEAILQHVPVRPADPDAPLQL QITSLDYSTYVGRIGVGRITRGRIKPGQPVVMRFGPEGDVLNRKINQVLSFQGLERVQVDSAEAGDIVL INGIEDVGIGATICAVEAPEALPMITVDEPTLTMNFLVNSSPLAGREGKFVTSRQIRDRLMKELNHNVA LRVKDTGDETVFEVSGRGELHLTILVENMRREGYELAVSRPRVVMQEIDGVKHEPYELLTVDLEDEHQG GVMEELGRRKGEMLDMVSDGRGRTRLEYRIPARGLIGFQSEFLTLTRGTGLMSHIFDSYAPVKEGSVGE RRNGVLISQDDGAAVAYALWKLQDRGRMFVKPGDALYEGMIIGIHSRDNDLVVNPIKGKQLTNVRASGT DEAVRLVPPIQMSLEYAVEFIDDDELVEVTPQSIRLRKRHLKEHERRRASREAEAG(SEQ ID NO:13)BipB BipB [Burkholderia pseudomallei] GenBank: ABO26356.1 >ABO26356.1 BipB [Burkholderia pseudomallei] MSSGVQGGPAAHANAYQTHPLRDAASALGTLSPQAYVDVVSAAQRNFLERMSQLASEQCDAQPAAHDAR LDDKPALRAPQERDAPPLGASDTGSRASGAAKLTELLGVLMSVISASSLDELKQRSDIWNQMSKAAQDN LSRLSDAFQRATDEAKAAADAAEQAAAAAKQAGADAKAADAAVDAAQKRYDDAVKQGLPDDRLQSLKAA LEQARQQAGDAHGRADALQADATKKLDAASALATQARACEQQVDDAVNQATQQYGASASLRTPQSPRLS GAAELTAVLGKLQELISSGNVKELESKQKLFTEMQAKREAELQKKSDEYQAQVKKAEEMQKTMGCIGKI VGWVITAVSFAAAAFTGGASLALAAVGLALAVGDEISRATTGVSFMDKLMQPVMDAILKPLMEMISSLI TKALVACGVDQQKAELAGAILGAVVTGVALVAAAFVGASAVKAVASKVIDAMAGQLTKLMDSAIGKMLV QLIEKFSEKSGLQALGSRTATAMTRMRRAIGVEAKEDGMLLANRFEKAGTVMNVGNQVSQAAGGIVVGV ERAKAMGLLADVKEAMYDIKLLGDLLKQAVDAFAEHNRVLAQLMQQMSDAGEMQTSTGKLILRNARAV (SEQ ID NO: 14) BipC BipC [Burkholderia pseudomallei] GenBank: ABO26357.1 >ABO26357.1 BipC [Burkholderia pseudomallei] MSIGVQSSGINISHAELSRLVDAGKSEQGDKAVRDDGRALARADAALAAVVGERVAARRDAVAGSGAQR VELARPKPDAQTRATDRRTVSGLEREHKRLAASQTPRVTGMHDALVQRHVSLDGAKAAHGEGVKRAAGD APRAAADAPQRFAFADDKAFDAMLALGAAMQKNVQSDLAMQGKLTMLAHDAMMSAAAQDRSIGAAQMTA AIAGGALQATTSLGGAMQQMKSLSTKSMSIEKELKPQAELKQFHAEQALELRGINKPVLSNDEVSHVKI KRDTGETVRHEIDHGGERMSDEHASVLAQEAPARQHRIDMHGMRHEENLVKASRQQMKGDLLQSGGQIG KNQIDGASAQQQGADRAEQKEDENAQQTAMAAASTRDEAAHRSREAAQKAIDAAKSQVANDNAVAAQVAGNLRT(SEQ ID NO: 15)BipD BipD [Burkholderia pseudomallei] GenBank: ABL67521.1 >ABL67521.1 BipD [Burkholderia pseudomallei] MNMHVDMGRALTVRDWPALEALAKTMPADAGAREMTDDDLRAAGVDRRVPEQKLGAAIDEFASLRLPDR IDGRFVDGRRANLTVFDDARVAVRGHARAQRNLLERLETELLGGTLDTAGDEGGIQPDPILQGLVDVIG QGKSDIDAYATIVEGLTKYFQSVADVMSKLQDYISAKDDKNMKIDGGKIKALIQQVIDHLPTMQLPKGA DIARWRKELGDAVSISDSGVVTINPDKLIKMRDSLPPDGTVWDTARYQAWNTAFSGQKDNIQNDVQTLVEKYSHQNSNFDNLVKMLSGAISTLTDAAKSYLQI(SEQ ID NO: 16)Omp3 OmpA family protein [Burkholderia pseudomallei] GenBank: ACN64870.1 >ACN64870.1 OmpA family protein [Burkholderia pseudomallei] MNKLSKLAFIAATAVMAASASAQSVPASRQAVNDNWVNGTGEWVWMNGTNELCWRDAFWTPATANAKCD GALVAQAPAPAPVAPVAPAITSQKITYQADTLFDFDKAVLKPAGKQKLDELAAKIQGMNVEVVVATGYT DRIGSDKYNDRLSLRRAQAVKSYLVSKGVPANKVYTEGKGKRNPVTGNTCKQKNRKQLIACLAPDRRVE VEVVGTQEVQKTTVPAQ (SEQ ID NO: 17) Omp7 OmpA family lipoprotein [Burkholderia pseudomallei] GenBank: ACN64871.1 >ACN64871.1 OmpA family lipoprotein [Burkholderia