Layered methods for the treatment of bacterial infection based on the combination of heterologous vaccination and antibiotic therapy

Abstract

In this disclosure, layered countermeasures based on the combination of pre -infection heterologous vaccination and post-infection antibiotic therapy were provided for the treatment of bacterial infection. Embodiments using layered countermeasures demonstrated efficacy and protection against infection from Yersinia pestis and Burkholderia pseudomallei.

Description

LAYERED METHODS FOR THE TREATMENT OF BACTERIAL INFECTION BASED ON THE COMBINATION OF HETEROLOGOUS VACCINATION AND ANTIBIOTIC THERAPY STATEMENT OF GOVERNMENTAL INTEREST Under paragraph 1(a) of Executive Order 10096, the conditions under which this invention was made entitle the Government of the United States, as represented by the Secretary of the Army, to an undivided interest therein on any patent granted thereon by the United States. BACKGROUND

[0001] The etiologic agent of plague, Yersinia pestis, is a globally distributed pathogen which is both a considerable public health risk and a potential biothreat agent. Plague is known as a fearsome menace of antiquity, as illustrated by the massive Black Death pandemic of the 14thcentury, with a fatality rate of more than 30%; yet it remains an important current zoonotic hazard (Baril et al. 2019, Bertherat 2016). In many countries, such as China, Madagascar, the United States of America, the Democratic Republic of the Congo, and elsewhere in South America and Africa, foci of Y. pestis persist, causing periodic outbreaks of disease (Baril, et al.2019, Bevins et al.2021). This situation is highlighted by the unusually large outbreak of bubonic and pneumonic plague of 2017 in Madagascar, where the majority of cases were the highly-lethal pneumonic form (Randremanana et al. 2019). Furthermore, Y. pestis is also categorized as a Tier 1 select agent by the US Department of Health and Human Services (Nelson et al. 2021), largely due its potential to be aerosolized and an acute (in some cases rapidly fatal) disease. Attempts to harness Y. pestis as a bioweapon have long been described, from the early use of plague corpses in the Middle Ages, to the experimental disseminations of plague in China by the infamous Japanese Unit 731 during World War II, and possibly beyond, as documented in detail (Alibek 1999, Cowdrey 1984, Williams and Wallace 1989, Worsham et al. 2018).

[0002] Burkholderia pseudomallei is a gram-negative bacterium that causes the disease melioidosis (Galyov et al., 2010; Brett et al., 2018). B. pseudomallei has been identified inSoutheast Asia, northern Australia, and in many other tropical areas around the world (Dance, 2000; Aardema et al., 2005; Inglis et al., 2006; Lo et al., 2009; Doker et al., 2014; Hogan et al., 2015; Jilani et al., 2016; Limmathurotsakul et al., 2016). The current consensus is that the number of melioidosis cases globally is likely grossly under reported, due in part to non-specific signs and symptoms resulting in difficulty with accurate diagnoses (Cheng and Currie, 2005; Peacock, 2006; Wiersinga et al., 2006, 2012).

[0003] In addition, this bacterium is known to be intrinsically resistant to several commonly used antibiotics, making effective treatment, which results in complete eradication of the bacteria, challenging (Moore et al., 1999; Wuthiekanun et al., 2005; Sarovich et al., 2012). B. pseudomallei can cause infections through cutaneous abrasions and lacerations, consumption of contaminated food or drinking water, and is known to be transmitted through an aerosolization process attributable to weather patterns, typically during monsoon season (Inglis et al., 2000; Currie and Jacups, 2003; Currie et al., 2010; Hassan et al., 2010; Limmathurotsakul et al., 2014b; Liu et al., 2015; Kaestli et al., 2016; Sanchez-Villamil et al., 2020).

[0004] Burkholderia pseudomallei has been a bacterium of concern to the United States Department of Defense for several reasons. It has been demonstrated that healthy individuals can be infected with B. pseudomallei and the bacteria can remain undetected for years or decades (Mays and Ricketts, 1975;; Ngauy et al., 2005). During the Vietnam conflict it was hypothesized that many U.S. military personnel would potentially be exposed to this bacterium while deployed to endemic areas which could result in disease as exposed individuals aged or developed co- morbidities (Patterson et al., 1967; Gilbert et al., 1968; Whelan et al., 1968; Greenberg, 1969;). Fortunately, the concerns regarding large numbers of latent or unidentified infections in personnel were never realized, however, this remains a concern for deployed individuals (Schully et al., 2019; Larson et al., 2020). Until recently, melioidosis in the U.S. has only been associated with foreign travel or exposure to imported exotic pets (Currie, 2003; Zehnder et al., 2014). However, in 2021 several fatal cases of melioidosis were associated with contaminated aromatherapy liquid manufactured in India and sold in the U.S. at a large national retailer (Centers for Disease Control and Prevention [CDC], 2021; Gee et al., 2022). Thus, this bacterium warrants further attention by both the public health and biodefense research communities.SUMMARY

[0005] Plague is an ancient disease caused by Yersinia pestis, a widely disseminated Tier 1 pathogen that poses significant public health and biothreat risks. The rapid course and high mortality of pneumonic plague limit the efficacy of antibiotic treatment and mandate the need for an effective, licensed, and readily available vaccine. New candidate vaccines are being developed; however, their efficacy in nonhuman primates, optimal vaccination schedule and immune response, duration of protection, and breadth of coverage against various virulent strains are inadequately understood. In this disclosure, homologous and heterologous vaccination schemes were explored using the sensitive BALB / c mouse models of bubonic and pneumonic plague challenged with Y. pestis strain C12, a derivative of wild-type strain CO92, which lacks the anti- phagocytic F1 capsule yet remains highly virulent. Protection against such nonencapsulated strains has been particularly elusive.

[0006] Therefore, the efficacy of live attenuated vaccine (LAV) derivatives of Y. pestis CO92 or C12 with a deletion of a type III secretion-associated gene (∆yscN) or of the pgm pigmentation locus and were cured of the pPst (PCP1) plasmid (CO92 pgm- pPst-) was tested. The LAVs were evaluated alone or accompanied by a dose of protein subunit vaccine (rF1V or rV). The most protective and immunogenic vaccination scheme, as tested under a variety of conditions in bubonic and pneumonic plague models, was heterologous vaccination with a LAV and the recombinant rF1V or rV protein subunit vaccine. Furthermore, in the heterologous scheme, different LAV and subunit vaccines could be substituted, affording flexibility in vaccine component selection. Also, a novel intervention strategy consisting of vaccination and post-exposure antibiotic treatment was evaluated. The layering of vaccination with the LAVs and post-exposure treatment with streptomycin was synergistic, extending the time after Y. pestis C12 challenge when treatment remained effective and affording a sparing of antibiotic.

[0007] Burkholderia pseudomallei, the gram-negative bacterium that causes melioidosis, is notoriously difficult to treat with antibiotics. A significant effort has focused on identifying protective vaccine strategies to prevent melioidosis. However, when used as individual medical countermeasures both antibiotic treatments (therapeutics or post-exposure prophylaxes) and experimental vaccine strategies remain partially protective. Here was demonstrated that when used in combination, current vaccine strategies (recombinant protein subunits AhpC and / or Hcp1 pluscapsular polysaccharide conjugated to CRM197 or the live attenuated vaccine strain B. pseudomallei 668 ΔilvI) and co-trimoxazole (trimethoprim / sulfamethoxazole) regimens can result in near uniform protection in a mouse model of melioidosis due to apparent synergy associated with distinct medical countermeasures. The results in this disclosure demonstrated significant improvement when examining several suboptimal antibiotic regimens (e.g., 7-day antibiotic course started early after infection or 21-day antibiotic course with delayed initiation). Importantly, this combinatorial strategy worked similarly when either protein subunit or live attenuated vaccines were evaluated.

[0008] In summary, layered and integrated medical countermeasures provide novel treatment options for melioidosis and plague as well as diseases caused by other pathogens that are refractory to individual strategies, particularly in the case of engineered, emerging, or re-emerging bacterial biothreat agents. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1. Survival curves of groups of vaccinated BALB / c mice challenged with Y. pestis strain C12. The mice were vaccinated with a single dose of CO92 ∆yscN (1.3 x 107CFU), C12 ∆yscN (1.7 x 107CFU), or both (Combo LAV), 1.54 x 107CFU total; the control group received KPhos buffer alone. All groups contained 10 mice. Twenty-eight days later the animals were exposed to 1.93 x 103CFU (214 LD50s) by the SC route (FIG. 1A) or with 1.71 x 106CFU (15 LD50s) by the aerosol route (FIG. 1B) and monitored for 21 days after challenge.

[0010] Figure 2. Survival curves of BALB / c mice vaccinated twice with the homologous LAV (days 0 and 23) and challenged 28 days later with Y. pestis strain C12 by the SC route (FIG. 2A) or by the aerosol route (FIG. 2B). The doses of C12 were 2.38 x 103CFU (264 LD50s) or 2.5 x 105CFU (3 LD50s), respectively. The mice were monitored for 21 days after challenge.

[0011] Figure 3. A comparison of protection elicited by homologous and heterologous vaccines against Y. pestis strain C12 challenge. The vaccine doses contained approximately 1 x 107CFU total Combo LAV (ranging from 0.76 – 0.88 x 107CFU) and / or 2 µg protein subunit vaccine, rF1V or rV.. Two vaccinations were given to the mice (n = 10 / group). Vaccinated mice received two doses 28 days apart and were exposed to C12 by the SC route (3.62 x 103CFU C12, 402 LD50s) 27 days later (FIG. 3A). Vaccinated mice received two doses 21 days apart and then exposed to7.93 x 105CFU of aerosolized C12 (10 LD50s) 28 days later (certain lines have been labeled with their respective group numbers, Group 8 is not visible behind group 4 and group 7) (FIG. 3B). Other groups of mice were vaccinated with two doses 21 days apart and then exposed to a larger challenge (1.65 x 106CFU, 22 LD50s) of aerosolized Y. pestis C1229 days later (FIG. 3C). The mice were monitored daily for 21 days after challenge. The vaccine regimens provided to the groups of mice are as follows:

[0012] Figure 4. The influence on survival after Y. pestis C12 challenge of extending the time between prime and boost doses of the vaccines. The study design, vaccines and their schedule of administration are detailed in (FIG.4A) and the table below. A cohort of mice was euthanized on day 119 in order to collect blood and organ homogenates for immunological assays and the remainder of the mice were exposed to aerosolized Y. pestis C12 on day 120. The LAV doses contained approximately 1 x 107CFU (0.86 – 1.34 x 107CFU) and 2 µg / dose of the subunit vaccines were delivered. The CO92 pgm- pPst- live vaccine is deleted of the 102 kB pgm locus and cured of the pPst plasmid. Each vaccine group had 8 mice. The mice were challenged 28 days after the final vaccination with 1.0 x 106CFU (13 LD50s) of aerosolized Y. pestis C12 and checked daily for 21 days after challenge (FIG. 4B) (certain lines have been labeled with their respective group numbers). NA = not applicable. The vaccine regimens provided to the groups of mice are as follows:

[0013] Figure 5. ELISpot assays for IFN-g-secreting splenocytes collected post-vaccination and detected after stimulation with rF1V (FIG. 5A) or temperature-shifted, killed whole cell TS C12 (FIG. 5B). The 12 groups are those described in the Figure 4 legend table. SFC – spot forming unit. The samples were obtained from 4 or 5 mice / group and the data expressed as GM and GSE.

[0014] Figure 6. The protection of BALB / c mice against lethal aerosol challenge with Y. pestis strain C12 by a vaccination and post-challenge antibiotic treatment strategy: The impact on survival of antibiotic dose and time of treatment. In each experiment, two groups of mice were vaccinated twice (20 days apart) with mutant CO92 ∆yscN (0.92 x107CFU and 0.48 x 107CFU / mouse, respectively), and two groups were unvaccinated. All mice were challenged with aerosolized Y. pestis C1228 days after the boost dose. The challenge doses were administered in two iterations, with doses of 5.6 x 105CFU / mouse (6 LD50s) for vaccinated groups and 3.11 x 105CFU (4 LD50s) for unvaccinated mice. The mice were treated with either 20 mg / kg streptomycin starting at 48 h (FIG. 6A) or 60 h (FIG. 6B) after challenge; or 40 mg / kg starting at 60 h after challenge (FIG. 6C). n = 8 mice / group (KPhos alone and vaccine alone) or 10 mice / group (streptomycin alone and vaccine + streptomycin).

[0015] Figure 7. The protection of BALB / c mice against lethal aerosol challenge with Y. pestis strain C12 by a vaccination and post-challenge antibiotic treatment strategy. The mice were vaccinated twice (23 days apart) Y. pestis C12 ∆yscN (0.48 x 107and 0.70 x 107CFU for doses 1 and 2 respectively); controls received KPhos alone. The mice were challenged 28 days later with 1.67 x 106CFU (22 LD50s)of aerosolized Y. pestis C12 (21 LD50s). They were either untreated or treated with 20 mg / kg streptomycin 60h after challenge with Y. pestis strain C12 four weeks after the boost dose. n = 9 mice / group (KPhos alone and vaccine alone) or 10 mice / group (streptomycin alone and vaccine + streptomycin).

[0016] Figure 8. The impact of combining prophylactic vaccine with a short course (7 days) of post-exposure co-trimoxazole therapy initiated early after exposure to aerosolized B. pseudomallei. (FIG. 8A) Mice were vaccinated with sham vaccine (PBS) before exposure to B. pseudomallei K96243. (FIG.8B) Mice were vaccinated with the live attenuated vaccine 6681ilvI before exposure to B. pseudomallei K96243 (21 h data are not visible; all mice survived through the end of study). (Fig. 8C) Mice were vaccinated with the subunit Hcp1 + conjugate vaccine before exposure to B. pseudomallei K96243 (6 h and 45 h data are not visible; all mice survived through the end of study). Antibiotics were initiated at the time indicated and were given every 12 h for 7 days. These data are representative graphic depiction of data included in Table 2; ∗P < 0.05 for survival compared to vaccine alone, actual P-values are indicated in Table 2.

[0017] Figure 9. The impact of combining prophylactic vaccine with a long course (21 days) of post-exposure co-trimoxazole therapy with a delayed initiation after exposure to aerosolized B. pseudomallei. (A) Mice were vaccinated with sham vaccine (PBS) before exposure to B. pseudomallei K96243. (B) Mice were vaccinated with the live attenuated vaccine 6681ilvI before exposure to B. pseudomallei K96243 (93 h data are not visible; all mice survived through the end of study). (C) Mice were vaccinated with the subunit Hcp1 + conjugate vaccine before exposure to B. pseudomallei K96243. Antibiotics were initiated at the time indicated and were given every 12 h for 21 days. These data are representative graphic depiction of data included in Table 3; ∗P < 0.05 for survival compared to vaccine alone, actual P-values are indicated in Table 3

[0018] Figure 10. Shows a graph of the SFC results of the 12 groups shown in FIG.4. DETAILED DESCRIPTION 1. Definitions

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although various methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. However, the skilled artisan understands that the methods and materials used and described are examples and may not be the only ones suitable for use in the invention. Moreover, as measurements are subject to inherent variability, any temperature, weight, volume, time interval, pH, salinity,molarity or molality, range, concentration and any other measurements, quantities or numerical expressions given herein are intended to be approximate and not exact or critical figures unless expressly stated to the contrary.

[0020] As used herein, the term “about” means plus or minus 20 percent of the recited value, so that, for example, “about 0.125” means 0.125 ±0.025, and “about 1.0” means 1.0 ±0.2.

[0021] As used herein, the term “attenuated” refers to the process of rendering certain pathogen virulence attributes needed to cause diseases less able to cause such disease symptoms.

[0022] As used herein, the term “treating” and its cognates refers to taking steps to obtain beneficial or desired results, including clinical results, including mitigating, alleviating or ameliorating one or more symptoms of a disease; diminishing the extent of disease; delaying or slowing disease progression; ameliorating and palliating or stabilizing a metric (statistic) of disease. The effect may be prophylactic in terms of completely or partially preventing a conditions or disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a condition or disease and / or adverse effect attributable to the condition or disease. “Treatment” refers to the steps taken. It can include any treatment of a condition or disease in a mammal, particularly in a human, and includes: (a) preventing the condition or disease or symptom thereof from occurring in a subject which may be predisposed to the condition or disease but has not yet been diagnosed as having it; (b) inhibiting the condition or disease or symptom thereof, such as, arresting its development; and (c) relieving, alleviating or ameliorating the condition or disease or symptom thereof, such as, for example causing regression of the condition or disease or symptom thereof.

[0023] As used herein, the term “prophylaxis” in the context of a disease refers to taking steps to obtain beneficial or desired results, including clinical results, and including steps to obtain total or partial prevention of a disease or condition. Vaccination for prophylaxis generally refers to administration of the composition in order to completely or partially prevent or reduce the chance of contracting a disease, inducing a condition, or the symptoms of, severity of, or duration of the disease or condition, including partially or completely inhibiting the symptoms, or ameliorating the symptoms or progression of the symptoms. Prophylaxis includes reducing the bacterial infection (bacterial titer), reducing reactions to bacterial toxins associated with the disease or condition.

[0024] As used herein, the term “subject” refers to any animal, optionally a mammal, and most optionally a human. Laboratory animals are included in this definition.

[0025] As used herein, the term “subject in need” refers to a subject, including a human patient, who suffers from melioidosis or plague or is in an environment which might expose the subject to melioidosis or plague. For example, such a subject in need includes, but is not limited to a person residing in an area where melioidosis or plague is endemic or a person living in an industrialized country (including military personnel) who travels to a less developed region where melioidosis or plague is endemic.

[0026] As used herein, the term “therapeutically effective amount” refers to an amount of a therapeutic agent, which, when administered to a subject, has the intended therapeutic effect. A therapeutic effect is an effect that treats the intended disorder or condition, including improving the disease, disorder or condition, or a symptom thereof, including reduction of a symptom or delaying the onset or reoccurrence of the disease, disorder, condition, or symptom. The full therapeutic effect does not necessarily occur by administration of one dose and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. In the context of a vaccine, a therapeutically effective amount is an amount that reduces the chance of occurrence or reduces the severity of a disease or condition in a subject currently not suffering from the disease or condition; or an amount that produces an immune response to an etiologic agent that is associated with protection against the disease or condition. When administered to a subject currently suffering from the disease or condition, a therapeutically effective amount is an amount that induces an immune response to an etiologic agent that is associated with protection against the disease or condition and ameliorates the disease or condition in the subject.

[0027] As used herein, the term “vaccine composition” refers to a composition designed to elicit an immune response, and it may include adjuvants. The vaccine composition can include adjuvants, preservatives (e.g., 2-phenoxyethanol), stabilizers (e.g., gelatin, amino acids, potassium, sodium, lactose), pH adjusters and buffers, tonicity adjuster (e.g., NaCl), surfactants and emulsifiers (e.g., Polysorbate 80 or Tween®), formaldehyde, and some vaccine contains antibiotics.

[0028] As used herein, the term “adjuvant” refers to ingredients used in some vaccines that induce a stronger immune response. Vaccines made from attenuated / weakened or killed / inactivated microorganisms contain naturally occurring adjuvants, however, subunit vaccines, toxoid vaccines, or other vaccines developed these days such as virus vector vaccine, plasmid DNA or mRNA vaccines include just small components of microorganisms instead of the entire virus or bacteria. Adjuvanted vaccines can cause more immunogenicity and local reactions (such as redness, swelling, and pain at the injection site) and more systemic reactions (such as fever, chills and body aches) than non-adjuvanted vaccines. The most common adjuvant is aluminum salts, such as aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate. Some commonly used adjuvant comprises Emulsigen-D (oil in water emulsion with mineral oil plus dimethyldioctadecyl ammonium bromide), MF59 (oil in water emulsion with squalene, Tween 80, Span 85, citrate buffer), ISCOMs (nanoparticles of cholesterol, phospholipids, saponins), ISCOMATRIX (combination of saponin, cholesterol, dipalmitoyl phosphatidylcholine), MetaStim (oil with emulsifier), Havlogen (carbopol), AS04 (alum adsorbed TLR agonist, monophosphoryl lipid A (MPLA)), AS03 (oil in water emulsion with squalene, Polysorbate 80, α-tocopherol), TS6 (light mineral oil plus multiple lipophilic and hydrophilic surfactants), Matrix-MTM (Saponins derived from the soapbark tree), and CpG (Cytosine phosphoguanine, a synthetic form of DNA that mimics bacterial and viral genetic material). (Tizard IR. Adjuvants and adjuvanticity. Vaccines for Veterinarians.2021:75–86, and https: / / www.cdc.gov / vaccinesafety / concerns / adjuvants.html)