pseudomallei] MTTRRVTMMSKKLRLAFAMLMIGALAACKSGVKLDEHANQGDAVSTQPNPENVAQVTVDPLNDPNSPLA KRSVYFDFDSYSVQDQYQALLQQHAQYLKSHPQRHILIQGNTDERGTSEYNLALGQKRAEAVRRALSLLGVGDAQMEAVSLGKEKPVALGHDEASWAQNRRADLVYQQ(SEQ ID NO: 18)Omp85 putative outer membrane protein [Burkholderia pseudomallei K96243] GenBank: CAH36153.1 >CAH36153.1 putative outer membrane protein [Burkholderia pseudomallei K96243] MLFKPHRFVPKTVAAAALAAHGLAAHATAPFVVQDIKIEGLQRVEAGSVFAYLPIKQGDTFTDDKASEA IRALYATGFFNDVRIATQGGVVIVQVQERPAIASIDFTGIKEFDKDNLNKALKAVGLSQGRYYDKALVD KAEQELKRQYLTRGFYAAEVSTTVTPVDANRVSILFAVAEGPSAKIRQINFIGNKAFKTSTLRDEMQLS TPNWFSWYTKNDLYSKEKLTGDLENVRSYYLNRGYLEFNIESTQVSISPDKKDMYLTVALHEGEPYTVS SVKLAGNLLDRQAELEKLVKIKPGDRFSAEKLQQTTKAIVDKLGQYGYAFATVNAQPEIDQATHKVGLT LVVDPSRRVYVRRINIVGNTRTRDEVVRREMRQLESSWFDSSRLALSKDRVNRLGYFTDVDVTTVPVEG TNDQVDVNVKVAEKPTGAITLGAGFSSTDKVVLSAGISQDNVFGSGTSLAVNVNTAKSYRTLTVTQVDP YFTVDGIKRITDVFYRTYQPLYYSTNSSFRIITAGGNLKFGIPFSETDTVYFGAGFEQNRLDVDSNTPQ SYQDYVNEFGRVSNTVPLTIGWSRDARDSALIPSRGYFTQANAEYGVPVGKIQYYKMDVQGQYYYSFAR GFILGLNFQAGYGNGIGNPYPIFKNYYAGGIGSVRGYEPSSLGPRDTKTNDPIGGSKMVVGNIELTFPL PGTGYDRTLRVFTFLDGGNVWGNAPGGTSTGANGLRYGYGIGLAWISPIGPLKLSLGFPLQKHEGDQYQ KFQFQIGTAF(SEQ ID NO: 19)OmpW outer membrane protein W [Burkholderia pseudomallei] GenBank: ALJ73329.1 >ALJ73329.1 outer membrane protein W [Burkholderia pseudomallei] MGDKHRRRRARRDWRAPVSCWLGATLLACAWSAHAQDSGAARWRDGADGIGFFPGGDAPGFDARAWGPV PGDARRAAASDARNGVAASAGSEATAAAPAADANAAPARKLTEERITLGERVAPVADAARRVRADGDDG IGFADAPGGPPAGGATPSAACDDGACVPDGGDAGRAPRRPPAGATPRFIAGVRYDRMPYELHPIDPERL PDLPEAQGPTLLEQLQGDDSNMIGVGWHYVLSTGRSTPVTTSTAALGIGSFANPGSAVSISNTNTPAFT FTHFFGEHVAAEIVAGIPPELTMRGHGSIGLPFDKIFPGVQGRLPLVDLGNTQSNPLGTTRAWLGSAVF KYYLGKREDRLRPYVGLGLSYTRFTNTNLNPVFAHKLASLGGLLSAGISLGDLQSLLTDSGALDRLLQA GANLILPNGVRATADVKSAWTPVFVVGANYQLTRQLSLSTALSYIPLKAAITVNINDTKGILASNTTTLSANVLLCTMLLNFRF(SEQ ID NO: 20)PotF spermidine / putrescine ABC transporter substrate-binding protein PotF [Burkholderia pseudomallei] GenBank: ANW57327.1 >ANW57327.1 spermidine / putrescine ABC transporter substrate-binding protein PotF [Burkholderia pseudomallei] MKRIAWLAAVLASLACAAARAAGGNVLNIYNWAEYFAPDTIAGFEKETGIKVRLDVYDSNEALQTKLTT GNSGYDLVFPSNDFLARQIQAGLYRKLDKSRLPNLTNLDPAIVARAAEVDPGNQYSVPYMQGTFGLGLN VAKVKQALGGPLPANTLELIFNPAYAAKLERCGIAFNDAGSEVFPLALRYIGRDPNTTDPRDYEAALDM MKKIRPTIRQFIATPVMNDLATGDVCVVTGYSGAVLVAARRAAEAKNGQQIVYSLPSAGAPFWFDSMAI PKGAAHADHALRFIDYILRPDVVAKISNKVMYPNPNRVATPLVDRRLTANPAIYPDAATMRTLWVKRPM PPQAMRMQTRYWTRFKTGY (SEQ ID NO: 21) OppA oligopeptide ABC transporter periplasmic oligopeptide-binding protein [Burkholderia pseudomallei] GenBank: CRY20041.1 >CRY20041.1 oligopeptide ABC transporter periplasmic oligopeptide- binding protein [Burkholderia pseudomallei] MKHTHAFAAVLAALALTIAPSAPAVTVASNVTLADQQDLTRQVPAEVESLDPAHIESWTGNTIGLDLFE