[0029] As used herein, the term “antibiotics” refers to medicines that treat bacterial infections in people and animals. They work by killing the bacteria or inhibits their growth. According to Merck Manual (https: / / www.merckmanuals.com / home / infections / antibiotics / overview-of-antibiotics), they can be classified into the classes of aminoglycosides, carbapenems, cephalosporins, fluoroquinolones, glycopeptides and lipoglycopeptides (such as vancomycin), macrolides (such as erythromycin and azithromycin), monobactams (aztreonam), oxazolidinones (such as linezolid and tedizolid), penicillins, polypeptides, rifamycins, sulfonamides, streptogramins (such as quinupristin and dalfopristin) and tetracyclines

[0030] As used herein, the term “aminoglycosides” refers to a class of antibiotics, which irreversibly inhibits proteins synthesis by binding to 30S-subunit ribosomal proteins, eventually leading to cell death. They (1) interfere with the initiation complex of peptide formation, (2) causemisreading of mRNA, and (3) breakup polysomes into nonfunctional monosomes. Aminoglycosides include streptomycin, neomycin, kanamycin, amikacin, gentamicin, tobramycin, netilmicin, and plazomicin. Spectinomycin is chemically related to aminoglycosides and works in a similar way, however it is not available in the United States. 2. Overview A. Plague by Y. pestis infection A.1. Introduction

[0031] The only vaccine for plague previously licensed in the United States was the immunogenic Plague Vaccine U.S.P, commonly known as the Cutter vaccine. It contained a formalin-killed suspension of virulent plague bacilli. It is no longer available but had been administered routinely to military personnel posted in Vietnam and certain other individuals, such as field personnel working in plague endemic areas and laboratory personnel working with Y. pestis (Meyer 1970). Although it was effective in reducing the occurrence of bubonic disease, as evidenced by the low incidence of plague in U.S. personnel situated in Vietnam, laboratory findings suggested that this vaccine might not protect against pneumonic plague (Sun 2016, Verma and Tuteja 2016).

[0032] Also, the major protective antigen in these vaccines was fraction 1 (F1), a surface capsule composed of the Caf1 protein encoded by the caf1 operon on plasmid pFra (pMT1). Such vaccines do not readily protect against genetically engineered or naturally occurring F1-negative strains, which can be highly virulent despite the absence of capsule (Andrews et al. 1999, Welkos et al. 1995, Worsham et al. 1995). A more recent human plague vaccine candidate is rF1V, a protein fusion of the strongly immunogenic F1 and LcrV (referred to as V), the low calcium response virulence protein. V is an essential anti-host virulence factor which is required for type III secretion system (T3SS)-mediated translocation of the toxic Yersinia outer protein effectors (Yops) into host cells. V also stimulates production of immunosuppressive cytokines (Heath et al. 1998, Mueller et al. 2005, Pettersson et al. 1999). The rF1V vaccine was shown to be efficacious in mice and some, but not all, species of nonhuman primates (Adamovicz and Worsham 2012, Mizel et al. 2009, Pitt 2004, Powell et al. 2005, Quenee et al. 2011, Verma and Tuteja 2016, Williamson and Oyston 2013). Furthermore, the protection afforded by rF1V againstF1-negative strains of Y. pestis relies solely on the V antigen component. Since there is evidence for V heterogeneity within Yersinia species (Anisimov et al. 2010, Daniel et al. 2019, Miller et al. 2012, Roggenkamp et al. 1997), the potential exists that naturally occurring or engineered strains harboring altered V antigens could overcome rF1V induced immunity (Verma and Tuteja 2016).

[0033] Thus, a more effective plague vaccine that can induce strong antibody and cell-mediated immune responses in various animal models of plague is needed. Recent candidates have involved various platforms, e.g., attenuated and recombinant Yersinia strains, live recombinant viral and bacterial vectors constructed in adenovirus, Salmonella, Yersinia pseudotuberculosis, or other microbes, defined antigen subunit vaccines, outer membrane vesicles, DNA, mRNA, and nanoparticle constructs, as reviewed in detail elsewhere (Aftalion et al.2023, Feodorova and Motin 2012, Kon et al. 2023, Rosario-Acevedo et al. 2021, Rosenzweig et al. 2021, Sun 2016, Verma and Tuteja 2016, Wang, et al.2013).

[0034] While a number of vaccine candidates have induced significant protection against defined wild-type strains of virulent Y. pestis in animal models, their optimal vaccination schedule and immune response, duration of protection, breadth of coverage, as well as the efficacy in non-human primates and human vaccinees, are inadequately understood. A.2. Embodiments of the invention

[0035] The vaccine efforts in this disclosure are focused on the modification and optimization of the candidate live attenuated vaccines (LAVs) and defined protein subunit vaccines (Bozue et al. 2012, Swietnicki et al. 2011). The LAV candidates evaluated in this study are highly attenuated due to a deletion in the yscN gene, or of the pigmentation locus (pgm) accompanied by the curing of the pPst plasmid. The yscN gene encodes for an ATPase which provides energy to the T3SS and is necessary for Yop effector translocation. It was previously demonstrated that this mutant strain does not secrete LcrV into culture supernatant in vitro but a small amount of LcrV can be found associated with whole-cell extracts in vitro using immunoassays (Bozue et al. 2012). The pgm mutation is a 102 kB deletion of the entire pgm locus that results in iron acquisition and storage deficiency (Fetherston et al. 1992, Pendrak and Perry 1991, Perry et al. 1990). The pPst plasmid encodes the plasminogen activator enzyme that activates host fibrinolysis and permits bacterial systemic dissemination (Ferber and Brubaker 1979, McDonough and Falkow 1989, Sodeinde and Goguen 1988).A.3. Summary of the results

[0036] Here, homologous and heterologous vaccination schemes were explored utilizing both the yscN- or pgm-pPst- mutants as standalone or paired with the rV or rF1V subunit vaccines in the sensitive BALB / c mouse model of protection against aerosol challenge with a F1 capsule-negative Y. pestis strain. Optimal protection against challenge with nonencapsulated Y. pestis was achieved by heterologous vaccination with a live strain and a protein subunit vaccine. In addition, here studies for a novel intervention strategy layering vaccination and post-exposure antibiotic treatment were initiated. Disease outcome improved and a delayed initiation of antibiotics was still protective when vaccination was integrated with post-exposure antibiotic treatment. The layering of medical countermeasures will expand the treatment options available for plague and potentially allow for vaccine and / or antibiotic dose-sparing. (a) F1-associated virulence of Y. pestis

[0037] Most wild-type strains of Y. pestis produce an outer capsule composed of a polymer of fraction 1 protein (F1) encoded by the caf1 operon of the pMT1 plasmid. F1 is a highly immunogenic and protective virulence factor which appears to have a role in preventing phagocytosis. However, the primary Y. pestis virulence factors that block uptake by phagocytosis are those encoded by the Type III secretion system (T3SS) (Du et al. 2002). In addition, strains with mutations preventing F1 synthesis frequently retain virulence in various mammalian models of plague, especially in mouse and guinea pig pneumonic plague models (Biryukov, et al. 2021, Davis et al.1996, Drozdov et al.1995, Du and Wang 2016, Friedlander et al. 1995, Welkos, et al. 1995, Worsham, et al.1995).

[0038] Nevertheless, F1-deficient strains are not as virulent as F1+ in certain situations, and their virulence may vary depending on the Y. pestis strain, exposure dose and route, and animal host, i.e., strain, sex, or source (Donavan et al. 1961, Friedlander, et al. 1995, Sebbane, et al. 2009, Weening et al.2011, Welkos et al.2004, Williams and Cavanaugh 1984). The F1 antigen appears to contribute to Y. pestis virulence in some mammalian models of bubonic and sometimes pneumonic plague (Donavan, et al. 1961, Du, et al. 2002, Du and Wang 2016, Samoilova, et al. 1996, Sha, et al. 2011, Weening, et al.2011, Williams and Cavanaugh 1984). (b) Heterologous vaccination with LAV and protein subunit vaccines of Y. pestis with CpG

[0039] In previous studies, the protective efficacy of several live attenuated mutant strains derived from Y. pestis strains CO92 or KIM6+ against bubonic and aerosol challenges of BALB / c mice with the wild-type encapsulated CO92 strain was characterized. Three of the LAV strains were confirmed to be safe in high doses (Cote, et al. 2021); and one dose of either F1+CO92 mutant (the ΔyscN or pgm- pPst- strain) protected BALB / c mice against infection by subcutaneous or inhalational routes to Y. pestis CO92. Full protection against CO92 in this model required induction of an immune response to F1 (Cote, et al. 2021). Two vaccines (CO92 ΔyscN, alone or combined with C12 ΔyscN) were down-selected for further analyses of protection against other virulent strains and especially those deficient in capsule synthesis. The ΔyscN strains do not secrete the V antigen due to the disrupted T3SS but do retain some V protein associated with the bacterial cells (Bozue et al.2012), and, in the case of the C12 derived vaccine strain, produces no F1. Thus, they allowed us to investigate protection afforded by potentially novel antigens, such as non-capsule surface antigens displayed by the C12 ΔyscN, e.g., Pla, Ail, LPS (Wang et al.2020).

[0040] The current work extended these studies by characterizing protection afforded by these LAVs, with or without the defined rF1V subunit vaccine, against challenge with the nonencapsulated and vaccine-resistant C12 strain of Y. pestis. It was found that the most protective vaccination scheme, as tested under various conditions and in both bubonic and pneumonic plague models, was heterologous vaccination with a live vaccine (Combo LAV composed of the C12 and CO92 ΔyscN mutants, or the CO92 pgm- pPst- strain) and a separate dose of a protein subunit vaccine. Although one or two identical doses of either ΔyscN mutant alone, or combined, provided significant protection against bubonic plague, they were poorly protective against pneumonic challenge (Figures 1 and 2). With the inclusion of a subunit rF1V vaccine, the heterologous vaccination approach was optimally protective against aerosol challenge regardless of changes in vaccination schedule and challenge conditions. The latter included the spacing between prime vaccine doses and subsequent booster dose(s) which was predicted to optimize antibody response (Castiglione et al. 2012). For instance, heterologous vaccination increased the survival rate or extended the survival time to a greater extent than did two doses of Combo LAV when mice were exposed to a significantly elevated C12 challenge. Additionally, in a heterologous scenario (Figures 3 and 4), a different LAV, i.e., the CO92 pgm- pPst- strain was as efficacious as the Combo LAV, when paired with rF1V booster. This situation requires further examination, but it potentially affords flexibility in selection of vaccine components.