GLARIDASGAVVPGVAQAWEHKAPDTWIFKLRRDAKWSNGQPVTAADFVYAWQRLADPKTGSKYTILVE FVKNASAIIAGKQPPGDLGIRAIDPYTIEVKTEVPVSYFPELTAMAPLTPVNKDAVAKFGDAWTRPKNI VSNGPYTLVDWQPNNRIVMAKSDKYWNARNVVIRKVTYLPIENDETALRMYQAGQIDYTYSIPAGGFGQ ISKQFGKELRPGLQLATYYYYLKNSDPALKDKRVREALAMVLDREILTSKITQAGEVPMYGLMPKGVKG VQRPFTPDWASWPMARRVDYAKNLLKQAGHGDANPLTFTLTYNTNDLHKKVALFAASEWRTKLGVTAKL ENVEFKVLMKQRHDGKVQVARDGWFADYNDAMTFFDLIRCGSSQNTVGYCNPKVDSLVAEANQKLDDGARAALLTQAHDLAMNDYPMVPLFQYSADRLVKSYVGGYTLTNYIDMRASQDMYLIKH(SEQ ID NO:22) BopA (Type III Effector) BopA protein [Burkholderia pseudomallei] GenBank: CRY24966.1 >CRY24966.1 BopA protein [Burkholderia pseudomallei] MINVGAFVASARSGARVVVGGDARGPVVSAARLGMKERLFAFLAHVPLLKHCDAVRRYAEQVRMENRRS LEVFVLALSKRYGPEGAKAAFDYGARRDGAPLDQRRVRNMVSIAEHFHGTGDAKPLARQMVFRSWECRG LDHPGHASLTIKNQADADAGRHVYEHVSWWPNQRLGSKEHFDRIEPKTLDGYRIDKRSEISSATEQRLR EGDAARRKILADGFKYANQDERHDARFFPRAGQKLDKDAEWGLSARKVYFPAIGFNHDRRDTDRPRAFV LFGLNEAAMLRDARTVKEGAKSGELKYRMISKKENCASMALRVLRAGGAEHFVPYTAAWISEDPNHAHA YALAVQARIDALNQRRADVERRCERLRDSASVRQAWRAFSEAGGASASPLAEDAGRGRASAHMRQARLD EHAREVERIGAYFAELSAGRSGKHRDRADAALADAMKRCAPSARDDVAALTRKASVLVETLGRHLDAPPPSDSSALRRLAAHAMIGRIEAFMAAAIAA(SEQ ID NO: 23)BimA (Autotransporter protein) BimA [Burkholderia pseudomallei] GenBank: ACF94992.1 >ACF94992.1 BimA [Burkholderia pseudomallei] MHAKASSSHAPDAPKPSSIATTLCRALASLSLGLSMDAEANPPEPPGGTNIPVPPPMPGGGANIPVPPP MPGGGANIPPPPPPPGGIGGATPSPPPLTPVNGNPGASTPTKTGLLKTLNRLSAELQNNPRVTEDVVDN VDAVIRNAVNLAPDANGDFSGRSAMPIEMAANAALRSLKKNPGDAGHAAPAYLPAERIGQLREKVRRTI EALESNRPPKPQPRSTPPQSTPPKPTQHPTAPNPNVPDASTPDASTPDASTPDASTPDASTPSRPAPAP RAGTGAPAASAATRAPAFANRVRKPNPAMPAASSHAIASDFASSNAFAIGDDSTAVGAQAIAFSEQSIA IGSRAIAAGARSIAVGTDATAAAPDSVALGSGSIAEREGTVSVGRDGHERQITHVASGTEPTDAVNVTQ LRAAMSNANAYTNQRIGDLQQSITDTARDAYSGVAAATALTMIPDVDRDKRVSIGVGGAVYKGHRAVAL GGTARINENLKVRAGVAMSAGGNAVGIGMSWQW (SEQ ID NO: 24) BPSL1897 hypothetical protein BPSL1897 [Burkholderia pseudomallei K96243] NCBI Reference Sequence: YP_108497.1 >YP_108497.1 hypothetical protein BPSL1897 [Burkholderia pseudomallei K96243] MTRALGRLRSPRRQRGATAIEFAILFPMFFLILYGIITYGMIFAAQQSLTLAATEGARAALNYQVAQTQ SAALGLRAAAACTAANNLTGWLSGATCTTSTNYTCSYDSTMYCIQVTLTYPYAANPLVPAVALFDAVLP TTLTSRATVQINPTNII(SEQ ID NO: 25)BPSL3369 acetaldehyde dehydrogenase [Burkholderia pseudomallei K96243] GenBank: CAH37382.1 >CAH37382.1 acetaldehyde dehydrogenase [Burkholderia pseudomallei K96243] MNHADMQHLNIEFPYRKQYGNFIGGEWVAPVGGEYFDNVSPVTGRPFTAIPRSREADIELALDAAHAAK AGWAAKGAAERANVLLRIADRMEANLTRLAVAETIDNGKPLRETTAADVPLAIDHFRYFAGCIRAQEGS IADIGGDMVAYHFHEPLGVVGQIIPWNFPLLMAAWKLAPALAAGNCVVLKPAEQTPASILVFAELIQDL LPPGVLNIVNGFGLEAGKPLASSKRIAKIAFTGETSTGRLIMQYASENLIPVTLELGGKSPNIFFADVM DRDDSYFDKALEGFAMFALNQGEVCTCPSRALVEESIYDRFIERALKRVEAIKQGHPLDSQTMIGAQAS AEQLEKILSYIDIGRGEGAQCLTGGERNVLGGELAEGYYVKPTVFRGHNKMRIFQEEIFGPVLAVTTFK TEEEALEIANDTLYGLGAGVWTRDGNRAYRFGRGIQAGRVWTNCYHAYPAHAAFGGYKQSGIGRETHKM MLDHYQQTKNLLVSYSEKPLGFF (SEQ ID NO: 26) BPSL2287 HesB family protein [Burkholderia pseudomallei K96243] GenBank: CAH36290.1 >CAH36290.1 HesB family protein [Burkholderia pseudomallei K96243] MAITLTEKAAQHVQKYLARRGKGLGLRLGVRTTGCSGLAYKLEYVDELTPEDQMFESHGVKVFVDPKSLAYIDGTELDFAREGLNEGFKFNNPNVKDECGCGESFRV(SEQ ID NO: 27)BPSL2765 putative OmpA family lipoprotein [Burkholderia pseudomallei K96243] GenBank: CAH36773.1 >CAH36773.1 putative OmpA family lipoprotein [Burkholderia pseudomallei K96243] MMSKKLRLAFAMLMIGALAACKSGVKLDEHANQGDAVSTQPNPENVAQVTVDPLNDPNSPLAKRSVYFD FDSYSVQDQYQALLQQHAQYLKSHPQRHILIQGNTDERGTSEYNLALGQKRAEAVRRALSLLGVGDAQMEAVSLGKEKPVALGHDEASWAQNRRADLVYQQ(SEQ ID NO: 28)VgrG5 Rhs element Vgr protein [Burkholderia pseudomallei 1710b] GenBank: ABA53677.1 >ABA53677.1 Rhs element Vgr protein [Burkholderia pseudomallei 1710b] MRLIELRSPLLDPDAVALSFVVHENLSQEPSYQLDLLSHDSNLDFDALLGSTLSADIDLGEGDIRTFNT HVFGGYDTGQMSGQYTYTLELRSWLSFLAENRNSRIFQDLSVPQIVEQVFQGHQRNGYRFELEGTYEPR EYCVQFQETDLNFVKRLLEDEGIYFWVEHEPDRHVVVISDTQRFEDLPLPNDTLEYLPDGEESRAIQGR EGVQRLQRTRRIKSNNVALRDFDYHAPSKQLDSDAQVEQQSLGGIPLEYYDYAAGYRDPEQGERLARLR LEAIQADAHALGGEANARALAVGRAFTLVGHPALSRNRRYYVTNSELTFIQDGPDSTSQGRNVAVKFRA LADDQPFRPLLVTKRPRVPGIQSATVVGPEMSEVHTDKLGRIRVHFHWDRYKTTEADASCWIRVTQAWA GKGWGVLAMPRVGQEVIVVYVDGDLDRPLATGIVYNGENPTPYDLPKDIRYTGLVTRSIKRAGGIPNAS QLTFDDQHGAERVMIHAERDLQQTVERNSSTSIAQDLNLSVKGTSTSVVGISVSFTGISVSYTGLSVSF TGVSARFTGVSTSFTGVSTSFTGVSTSFTGVDTSFTGVSTGFKGVDTSFTGVATSMVGVSTSITGSSNS VTGVSNSMTGISSSWKDVSMSTTGQSESITGVSLSYTGTSNSMTGTSTSVTGTSTSITGTSMSNTGSST SITGTSMSTTGSSVSTTGSSMSATGSSVGTTGSSVSTTGSKMSVTGFSFSYTGASYEDVGVDLKKLGMQTKN(SEQ ID NO: 29)Burkholderia mallei polypeptide sequences BMA_A0768 mannitol dehydrogenase family protein mannitol dehydrogenase family protein [Burkholderia mallei ATCC 23344] GenBank: AAU46944.1 >AAU46944.1 mannitol dehydrogenase family protein [Burkholderia mallei ATCC 23344] MPLLSSDHCRALPPEVSRPRYDRRALRTGIVHLGLGAFHRAHQACYTETLVERGDLRWGIAGVELRRRH TVERLAAQDHLYSVTERAGDAARTRVVGAVHRTLFAPQALATLLGLIADPSVSIVSLTVTEKGYYRRPG GGGLDLDDPAIRRDLAQPHAPSTTLGVLAAGIRLRAAHAPLSVLSCDNMPSNGDTLRALLAQYAEQTDG ALARRIRCDVAFPNTMVDRIVPAATPESLDWVQSRIGVRDEAAIVCEPFAQWVFEDRFAGARPRWEDAG ALVAADVRPYEKMKLRLLNGSHSAIAYAGQLRGRRTVSDAMADPLIDALARGVMTRELLATLDVPAGYD VRAYCASLIERFRNPALAHRTAQIATDGTQKVPLRWLPALAESAAAGVERPFLERSLAMWLHYVEVARD ESGRPLVLEDPGAQALAARLHGAPGATDAVRAALGLIASRDAARWPEALTARVGAHLETVRTRGTDALL RPLLDA(SEQ ID NO: 30)BMA_2821ABC Transporter ATP binding protein glutathione ABC transporter ATP-binding protein [Burkholderia mallei] GenBank: KKM47108.1 >KKM47108.1 glutathione ABC