[0041] In efforts to improve the rF1V vaccine, Amemiya et al. and Biryukov et al. showed that the addition of TLR agonist CpG2006 to rF1V augmented vaccine efficacy and immunogenicity (Amemiya, et al. 2009, Biryukov, et al. 2021). A similarly protective role of CpG in mice vaccinated with LAVs or protein subunit combination vaccines was examined (Figure 3). Our results suggested that CpG enhanced protection afforded by the heterologous Combo LAV and rF1V vaccine against Y. pestis C12 aerosol challenge (Figure 3B and C). Although, CpG might not be required for maximal protection against bubonic plague model described here, there may be scenarios where the addition of CpG could improve disease outcome (Figure 3A). (c) Quantity assessment of bacterial loads in tissues

[0042] The impact of vaccination on accelerating bacterial clearance or inhibition of dissemination in vivo was also determined by quantitatively assessing bacterial loads in tissues three days after the aerosol challenge. Organ and blood levels of Y. pestis C12 in mice vaccinated with the heterologous vaccine (Combo LAV prime and rF1V + CpG boost) were lowest compared to those in the other control and vaccinated groups. There appeared to be a relationship between CFU and protection, i.e., lower CFU load in the lungs at day 3 was associated with vaccines protective efficacy, albeit a statistical correlation was precluded due to low numbers of samples. Furthermore, survival rates of the two groups vaccinated with Combo LAV plus rF1V (+ / - CpG) were not significantly different (90% and 50%, respectively), yet the mean TTM post-challenge of the group receiving the CpG immunostimulant was increased significantly compared to that of most other groups. (d) Immune responses

[0043] Also, the associations between the extent of protection and the immune responses elicited by the vaccine was evaluated. The heterologous vaccination groups with the highest survival rates had the greatest serum titers to rF1V, rV, and to the CO92 whole cell antigens, and their splenocytes secreted higher levels of IFN-γ after rF1V stimulation in ELISpot assays. These findings confirm the strong immunogenicity of rF1V in BALB / c mice as demonstrated in our initial vaccine studies using the live attenuated plague strains (Cote, et al. 2021); full protection against F1+Y. pestis CO92 required induction of a strong humoral anti-F1 response. The consistently greater protection against aerosol exposure to Y. pestis C12 of the heterologous, rF1V- containing vaccines compared to homologous single or Combo LAVs (Figure 3B-C and Figure4) supports a role for rF1V-associated immune responses in vaccine efficacy against C12. Importantly, although the ∆yscN based vaccines (and in general most live vaccines designed to ameliorate plague) do not generate a robust antibody response against secreted V antigen, there appears to be enough V protein associated with the cell that can act as a prime vaccination against this antigen. Once boosted with rF1V (+ / - CpG) there is an appreciable increase in anti-rV titers. These titers are greater than those observed in mice receiving a single dose of rF1V (+ / - CpG), but this increase is not statistically significant. This lack of statistical significance in anti-rV titers among groups 3-6 (Table 2 and Figure 3B) is an important point of discussion as it implies that there are other immunogens that were provided by the LAV prime vaccination that are ultimately increasing vaccine efficacy in the mice (survival of group 4 compared to groups 5 and 6 in Figure 3B) and protection could not be correlated with anti-rV titers alone. Accordingly, the immune response generated by our LAVs in the mouse model was explored.

[0044] In a previous study with our LAVs (alone), cell-mediated immunity involving elevated Th17- and Th2- associated cytokines was associated with protection against the encapsulated CO92 strain (Cote, et al. 2021). The findings were obtained using splenocytes collected before the aerosol challenge. Although an association with protection against the C12 strain was not clearly observed with stimulated splenocytes collected pre-exposure from the extended boost time study (Figure 5), none of the vaccines included CpG and none were significantly protective in this high dose challenge study. In contrast, with some exceptions, protection was associated with overall down-regulation of cytokines in tissue extracts obtained after challenge with C12 in heterologously vaccinated mice relative to KPhos vaccinated mice. This controlled cytokine response potentially mitigates excessive Th17 and neutrophil migration (CXCL1, CXCL2) and activation (G-CSF, IL-17A, IL-1B, IL-6, IL-22) which may contribute to reduced inflammation related destruction of lung tissue. In addition, inability to control the infection may predispose mice to excessive alveolar macrophage and eosinophil activation and recruitment via colony- stimulating factors (G-CSF, M-CSF, GM-CSF). Excessive GM-CSF production by lung epithelium may also be a direct correlate of lung damage and act as a marker of futile effort to repair and restore epithelial barrier function (Chen et al.2016, Rosler and Herold 2016, Standiford et al.2012, Trapnell and Whitsett 2002).

[0045] Thus, it was intended to investigate a protective role for vaccine-associated dampening of the cytokine storm induced by exposure to virulent F1+and F1- Y. pestis strains. It is predicted that a fine-tuned modulation between a protective and damaging extent of cytokine expression is required for optimal vaccine efficacy. Cytokine expression which was either excessive or overly suppressed was associated with poor protection, as suggested for example by the cytokine profiles of restimulated splenocytes described in Table 6. Of the two most protective vaccine groups (CO92 pgm- pPst- + rF1V and CO92 ∆yscN + rF1V) that afforded the same level of protection, there appeared to be a distinct immune response that induced a relatively low anti-TS C12 antibody response in both groups. Controlled upregulation of IFN-γ, IL-17A, IL-2 and IL-4 in the CO92 pgm- pPst- + rF1V vaccinated mice appeared to correlate with protection. While controlled upregulation of Th2- (IL-4, IL-5, IL-13), Th17 (IL-17A, IL-22) along with the pleiotropic IL-2 and IL3 cytokines was associated with CO92 ∆yscN + rF1V vaccinated mice. It is important to note that our cytokine data are collected from a single time-point and these expression levels and their correlation with protection may differ throughout the course of infection. However, the anti- rF1V and anti-CO92 antibody responses were highest in the two most protective vaccines and maybe used as correlates of protection. The cytokine response against TS C12 was much more pronounced with vaccines that included CO92 ∆yscN relative to CO92 pgm- pPst- and yet the level of protection was the same. Following rF1V stimulation the number of IFN-γ secreting splenocytes after 24 h was relatively high in all rF1V vaccine groups but after 48 h the levels of IFN-γ were substantially higher in all CO92 ∆yscN vaccine groups, especially in groups that were also administered rF1V. The short half-life of IFN-γ maybe an indicator of this large discrepancy between the two timepoints for the different vaccine formulations, such that CO92 pgm- pPst- + rF1V vaccine group produces a strong yet transient response that recedes by 48 h relative to the CO92 ∆yscN + rF1V that appears to be longer in duration and more intense (Lortat-Jacob et al. 1996). Furthermore, TS C12 antigen stimulated a greater IFN-γ response in splenocytes from mouse groups vaccinated with CO92 ∆yscN mutant (+ / - rF1V: groups 9 – 12) and not CO92 pgm- pPst- (+ / - rF1V: groups 2 - 7). (e) Advantages of heterologous vaccination of Y. pestis

[0046] In addition to the enhanced efficacy and potential modularity of our combination vaccine approach against both F1+and F1- Y. pestis strains, an advantage inherent with the ΔyscN mutantLAVs is their inclusion of antigens some of which are not expressed in other live vaccines, i.e., potential antigens encoded by the chromosomal 102 Kb pgm locus, by other Y. pestis chromosomal genes, and / or by plasmids pMT1 (pFra) and pPcP1 (pPst). However, the CO92 pgm- pPst- strain was also highly protective, possibly due in part to an immune response to the secreted T3SS YOPs.

[0047] The V antigen is an essential component of the Y. pestis T3SS and an indispensable virulence factor of Y. pestis; thus, it is an obvious target for countermeasure development. However, although V-specific immunity has been correlated with protection, the adaptive immune responses to V are complicated and may not be essential to an effective vaccine. In our previous work (Cote, et al.2021), the ∆yscN vaccines did not induce a significant anti-V antibody response, as expected due to the requirement of the YscN ATPase for a functional T3SS (Bozue, et al.2012). In this current work was noted a significant increase in anti-rV titers when the F1-V subunit vaccine was used as a booster. However, the lack of a robust anti-V response in animals or humans vaccinated with live attenuated F1+and F1- Y. pestis strains has been documented repeatedly (Bozue, et al.2012, , Cote, et al.2021, Demeure et al.2019, Feodorova et al.2020, Qiu et al.2010, Quenee, et al. 2008, Sun et al. 2014, Williamson et al. 1995). The basis of this observation has not been fully discerned but may be related to the V-producing strain of live vaccine and possibly the animal host. The potential role of the immune-modulatory activity of V in the suppression of the host immune responses in context of LAVs remains to be clarified However, our data suggest that a LAV followed by a rF1V booster can overcome the low V titers typically observed with LAVs and this heterologous strategy may represent a superior vaccination strategy. (f) Combination of pre-exposure vaccination and post-exposure antibiotic treatment

[0048] To maximize protection against infection, the efficacies of pre-exposure vaccination and post-exposure antibiotic treatment were also compared alone and combined, against inhalational challenge with Y. pestis C12 strains. A layered defense strategy employing vaccination with the ∆yscN mutant of the wild-type or F1-deficient strain, separately or combined, and treatment with streptomycin provided optimal protection and was clearly synergistic (Figures 6 and 7). These promising results predict that the “pre-post” strategy will extend the time post-exposure when antibiotic administration must be initiated to be successful. Our ongoing studies focus on assessing the limits of this strategy as well as its impact on vaccination with the heterologous LAV / subunit constructs.(g) Advantages of combining vaccination strategies with antibiotic regimens

[0049] The layered countermeasure strategy offers several attractive advantages to single medical countermeasure strategies. As observed in animal studies, the efficacy of antibiotic treatment of Warfighters exposed to a bolus of aerosolized Y. pestis is predicted to be enhanced in prophylactically vaccinated compared to unvaccinated individuals. Vaccination is also expected to extend the window of time post-exposure during which antibiotics are effective, i.e., before reaching the “point of no return.” Concomitantly, for vaccinees with waning immunity, post- exposure antibiotic treatment could potentially enhance the protective effects of the vaccine. This might be especially important in a biothreat scenario where the limited time before potential exposure allowed only a single administration of a plague vaccine. Finally, strategies employing layered, or combination countermeasures afford vaccine antigen / antibiotic dose sparing and potentially allows use of second-line antibiotics as needed, e.g., in situations involving naturally or engineered antibiotic-resistant challenge strains (Klimko et al.2022b, Zauberman et al.2019).