transporter ATP-binding protein [Burkholderia mallei] MSASRAAPSLPDARVLAVDGLTVTFRREDAAFVAVRDLSFHVDRGETLAIVGESGSGKSVTSLALMRLV EHGGGAIAGGAIALRRRGGAVLDLARATPSTLRTVRGADVAMIFQEPMTSLNPVFTVGDQISEAIALHQ HKSAGEARAETLRLLDLVRIPEARRVFARHPHQLSGGMRQRVMIAMALSCRPALLIADEPTTALDVTIQ AQILQLIRGLQDEMDMGVIFITHDMGVVAEVADRVLVMYRGEKVEEGACDAIFAAPSHPYTKALLAAVP RLGSMRGTDAPAKFPLLRFDPAAGDALVVAGGDATAASGDAARESVLFVDSDAAAASAASTASTASAAS AASAAPTACARPAIDAGAPPLLRVRELVTRFPVKSGVFGRVSQYVHAVERVSFELRAGETLALVGESGC GKSTTGRSLLRLVERVSGSIEFEGREIGALKGRELQALRRNIQFIFQDPFASLNPRLTVGFSIMEPLLV HGVASGRQAQARVDWLLERVGLPADAARRYPHEFSGGQRQRIAIARALALNPKVVVADESVSALDVSVQ AQIVNLMLDLQRELGVAYLFISHDMAVVERISHRVAVMYLGQIVEIGPRRAVFETPRHPYTKKLMSAVPIADPACRHAPRTLPADELPSPIRALGDEPEVAPLVAVGPAHFVAEHRVGGAY(SEQ ID NO: 31)BMA_0816 maltooligosyl trehalose synthase maltooligosyl trehalose synthase, putative [Burkholderia mallei ATCC 23344] GenBank: AAU49513.1 >AAU49513.1 maltooligosyl trehalose synthase, putative [Burkholderia mallei ATCC 23344] MKPRATLRLQLHAGFTFDDAAAHVGYFARLGVSHLYLSPITAAEPGSRHGYDVIDYSTVNPELGGEAAF VRLIDALRRRGMGAIVDIVPNHMGVGGSSNRWWNDVLEWGARSRFARHFDIDWHASDPALQRKVLLPCL GRPYGEALAAGDIALRADAAHGRFAIACAGRTLPVQIGAYPDILRAANRSDLNALAERFDAPGARPSNH ARLDAAHAALRDYAAARGPGALDAVLHGFDPRIARSREMLHRLLEQQHYRLAWWRTATDEINWRRFFDI STLACMRIEDAAVFDDVHALLWRLYAAGLVDGVRIDHVDGLADPRGYCRQLRGRLAALRDGEPYIVVEK ILAPDERLPEDWRVDGTTGYDFMNDVSALLHDAAGAAPLAALWADMTGAETTFAREALDGKRRVLARQF AAEHERVARAMHRLARASRDARDFALNPIRRAVAELAIRLPVYRLYPSAGAPQRTDRALLAGAWQAARS AIAPADRAALDYVAATLGLPGVARAVAGLGDPARLAARVGFAQLTAPLAAKGVEDTACYRYGRLLSRNE VGAHADALSLAPGAFHTRNRRRRRTFPGALLATATHDHKRGEDARARLAVLSEAHRAWRAAALDWAAFN APHHHGAPAAADRIPGPAAEAMLYQTLVGAWPPALAPDDAPGLAALTDRVERWQLKALREAKRDTDWLE PNLGYEAGCAAFLRAIMTPRGPDDFAHRLHRLVARIAPAGIVNSLSQAALRLLSPGVPDLYQGAQTWDH TLVDPDNRADVPFARYAAQRIDAPVAAYLRDWADGRVKHALIGRLLALRAAHPETFAAGAYVPLHVRGT RRGHALAFARRDASTTIVVIATRLAYPLLGDAPARPCVEAACWADTAVGLAPGFAGPWRDMLNDGTLDAPSGMLPLAAALAHLPVAVLIREGGAADTPRRGA(SEQ ID NO: 32)GroEL molecular chaperone GroEL [Burkholderia mallei ATCC 23344] NCBI Reference Sequence: YP_103588.1 >YP_103588.1 molecular chaperone GroEL [Burkholderia mallei ATCC 23344] MAAKDVVFGDSARAKMVEGVNILANAVKVTLGPKGRNVVLERSFGGPTVTKDGVSVAKEIELKDKLQNM GAQMVKEVASKTSDNAGDGTTTATVLAQSIVREGMKYVASGMNPMDLKRGIDKAVAAAVEELKKISKPC TTNKEIAQVGAISANSDSSIGDRIAEAMDKVGKEGVITVEDGKSLADELDVVEGMQFDRGYLSPYFINN PDKQVAVLENPFVLLHDKKVSNIRDLLPVLEQVAKAGRPLLIIAEDVEGEALATLVVNNIRGILKTVAV KAPGFGDRRKAMLEDIAILTGGQVIAEETGLTLEKATLAELGQAKRIEVGKENTTIIDGAGEAVNIEAR VKQIRTQIEEATSDYDREKLQERVAKLAGGVAVIKVGAATEVEMKEKKARVEDALHATRAAVEEGIVPG GGVALIRARTAIASLTGVNADQNAGIKIVLRAMEEPLRQIVTNGGEEASVVVAAVAAGKGNYGYNAATG EYVDMVEAGVVDPTKVTRTALQNAASVAGLLLTTDAAVAELPKEDAPMPGGMPGGMGGMGMGMGMDM (SEQ ID NO: 33) References 1 Jia, Q. et al. Replicating bacterium-vectored vaccine expressing SARS-CoV-2 Membrane and Nucleocapsid proteins protects against severe COVID-19-like disease in hamsters. NPJ Vaccines 6, 47, doi:10.1038 / s41541-021-00321-8 (2021). 2 Jia, Q. et al. Replicating bacterium-vectored vaccine expressing SARS-CoV-2 Membrane and Nucleocapsid proteins protects against severe COVID-19 disease in hamsters. bioRxiv, doi:10.1101 / 2020.11.17.387555 (2020). 3 Jia, Q. & Horwitz, M. A. Live Attenuated Tularemia Vaccines for Protection Against Respiratory Challenge With Virulent F. tularensis subsp. tularensis. Frontiers in cellular and infection microbiology 8, 154, doi:10.3389 / fcimb.2018.00154 (2018). 4 Jia, Q. et al. Single vector platform vaccine protects against lethal respiratory challenge with Tier 1 select agents of anthrax, plague, and tularemia. Scientific reports 8, 7009, doi:10.1038 / s41598-018-24581-y (2018). 5 Jia, Q. et al. Francisella tularensis Live Vaccine Strain deficient in capB and overexpressing the fusion protein of IglA, IglB, and IglC from the bfr promoter induces improved protection against F. tularensis respiratory challenge. Vaccine 34, 4969-4978, doi:10.1016 / j.vaccine.2016.08.041 (2016). 6 Jia, Q., Bowen, R., Sahakian, J., Dillon, B. J. & Horwitz, M. A. A heterologous prime-boost vaccination strategy comprising the Francisella tularensis live vaccine strain capB mutant and recombinant attenuated Listeria monocytogenes expressing F. tularensis IglC induces potent protective immunity in mice against virulent F. tularensis aerosol challenge. Infect Immun 81, 1550-1561, doi:10.1128 / IAI.01013-12 (2013). 7 Jia, Q. et al. A Francisella tularensis live vaccine strain (LVS) mutant with a deletion in capB, encoding a putative capsular biosynthesis protein, is significantly more attenuated than LVS yet induces potent protective immunity in mice against F. tularensis challenge. Infect Immun 78, 4341-4355, doi:10.1128 / IAI.00192-10 (2010). 8 Burtnick, M. N. et al. The cluster 1 type VI secretion system is a major virulence determinant in Burkholderia pseudomallei. Infect Immun 79, 1512-1525, doi:10.1128 / IAI.01218-10 (2011). Harland, D. N. et al. Identification of a LolC homologue in Burkholderia pseudomallei, a novel protective antigen for melioidosis. Infect Immun 75, 4173-4180, doi:10.1128 / IAI.00404-07 (2007). Whitlock, G. C. et al. Protective response to subunit vaccination against intranasal Burkholderia mallei and B. pseudomallei challenge. Procedia in vaccinology 2, doi:10.1016 / j.provac.2010.03.013 (2010). Whitman, L. et al. Rapid, scarless cloning of gene fragments using the electra vector system. Genet. Eng. Biotechnol 33, 10.1089 (2013). Silva, E. B. et