[0050] Further efforts are being pursued to optimize the vaccination and integrated prophylactic / post-exposure countermeasure strategies. These include a determination of the latest time post-exposure to aerosolized bacteria when antibiotic treatment can rescue the mice and the lowest effective dose of antibiotic; the optimal heterologous vaccine composition, order of delivery, and schedule; the most synergistic antibiotic(s) for various vaccine strategies; and, in the context of single vaccination, the longest interval between vaccination and challenge after which the antibiotic treatment remains more effective than in unvaccinated treated individuals. BALB / c mice are an appropriate early model for both pathogenesis research and medical countermeasure development; however, due to differences in LD50 estimations and other inherent differences their recapitulation of human disease remains equivocal and thus, follow on work will also be required in a non-human primate model of pneumonic plague. Additionally, plague vaccine candidates have generally been evaluated using only one virulent strain. However, many distinct strains of virulent Y. pestis have been isolated from multiple geographical sources (Vogler, et al.2016), and analyses of vaccines for their performance against such strains are needed. B. Melioidosis by Burkholderia pseudomallei B.1. Introduction

[0051] Also, such combination countermeasure strategies are not restricted to one pathogen or model. The potential broad applicability of a similar protection strategy was also exemplified in the following with Burkholderia pseudomallei, the etiologic agent of melioidosis (Klimko et al. 2022a). B. pseudomallei is intrinsically resistant to many commonly used antibiotics, and infections with it are notoriously difficult to eradicate. Effective antibiotic treatment regimens are complex and of long duration.

[0052] The current antibiotic regimen recommended by the U.S. Centers for Disease Control and Prevention based upon the Darwin melioidosis treatment guidelines consists of at least 2 weeks of intravenous antibiotics (ceftazidime administered every 6–8 h or meropenem administered every 8 h) followed by 3–6 months of oral antimicrobial therapy (co-trimoxazole taken every 12 h or amoxicillin / clavulanic acid taken every 8 h) (Pitman et al., 2015; Sullivan et al., 2020). These guidelines also recommend intravenous antibiotic administration for up to 8 weeks depending upon the extent of infection. For example, Sullivan et al. (2020) recommend 2 weeks of intravenous treatment if a patient presents with unilobar pneumonia but not bacteremia. However, 4 weeks of intravenous antibiotic treatment is recommended for patients exhibiting both bacteremia and pneumonia. Six weeks and 8 weeks of intravenous antibiotic treatment is recommended for cases involving osteomyelitis or central nervous system involvement, respectively. It is important to note that at times abiding by these guidelines may be difficult or impossible due to different medical standards and economic considerations in many areas of the world where B. pseudomallei is endemic. The stringency of the melioidosis treatment guidelines underscores the complexity of the bacterial pathogenesis, the non-uniformity of disease progression, and the difficulty associated with disease eradication.

[0053] Additionally, the clinical presentation of B. pseudomallei infection is extremely diverse and can include abscess / pyogranulomas in multiple organs, osteomyelitis, and primary or secondary pneumonia. Mouse models have partially recapitulated some of the diverse clinical manifestations of human melioidosis. BALB / c mice are considered a model for acute melioidosis, while C57BL / 6 mice are less susceptible to B. pseudomallei and tend to develop characteristics that are like chronic forms of melioidosis (Leakey et al., 1998;).

[0054] In the absence of distinct clinical manifestations, many cases of infection with B. pseudomallei are difficult to diagnose and the disease is often referred to as the “great mimicker.” Patients in endemic areas can be infected multiple times (or by multiple strains in rare instances) and individuals traveling to endemic areas have been documented to have no disease presentation until years or decades after the primary encounter with the bacterium (Mays and Ricketts, 1975;; Ngauy et al., 2005; Limmathurotsakul et al., 2007; Pitt et al., 2007). Further complicating treatment plans are the known risk factors for susceptibility to B. pseudomallei infection (e.g., diabetes and heavy alcohol consumption) and the disparate quality of medical care available in some geographic locations where B. pseudomallei is known to be or suspected to be endemic (Hassan et al., 2010).

[0055] Significant effort has been spent on the identification, characterization, and optimization of protective vaccine antigens that will prevent or ameliorate B. pseudomallei infections and melioidosis disease. Various vaccine strategies including live attenuated vaccine strains, OMVs, and protein subunit / polysaccharide conjugate combinations have produced robust immune responses that have been protective in both mouse and non-human primate models of melioidosis (Atkins et al., 2002; Amemiya et al., 2019; Biryukov et al., 2022). However, even with these successes in vaccine strategies, there remains the possibility that survivors of the acute phase of the infection could continue to harbor the bacteria that could reemerge later.

[0056] There has been equally impressive progress toward the understanding and evaluation of antibiotic regimens that are protective.However, diagnosed B. pseudomallei infections continue to warrant extended intravenous antibiotic regimen followed by oral antibiotics for months. B.2. Embodiments of the invention

[0057] Consequently, it is believed that B. pseudomallei infections will be optimally treated with layered and integrated medical countermeasures.

[0058] In this disclosure, co-trimoxazole was selected as the antibiotic paired with our vaccines in C57BL / 6 mice. This antibiotic has been previously shown to be successful in the more susceptible BALB / c mouse models of melioidosis (Ulett et al., 2003; Sivalingam et al., 2008; Barnes et al., 2013, 2017) and continues to be an important component for patient treatment plans (Sullivan et al., 2019, 2020).

[0059] Here is reported the improvement of disease outcome observed when suboptimal antibiotic regimens were used in combination with current experimental vaccines. A detailed description of the vaccine candidates used in this report and their resulting immune responses is described by Biryukov et al. (2022). B.3. Summary of the results

[0060] The data described here provide proof-of-concept that partially protective B. pseudomallei vaccination strategies can synergize with suboptimal antibiotic regimens resulting in nearly 100% survival in a C57BL / 6 mouse model of melioidosis. In Examples, several studies are detailed that demonstrate the utility of a combination of medical countermeasures in the C57BL / 6 mouse model of inhalational B. pseudomallei infection. The antibiotic co-trimoxazole treatment significantly augmented the protection afforded by vaccines of recombinant protein subunit conjugates or a LAV strain for the prevention of melioidosis. (a) Combination of pre-exposure vaccination and post-exposure antibiotic treatment

[0061] In the C57BL / 6 mouse model of B. pseudomallei infection, combining vaccination strategies with antibiotic regimens demonstrated a clear advantage over mice that were only vaccinated or mice that only received co-trimoxazole. In this disclosure, co-trimoxazole was initiated, given every 12 h, starting at approximately 21, 45, or 69 h post exposure to aerosolized B. pseudomallei for 21 days (Table 1). Treatment with co-trimoxazole alone at 69 h post-infection only protected 30% of the animals. However, if vaccinated mice were treated with co-trimoxazole starting at 69 h post-infection, 90% of the mice were protected through day 82.

[0062] To test the limits of this combination therapy and to examine if the protection afforded by vaccines could allow for a shortened antibiotic treatment course, the co-trimoxazole treatment of 21 days was reduced to 7 days and the treatment was initiated at time points ranging from approximately 9–69 h post-infection. In the absence of vaccination, no mice survived the infection after the shortened course of co-trimoxazole when the treatment was initiated 69 h post-exposure (Table 2), however, vaccinated mice receiving co-trimoxazole treatment starting at 69 h post- exposure led to 80% or greater survival through day 70.

[0063] Finally, a 21-day co-trimoxazole regimen but with a delayed initiation at either approximately 93 h or 117 h post-infection was tested. As described in Figure 2 and Table 3, using this antibiotic regimen and under these experimental conditions, uniform lethality occurred by day 5 post-exposure. However, vaccinated mice receiving these delayed antibiotic treatments exhibited 90% or greater survival rates and most of the mice surviving through day 85 had no evidence of residual bacteria in lungs or spleens. It is important to note that the animals were sampled that survived through the end of study for bacterial burden, and that a serial sampling experiment was not performed. Additionally, it is imperative to reiterate that spleens and lungs were sampled and that the animals were examined for gross signs of pyogranuloma formation in other tissues. Due to the significant heterogeneity of clinical presentation of melioidosis and the propensity of B. pseudomallei to enter a persister or non-culturable state (Pumpuang et al., 2011; Li et al., 2014; Lewis and Torres, 2016), complete sterility in the surviving animals could not be verified. (b) Advantages of combining vaccination strategies with antibiotic regimens

[0064] Once proven safe and immunogenic, licensed B. pseudomallei vaccines should be encouraged in endemic areas with high clinical incidence. These vaccination strategies will, at the very least, ameliorate the acute form of the disease, but possibly and perhaps more importantly allow for shorter courses of effective antibiotics or provide larger windows of opportunity to initiate effective antibiotic therapy.

[0065] In addition to improved patient outcomes, the combinatorial strategies of vaccines and antibiotics offer the secondary advantage of potentially reducing the emergence of antimicrobial resistant isolates. The data generated in the disclosed mouse models of inhalational melioidosis suggest that vaccinated patients would respond better to antibiotic therapy. Shortened antibiotic durations in vaccinated individuals may be protective and could lead to reduced numbers of antibiotic resistant bacterial isolates (B. pseudomallei or normal flora) arising after exposure to prolonged or less-effective antibiotic regimens (Sunde et al., 1998; Vatopoulos et al., 1998; Rice, 2008; Fair and Tor, 2014; Ventola, 2015a,b). Even if a vaccine was partially protective, vaccinated individuals could have lessened clinical symptoms or fewer secondary bacterial infections that would have likely resulted in unnecessary antibiotic regimens (Lewnard et al., 2020; Rodgers et al., 2021). Thus, patient outcomes should be considerably improved if individuals are immunized,and appropriate antibiotic therapy is initiated as soon as possible after possible exposure to B. pseudomallei. C. Methods of Use

[0066] This invention provides layered treatment methods for refractory infectious diseases such as plague, in particular pneumonic plague, caused by Y. pestis, and melioidosis caused by B. pseudomallei, using strategies of pre-infection vaccination and post-infection antibiotic therapy.