al. Correlates of immune protection following cutaneous immunization with an attenuated Burkholderia pseudomallei vaccine. Infect Immun 81, 4626-4634, doi:10.1128 / IAI.00915-13 (2013). Becker, S., Lochau, P., Jacob, D., Heuner, K. & Grunow, R. Successful re- evaluation of broth medium T for growth of Francisella tularensis ssp. and other highly pathogenic bacteria. Journal of microbiological methods 121, 5-7, doi:10.1016 / j.mimet.2015.11.018 (2016). Pavlovich, N. V. & Mishan'kin, B. N. [Transparent nutrient medium for culturing Francisella tularensis]. Antibiot Med Biotekhnol 32, 133-137 (1987). Geertsma, E. R. & Dutzler, R. A versatile and efficient high-throughput cloning tool for structural biology. Biochemistry 50, 3272-3278, doi:10.1021 / bi200178z (2011). Lapid, C. & Gao, Y. PrimerX: Automated design of mutagenic primers for site- directed mutagenesis, <https: / / www.bioinformatics.org / primerx / > ( Garcia-Nafria, J., Watson, J. F. & Greger, I. H. IVA cloning: A single-tube universal cloning system exploiting bacterial In Vivo Assembly. Scientific reports 6, 27459, doi:10.1038 / srep27459 (2016). Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature methods 6, 343-345, doi:10.1038 / nmeth.1318 (2009). 19 Waldo, G. S., Standish, B. M., Berendzen, J. & Terwilliger, T. C. Rapid protein- folding assay using green fluorescent protein. Nat Biotechnol 17, 691-695, doi:10.1038 / 10904 (1999). 20 Chen, X., Zaro, J. L. & Shen, W. C. Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev 65, 1357-1369, doi:10.1016 / j.addr.2012.09.039 (2013). 21 Klein, J. S., Jiang, S., Galimidi, R. P., Keeffe, J. R. & Bjorkman, P. J. Design and characterization of structured protein linkers with differing flexibilities. Protein Eng Des Sel 27, 325-330, doi:10.1093 / protein / gzu043 (2014). 22 Monk, I. R., Gahan, C. G. & Hill, C. Tools for functional postgenomic analysis of listeria monocytogenes. Appl Environ Microbiol 74, 3921-3934, doi:10.1128 / AEM.00314-08 (2008). 23 Inoue, H., Nojima, H. & Okayama, H. High efficiency transformation of Escherichia coli with plasmids. Gene 96, 23-28, doi:10.1016 / 0378- 1119(90)90336-p (1990). 24 Gasteiger, E. et al. ExPASy: The proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res 31, 3784-3788, doi:10.1093 / nar / gkg563 (2003). 25 Propst, K. L., Mima, T., Choi, K. H., Dow, S. W. & Schweizer, H. P. A Burkholderia pseudomallei deltapurM mutant is avirulent in immunocompetent and immunodeficient animals: candidate strain for exclusion from select-agent lists. Infect Immun 78, 3136-3143, doi:10.1128 / IAI.01313-09 (2010). 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. 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.