[0067] First, for prophylactic vaccination, heterologous vaccination strategies are provided using combination of live attenuated bacteria and their recombinant protein subunits, which can be administered to a subject as a vaccine composition. Attenuated bacteria and their protein subunits work as antigens to elicit immune responses. Immune responses induced by a live vaccine are similar to the natural infection in that the immune responses progress through the innate and antigen-specific immune responses so that humoral, cellular, and memory immune responses are developed. Immunity is generally long-lasting so that just 1 or 2 doses of most live vaccines can give a lifetime of protection against a microbe and the disease it causes. However, live vaccines have problems: the vaccines may still be dangerous for immune suppressed people, and the vaccine may become virulent again. To reduce such risk and still induce strong immunogenicity, a heterologous vaccination can be recommended, which is a combination of a LAV and killed / inactivated bacterial antigen or protein subunit antigens, which usually elicit antibody- mediated humoral immune response without inducing innate immune response, but without the risk of infection by the vaccine. These vaccines are usually administered with adjuvants such as aluminum salts or CpG that boosts their immunogenicity.

[0068] Although in this disclosure, certain strains of bacteria and purified recombinant protein subunits are presented as examples, the scope of the invention may extend to include any components of the infectious agents, including toxoids or polysaccharides, as well as DNA (e.g., plasmid or viral vector) or mRNA vaccines producing the recombinant protein subunits inside the subject’s body. In addition, the bacterial strains that can be used for this layered treatment of infectious diseases may comprise other non-virulent, attenuated or inactivated strains of Y. pestis or B. pseudomallei than the strains used in this disclosure. Inactivated bacterial vaccine can be produced by chemicals (e.g., formalin), irradiation, or heat treatment of bacteria.

[0069] In some embodiments, subjects are vaccinated with an LAV or LAVs of Y. pestis or B. pseudomallei once or twice, but if necessary they may have more booster vaccination. The bacterial strain in the first vaccine composition and that in the second vaccine composition may be the same or different. In some embodiments, the vaccine composition comprises only a LAV or LAVs. In other embodiments, the vaccine composition comprises a LAV and a subunit. In other embodiments, the vaccine composition comprises only protein subunit or subunits with or without CpG. In other embodiments, the first vaccine composition comprises a LAV / LAVs, and the second vaccine composition comprises a subunit / subunits with or without CpG. In other embodiments, the first vaccine composition comprises a subunit / protein subunits with or without CpG, and the second vaccine composition comprises a LAV / LAVs. In other embodiments, the first vaccine composition comprises CpG, and the second vaccine composition comprises a subunit / subunits, and / or a LAV / LAVs. In other embodiments, the vaccine composition comprising a LAV / LAVs and / or a subunit / subunits can be administered more than two times, in particular three times, or if necessary more than three times, in particular 4 or 5 times.

[0070] Second, for the treatment of bacterial infection after exposure to the pathogen, a therapeutically effective amount of an antibiotic / antibiotics is applied topically to the infection site or systemically, e.g., i.v. or i.m. injection (streptomycin, co-trimoxazole) or oral uptake (co- trimoxazole (trimethoprim / sulfamethoxazole)). Streptomycin is an example of aminoglycoside antibiotics, and thus other members of the aminoglycoside antibiotics can be considered for the post-infection treatment of Y. pestis. Trimethoprim, a trimethoxybenzylpyrimidine, is a selective inhibitor of bacterial dihydrofolic acid reductase, and it can be replaced with another benzylpyridine, pyrimethamine, to be used in combination with sulfamethoxazole, which can be administered through intravenous injection or oral tablet for the treatment of B. pseudomallei.

[0071] In a specific embodiment, provided is a method of prophylactic or therapeutic treatment of bubonic- and / or pneumonic plague caused by Yersinia pestis infection in a subject; the method comprising steps of: -before infection, optionally at least about 28 (±3) days before infection, administering a therapeutically effective amount of a vaccine composition to the subject comprising at least one live attenuated vaccine (LAV) selected from Y. pestis mutant C12 ∆yscN, CO92 ∆yscN, or CO92 pgm- pPst-, in particular combination of C12 ∆yscN and CO92 ∆yscN, or CO92 pgm- pPst-, and-before 48-60 hours after infection, administering to the subject an antibiotic treatment to an effective amount of an antibiotic or antibiotics. According to another specific embodiment, provided is a method of prophylactic or therapeutic treatment of bubonic- and / or pneumonic plague caused by Yersinia pestis infection in a subject; the method comprising steps of, -administering to the subject a therapeutically effective amount of a first vaccine composition comprising a recombinant protein subunit of rF1V or rV with or without CpG, combination of rF1V and rV with or without CpG, or at least one LAV selected from Y. pestis mutant C12 ∆yscN, CO92 ∆yscN, or CO92 pgm- pPst-, in particular combination of CO92 ∆yscN and C12 ∆yscN, or CO92 pgm- pPst-, and -between 20-90 days, in particular between 20-46 days, more in particular between 20-28 days, after the first vaccination, and before infection, optionally at least about 28 (±3) days before infection, administering to the subject a therapeutically effective amount of a second (a first booster) vaccine composition comprising CpG, a recombinant protein subunit of rF1V or rV with or without CpG, combination of rF1V and rV with or without CpG, and / or at least one LAV selected from Y. pestis mutant C12 ∆yscN, CO92 ∆yscN, or CO92 pgm- pPst-, wherein if the first vaccine composition comprises only subunit(s) of rF1V and / or rV, the second vaccine composition comprises at least one LAV selected from Y. pestis mutant C12 ∆yscN, CO92 ∆yscN, or CO92 pgm- pPst-, and -optionally, before 48-60 hours after infection, administering to the subject an antibiotic treatment with an effective amount of an antibiotic or antibiotics. In yet another embodiment, provided is a method of prophylactic or therapeutic treatment of bubonic and / or pneumonic plague caused by Yersinia pestis infection in a subject; the method comprising steps of, -administering to the subject a therapeutically effective amount of a first vaccine composition comprising a recombinant protein subunit of rF1V or rV with or without CpG, combination of rF1V and rV with or without CpG, and / or at least one LAV selected from Y. pestismutant C12 ∆yscN, CO92 ∆yscN, or CO92 pgm- pPst-, in particular combination of CO92 ∆yscN and C12 ∆yscN, or CO92 pgm- pPst-, -between 20-90 days, in particular between 20-46 days, more in particular between 20-28 days, after the first vaccination, administering to the subject a therapeutically effective amount of a second (a first booster) vaccine composition comprising CpG, a recombinant protein subunit of rF1V or rV with or without CpG, combination of rF1V and rV with or without CpG, or at least one LAV selected from Y. pestis mutant C12 ∆yscN, CO92 ∆yscN, or CO92 pgm- pPst-, and -between 20-90 days, in particular between 20-46 days, more in particular between 20-28 days, after the second vaccination, and before infection, optionally at least about 28 (±3) days before infection, administering to the subject a therapeutically effective amount of a third (a second booster) vaccine composition comprising CpG, a recombinant protein subunit of rF1V or rV with or without CpG, combination of rF1V and rV with or without CpG, or at least one LAV selected from Y. pestis mutant C12 ∆yscN, CO92 ∆yscN, or CO92 pgm- pPst-, wherein if the first and second vaccine compositions do not comprise a LAV or LAVs, the third composition comprises at least one LAV selected from Y. pestis mutant C12 ∆yscN, CO92 ∆yscN, or CO92 pgm- pPst-, and, -optionally, before 48-60 hours after infection, administering to the subject an antibiotic treatment with an effective amount of an antibiotic or antibiotics.

[0072] One example of an antibiotic useful in accord with the teachings here includes, but is not limited to, is an aminoglycoside antibiotic or combination thereof, in particular streptomycin. Typically, a dose of antibiotic includes, but is not limited to 10-100 mg / kg / dose, in particular 20- 40 mg / kg / dose. Typically, the antibiotic is administered through intramuscular or intravenous injection every 6 hours for 5-30 days.

[0073] Concerning dosing of a vaccine, in one example, a single dose volume of the vaccine composition is 0.1-0.5 mL, and is formulated for administration through a subcutaneous, intradermal or intramuscular route.

[0074] In one example of a therapeutically effective amount of a LAV or a total amount of LAVs in a vaccine composition, the amount is between 0.1 - 10.0 × 106-7CFU per kg body weight. Inan example of a therapeutically effective amount of a recombinant protein subunit rF1V or rV, or combination of both subunits in the vaccine composition, the amount is between 0.1-10 µg per kg body weight.

[0075] In one example concerning an amount of CpG administered, this may involve between 0.1-30 µg per kg body weight.

[0076] According to another example, provided is a method of prophylactic or therapeutic treatment of melioidosis caused by Burkholderia pseudomallei infection in a subject; the method comprising steps of: -before infection, optionally at least about 32 (±3) days before infection, administering to the subject a therapeutically effective amount of a live attenuated vaccine (LAV) composition of B. pseudomallei, 668 ΔilvI two times, optionally on day 0 and day 21-24, and -before 117 hours, in particular 69 hours, in more particular 45 hours, after infection, administering to the subject an antibiotic treatment with an effective amount of an antibiotic or antibiotics.

[0077] In a further embodiment, provided is a method of prophylactic or therapeutic treatment of melioidosis caused by Burkholderia pseudomallei infection in a subject; the method comprising steps of: -before infection, optionally at least about 32 (±3) days before infection, administering to the subject a therapeutically effective amount of recombinant protein subunit vaccine composition comprising AhpC and / or Hcp1 three times, optionally on day 0, day 21-24, and day 35-38; and before 117 hours, in particular 69 hours, in more particular 45 hours, after infection, administering to the subject an antibiotic treatment with an effective amount of an antibiotic or antibiotics.

[0078] In yet another embodiment, disclosed is a method of prophylactic or therapeutic treatment of melioidosis caused by Burkholderia pseudomallei infection in a subject; the method comprising steps of; -before infection, optionally at least about 32 (±3) days before infection, administering to the subject a first therapeutically effective amount of a live attenuated vaccine (LAV) composition of B. pseudomallei, 668 ΔilvI or recombinant protein subunit vaccine composition comprisingAhpC and / or Hcp1, administering to the subject a second therapeutically effective amount of a live attenuated vaccine (LAV) of B. pseudomallei, 668 ΔilvI or recombinant protein subunit vaccine composition comprising AhpC and / or Hcp1, before 90 days optionally before 60 days, more optionally before 30 days after the first vaccination, wherein the second vaccine composition comprises a LAV or protein subunit(s) not included in the first vaccine composition, and, optionally, -before 117 hours, in particular 69 hours, in more particular 45 hours, after infection, administering to the subject an antibiotic treatment with an effective amount of an antibiotic or antibiotics.

[0079] For the above methods directed to prophylactic or therapeutic treatment of melioidosis caused by Burkholderia pseudomallei, the recombinant protein subunit vaccine composition can be administered with adjuvant, which comprises capsular polysaccharides (CPSs) conjugated to Cross-Reactive-Material-197 (CRM197, a non-toxic mutant of diphtheria toxin as a carrier protein), CpG oligodeoxynucleotides, and / or an aluminum hydroxide wet gel suspension, wherein the content of the CPS conjugated to CRM197 is 0.1-10 μg per kg body weight, the content of CpG ODN is 0.1-20 μg per kg body weight and the content of the aluminum hydroxide wet gel suspension is 0.1-500 μg per kg body weight.

[0080] In one example related to prophylactic or therapeutic treatment of melioidosis caused by Burkholderia pseudomallei, the antibiotic used may be co-trimoxazole (trimethoprim / sulfamethoxazole). In a specific example, a dose of co-trimoxazole is trimethoprim 160 mg and sulfamethoxazole 800 mg, and wherein the co-trimoxazole is administered through intramuscular injection or oral tablet every 12 hours for 7-21 days. In another example, a single dose volume of the vaccine composition is 0.1-0.5 mL, and wherein the vaccine composition is administered through subcutaneous, intradermal or intramuscular route. In another example, a therapeutically effective amount of LAV in the first vaccine composition is between 0.1 - 10.0 × 106-7CFU per kg body weight, and a therapeutically effective amount of LAV in the second vaccine composition is between 0.1 - 10.0 × 106-7CFU per kg body weight. In another example, a therapeutically effective amount of LAV in the vaccine composition is between 0.1-10 µg per kg body weight. A therapeutically effective amount of a recombinant protein subunit AhpC and / or Hcp1, orcombination of both subunits in the vaccine compositions may be, but not limited to, between 0.1- 10 µg per kg body weight.

[0081] According to further embodiments, provided are method of prophylactic or therapeutic treatment of bubonic- and / or pneumonic plague caused by Yersinia pestis infection in a subject; the method comprising steps of, -administering to the subject a therapeutically effective amount of a first vaccine composition comprising a recombinant protein subunit of rF1V or rV with or without CpG, combination of rF1V and rV with or without CpG, or at least one LAV selected from Y. pestis mutant pgm-, pPst- Δcaf1 and / or pgm-, pPst-, ΔyopD / Δcaf1, -between 20-90 days, in particular between 20-46 days, more in particular between 20-28 days, after the first vaccination, and before infection, optionally at least about 28 (±3) days before infection, administering to the subject a therapeutically effective amount of a second (a first booster) vaccine composition comprising CpG, a recombinant protein subunit of rF1V or rV with or without CpG, combination of rF1V and rV with or without CpG, and / or at least one LAV selected from Y. pestis mutant pgm-, pPst-, Δcaf1 and / or pgm-, pPst-, ΔyopD / Δcaf1, wherein if the first vaccine composition comprises only subunit(s) of rF1V and / or rV, the second vaccine composition comprises at least one LAV selected from Y. pestis mutant pgm-, pPst- , Δcaf1 and / or pgm-, pPst-, ΔyopD / Δcaf1, and, optionally, -before 48-60 hours after infection, administering to the subject an antibiotic treatment with an effective amount of an antibiotic or antibiotics.

[0082] According to a further embodiment, provided is method of prophylactic or therapeutic treatment of bubonic and / or pneumonic plague caused by Yersinia pestis infection in a subject; the method comprising steps of, -administering to the subject a therapeutically effective amount of a first vaccine composition comprising a recombinant protein subunit of rF1V or rV with or without CpG, combination of rF1V and rV with or without CpG, and / or at least one LAV selected from Y. pestis mutant pgm-, pPst-, Δcaf1 and / or pgm-, pPst-, ΔyopD / Δcaf1,-between 20-90 days, in particular between 20-46 days, more in particular between 20-28 days, after the first vaccination, administering to the subject a therapeutically effective amount of a second (a first booster) vaccine composition comprising CpG, a recombinant protein subunit of rF1V or rV with or without CpG, combination of rF1V and rV with or without CpG, or at least one LAV selected from Y. pestis mutant pgm-, pPst-, Δcaf1 and / or pgm-, pPst-, ΔyopD / Δcaf1, -between 20-90 days, in particular between 20-46 days, more in particular between 20-28 days, after the second vaccination, and before infection, optionally at least about 28 (±3) days before infection, administering to the subject a therapeutically effective amount of a third (a second booster) vaccine composition comprising CpG, a recombinant protein subunit of rF1V or rV with or without CpG, combination of rF1V and rV with or without CpG, or at least one LAV selected from Y. pestis pgm-, pPst-, Δcaf1 and / or pgm-, pPst-, ΔyopD / Δcaf1, -wherein if the first and second vaccine compositions do not comprise a LAV or LAVs, the third composition comprises at least one LAV selected from Y. pestis mutant pgm-, pPst-, Δcaf1 and / or pgm-, pPst-, ΔyopD / Δcaf1, and, optionally, -before 48-60 hours after infection, administering to the subject an antibiotic treatment with an effective amount of an antibiotic or antibiotics.

[0083] Other embodiments relate to a method of inducing an immune response against Y. pestis comprising administering a therapeutically effective amount of a vaccine composition comprising a recombinant protein subunit of rF1V or rV with or without CpG, combination of rF1V and rV with or without CpG, and / or at least one LAV selected from Y. pestis mutant C12 ∆yscN, CO92 ∆yscN, or CO92 pgm- pPst-, in particular combination of CO92 ∆yscN and C12 ∆yscN, or CO92 pgm- pPst-. In a specific embodiment, the composition comprises at least one LAV selected from Y. pestis mutant C12 ∆yscN, CO92 ∆yscN, or CO92 pgm- pPst-, in particular combination of CO92 ∆yscN and C12 ∆yscN, or CO92 pgm- pPst-.

[0084] In a further embodiment, provided is a method of inducing an immune response against Y. pestis comprising administering a therapeutically effective amount of a composition comprising a recombinant protein subunit of rF1V or rV with or without CpG, combination of rF1V and rV with or without CpG, and / or at least one LAV selected from Y. pestis mutant pgm-, pPst-, Δcaf1 orpgm-, pPst-, ΔyopD / Δcaf1. In another embodiment, provided is a composition comprising at least one LAV strain selected from pgm-, pPst-, Δcaf1 or pgm-, pPst-, ΔyopD / Δcaf1. (a) Vaccine Composition and Administration

[0085] Vaccine compositions of the present invention may be administered to any subject, including all vertebrates, in particular mammals, more in particular human who live in the region where plague or melioidosis is endemic or who plan to travel in such a region. The vaccine can be administered as a prophylactic, but if necessary it can be used for treatment purposes.

[0086] In a specific embodiment, provided is a vaccine composition comprising a recombinant protein subunit of rF1V or rV with or without CpG, combination of rF1V and rV with or without CpG, and / or at least one LAV selected from Y. pestis mutant C12 ∆yscN, CO92 ∆yscN, or CO92 pgm- pPst-, in particular combination of CO92 ∆yscN and C12 ∆yscN, or CO92 pgm- pPst-. In a specific embodiment, the composition comprises at least one LAV selected from Y. pestis mutant C12 ∆yscN, CO92 ∆yscN, or CO92 pgm- pPst-, in particular combination of CO92 ∆yscN and C12 ∆yscN, or CO92 pgm- pPst-.

[0087] A composition comprising a recombinant protein subunit of rF1V or rV with or without CpG, combination of rF1V and rV with or without CpG, and / or at least one LAV selected from Y. pestis mutant pgm-, pPst-, Δcaf1 or pgm-, pPst-, ΔyopD / Δcaf1. In another embodiment, provided is a composition comprising at least one LAV strain selected from pgm-, pPst-, Δcaf1 or pgm-, pPst-, ΔyopD / Δcaf1.

[0088] The vaccine composition can include adjuvants, preservatives (e.g., 2-phenoxyethanol), stabilizers (e.g., gelatin, amino acids, potassium, sodium, lactose), pH adjusters / buffers, tonicity adjuster (e.g., NaCl), surfactants and emulsifiers (e.g., Polysorbate 80 or Tween®), formaldehyde, and some vaccine can contain antibiotics.

[0089] In certain embodiments of the invention, the vaccine compositions discussed herein are formulated and administered as a pharmaceutical composition that contains the vaccine and a pharmaceutically acceptable carrier, and optionally one or more additional active agents. Optionally, the pharmaceutical compositions comprise a therapeutically effective amount of vaccine. A pharmaceutically acceptable carrier refers to any convenient compound or group of compounds that is not toxic and that does not destroy or significantly diminish thepharmacological or immunological (vaccine) activity of the agent with which it is formulated. Such pharmaceutically acceptable carriers or vehicles encompass any of the standard pharmaceutically accepted solid, liquid, or gaseous carriers known in the art, such as those discussed in the art.

[0090] A suitable carrier depends on the route of administration contemplated for the pharmaceutical composition. Routes of administration are determined by the person of skill according to convenience, the health and condition of the subject to be treated, and the location and stage of the condition to be treated. Such routes can be any route which the practitioner deems to be most effective or convenient using considerations such as the patient, the patient’s general condition, and the specific condition to be treated.

[0091] In some embodiments, the live attenuated vaccines of the invention optionally can be administered by the oral (e.g., sublingual), intranasal or parenteral route (e.g., subcutaneous, intradermal, or intramuscular routes). The subunit vaccines of the invention can be administered through parenteral route or intranasal route in a liposome. Other routes of administration can include, but are not limited to local or parenteral, including: intravenous, intra-arterial, intrathecal, intraperitoneal, or local injection; rectal or vaginal suppository, topical, buccal, transdermal, inhalation, transmucosal, wound covering, and the like. The administration can also be given by transfusion or infusion, and can be administered by an implant, an implanted pump, or an external pump, or any device known in the art.

[0092] Therefore, the forms which the pharmaceutical composition can take will include any form suitable or convenient for any of those routes of administration. Such product forms or dosage forms of the vaccine compositions include, but are not limited to: powders or granules for dilution, pre-filled syringes, liquids for injection, suspensions for injection, oral solutions, oral suspensions, oral emulsions, nasal spray solutions, tablets, capsules, caplets, lozenges, dragees, pills, granules, powders for inhalation, vapors, gases, rectal suppositories, vaginal suppositories, creams, lotions, oils, ointments, suspensions, emulsions, lipid vesicles, and the like. Optionally, each dosage form contains an effective amount of vaccine.