Claims

CLAIMS:

1. A composition of matter comprising at least one immunogenic fusion protein comprising antigenic epitopes present in: at least one Burkholderia pseudomallei or Burkholderia mallei protein selected from HCP-1 and HCP-2; and at least one Burkholderia pseudomallei or Burkholderia mallei protein selected from HCP-6 and LolC, wherein the composition comprises an attenuated Francisella tularensis live vaccine strain (LVS) expressing the at least one fusion protein.

2. The composition of claim 1, wherein: an immunogenic fusion protein comprising HCP-1 and / or HCP-2 in combination with HCP-6 and / or LolC is expressed at levels that are at least 50% greater than expression levels of control immunogenic fusion proteins comprising HCP-1 and / or HCP-2 in the absence of HCP-6 and / or LolC; the immunogenic fusion protein comprises antigenic epitopes present in HCP6, HCP1 and HCP2; and / or the immunogenic fusion protein comprises antigenic epitopes present in HCP6, HCP1 and LolC.

3. The composition of claim 1, wherein the composition comprises an attenuated Listeria monocytogenes vector that: does not express a functional InlB protein; does not express a functional actA protein; and / or expresses prfA protein having a G155S substitution mutation.

4. The composition of claim 1, wherein the composition comprises at least two fusion proteins.

5. A method of generating an immune response to a Burkholderia pseudomallei in a mammal comprising administering to the mammal a composition of claim 1 such that an immune response to Burkholderia pseudomallei is generated.

6. The method of claim 5, wherein the mammal is immunized with the composition intranasally, subcutaneously, intradermally, intramuscularly or orally.

7. The method of claim 5, wherein the composition is administered subcutaneously.

8. The method of claim 5, wherein the composition is administered intradermally.

9. The method of claim 5, wherein the mammal is a human.

10. Use of the composition of claim 1 as a vaccine to generate an immune response to Burkholderia pseudomallei or Burkholderia mallei in a mammal.

11. A method of generating an immune response to a Burkholderia mallei in a mammal comprising administering to the mammal a composition of claim 1 such that an immune response to Burkholderia mallei is generated.

12. The method of claim 11, wherein the mammal is immunized with the composition intranasally, subcutaneously, intradermally, intramuscularly or orally.

13. The method of claim 11, wherein the composition is administered subcutaneously.

14. The method of claim 11, wherein the composition is administered intradermally.

15. The method of claim 11, wherein the mammal is an equine.

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