[0093] Any pharmaceutically acceptable carrier and / or excipient known in the art is contemplated for use with the invention. Carriers can include, for example, solvents, diluents, other carriers, and the like to contain the active ingredient(s) in solid, liquid, or gas form.Common carriers in solid form include starch (e.g., corn starch, potato starch, rice starch), celluloses (e.g., microcrystalline cellulose, methylcellulose, and the like), sugars (e.g., lactose, sucrose, glucose, fructose, and the like), clays, minerals (e.g., talc, and the like), gums, waxes, and the like. Common carriers in liquid of semi-liquid form include gels, lipids (e.g., lipid vesicles or nanoparticles), oils, polyethylene glycols, glycerine, propylene glycol, emulsifiers, organic solvents (e.g., ethanol), aqueous solvents (e.g., water, saline solutions, electrolyte solutions, lactated saline solutions), suspending agents, and the like.

[0094] The vaccine composition or pharmaceutical compositions containing the vaccine can be provided in containers such as sachets, envelopes, blister packs, boxes, ampoules, vials, bottles, pre-filled syringes, bags, sprayers, inhalers, and the like.

[0095] Extended and sustained release of compositions also are contemplated for use with and in the inventive embodiments. Thus, suitable carriers can include any of the ingredients known to achieve a delayed release, extended release or sustained release of the active components.

[0096] The vaccines of the invention, in certain embodiments, are formulated as a lyophilized vaccine powder (representing a dry mix of the six individual lyophilized vaccines strains), presented in a sachet that represents one dose of vaccine, and optionally packaged with a sachet or other package of buffer powder. In some cases, the vaccine can be provided in 5-dose, or 10- dose packages. In certain formulations of the vaccines, the dose is prepared for oral administration, by suspending the contents of the sachet in water, for example about 100 mL of water, optionally with buffer powder contained in an accompanying sachet. Once reconstituted, the resulting “cocktail” of vaccine strains in buffer solution is delivered orally. Optionally, the sachet containing the vaccine contains suitable buffers so that reconstitution is performed with water alone, or the sachet is reconstituted in a buffer solution. This embodiment of vaccine is suitable for a travelers’ vaccine, for distribution to at-risk populations and for distribution in the developing world or to military personnel. Different embodiments of the dosage forms include, but are not limited to an individual (single use) dose containing the mix of strains and a multi- pack of individual doses containing the mix of strains. Either embodiment can optionally be accompanied by a sachet of buffer powder or a buffer solution. An additional embodiment includes a multi-dose package of individual dosage sachets, such as a 5-dose or a 10-dose presentation.(b) Doses and Regimens

[0097] Treatment regimens include a single administration of the vaccine or a course of an initial administration and one or more “booster” administrations. Optionally, the administration is determined by medical personnel. The initial administration is given to the subject prior to exposure to conditions where plague or melioidosis is endemic, but may be given after exposure, or after symptoms of plague or melioidosis occur. Booster administrations are given when needed. Examples of possible doses of vaccines may vary from about 107CFU to about 1012CFU dry attenuated bacteria, or as recommended by medical personnel, optionally about 108CFU to about 1010CFU, or about 108CFU to about 1010CFU, or about 108CFU to about 109CFU, or any dose suitable as determined by the practitioner, depending on the size, exposure, general health, age, or other factors. Doses of subunit vaccines may vary from about 10 µg-1 g / body weight EXAMPLES

[0098] This invention is not limited to the particular processes, compositions, or methodologies described, as these may vary. The terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention. All publications mentioned herein, are incorporated by reference in their entirety; nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Example 1. Yersinia pestis and bubonic / pneumonic plague 1.1. Materials and Methods 1.1.1. Bacterial strains, media, and growth conditions

[0099] The wild-type and mutant derivatives of Y. pestis and their construction and characteristics were described previously (Cote et al. 2021) and in the current study. These strains included the virulent F1-negative (nonencapsulated) derivative of CO92, C12 (Worsham, et al. 1995). Therecombinant strains had a mutation in the virulence-associated gene yscN or had a deletion of the entire pgm locus and were cured of the pPst (PCP1) plasmid encoding plasminogen activator (CO92 pgm- pPst-) (Bozue et al. 2014, Bozue, et al. 2012, Swietnicki, et al. 2011, Welkos et al. 2002, Welkos et al.1997). Y. pestis strains were grown on 5% sheep blood agar plates (SBAP) or tryptose blood agar (TBA) base slants for approximately 48 h at 30°C. Liquid cultures of Y. pestis C12 used for mouse challenge studies were prepared in heart infusion broth (HIB) medium supplemented with 0.2% xylose (HIBX); and the live attenuated vaccine suspensions were prepared with broth cultures incubated in HIBX supplemented with 2.5 mM CaCl2, as described below (Cote, et al. 2021). A solution of 10 mM potassium phosphate, pH 7.3–7.4 (KPhos) was used to prepare and dilute bacterial inocula. Challenge doses were determined by serial dilutions in KPhos buffer and plating on SBA. Bacteriological media were obtained from Thermo Fisher- Remel (Rockville, MD).

[0100] The rF1V vaccine was described previously (Amemiya et al. 2009, Biryukov et al. 2021, Heath, et al. 1998). Toll-like receptor 9 (TLR9) oligonucleotide (ODN) CpG ODN 2006 (CpG2006) was purchased from InvivoGen (San Diego, CA) and reconstituted in accordance with manufacturer recommendations. The purified recombinant V (rV) protein was obtained from BEI resources (Manassas, VA). Alhydrogel was sourced from InvivoGen. Streptomycin for injection was obtained from X-Gen Pharmaceuticals, Inc. (Horseheads, NY). 1.1.2. Preparation of candidate vaccines for injection

[0101] The day prior to vaccination, flasks were inoculated with a suspension of colonies from a freshly inoculated SBAP and incubated for 48 h at 28–30°C with shaking at 200 rpm. The next day, the cultures were adjusted to an OD600 of 0.1 in fresh medium and incubated to the OD600 determined to produce the target CFU concentration, which was 107CFU in doses of 0.2 mL (Cote, et al. 2021). To confirm the actual delivered dose of bacteria, the final suspensions were diluted and plated for viable counts. All plates were incubated at 28–30 °C for approximately 48 h before counting.

[0102] The doses of rF1V vaccine contained 2 µg rF1V in the presence or absence of 5 µg CpG2006 (CpG) and included Alhydrogel (250 µg) in a total volume of 0.1 mL (Biryukov, et al. 2021). In the studies indicated, 2 µg of rV with or without 5 µg CpG, was administered in a total volume of 0.1 mL.1.1.3 Animals and vaccination studies

[0103] The animal research was conducted under an animal use protocol approved by the USAMRIID Institutional Animal Care and Use Committee (IACUC) in compliance with the Animal Welfare Act, PHS Policy, and other Federal statutes and regulations relating to animals and experiments involving animals. The facility where this research was conducted is accredited by the AAALAC International and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). Female BALB / c mice were obtained from Charles River (Frederick, MD) and were 7-10 weeks of age at time of vaccination; all groups contained ten mice, with exceptions specified.

[0104] Mice vaccinated with a single dose of vaccine were injected subcutaneously and exposed four weeks later by the aerosol or subcutaneous (SC) route to a lethal dose of Y. pestis C12. As indicated, mice vaccinated twice were administered the second dose 20-28 days after the initial vaccine dose. Sera and spleens were collected from a cohort of mice to assess immune responses to the vaccines. Mice were challenged 27-29 days post final vaccination. 1.1.4. Exposure of vaccinated mice to virulent Y. pestis C12

[0105] Mice were exposed to aerosolized (pneumonic) or SC (bubonic) challenge doses of virulent Y. pestis C12. For bubonic challenge, bacteria were harvested from TBA slants. Mice exposed by the SC route were inoculated with 0.2 mL volumes of the suspension in KPhos (Bozue, et al.2014, Bozue, et al. 2012). The bacteria used for aerosol studies were prepared by using colonies from freshly inoculated TBA slants which were suspended in HIBX to an initial OD620of approximately 0.01 and incubated for approximately 24 h at 28–30°C. For aerosol exposures, the cultures were harvested by centrifugation and suspended in HIB medium (no xylose) to the concentration yielding the number of LD50doses indicated in the tables. Exposure to aerosolized bacteria was accomplished as previously described (Bozue, et al. 2014, Bozue, et al. 2012, Trevino et al.2018). Briefly, mice were transferred to wire mesh cages and were placed in a whole-body aerosol chamber within a class three biological safety cabinet located inside a BSL-3 laboratory. Mice were exposed to aerosols of Y. pestis strain C12 created by a three-jet collison nebulizer. Samples were collected from the all- glass impinger (AGI) vessel and analyzed by performing CFU calculations to determine the inhaled dose of Y. pestis.1.1.5. Treatment of vaccinated mice

[0106] A solution of streptomycin was prepared in water for injection (Corning, Manassas, VA) and a dose of 20 or 40 mg / kg administered to mice by the intraperitoneal (IP) route every 6 h for 5 days. Mice were injected with the antibiotic beginning 48 h or 60 h after exposure to aerosolized Y. pestis C12. 1.1.6. Bacteriology

[0107] The tissues collected from necropsied mice included lung, spleen, and blood. They were weighed, homogenized with disposable PRECISION™ homogenizers (Covidien, Dublin, Republic of Ireland), and the CFU of the homogenate were determined on SBA plates. Undiluted homogenate and ten-fold dilutions in KPhos were plated in duplicate to determine sterility. The limit of detection (LOD) was approximately 100 CFU / mL blood or 5 CFU / organ. After CFU determinations, samples were radiation-inactivated, sterility checked, and stored at -80°C for immunological analyses. 1.1.7. Humoral immune response assays

[0108] Immunoglobulin (Ig) IgG antibody responses to the vaccines were determined by semi- quantitative endpoint ELISA using sera from vaccinated BALB / c mice, as previously described (Biryukov, et al. 2021, Cote, et al. 2021). The sera were collected as terminal blood collections from axillary vessels and titrated against several capture antigens: the rF1V recombinant fusion protein, the rV protein, and γ-radiation inactivated whole cells of Y. pestis strains temperature switched (TS) CO92 or TS C12. The strains had been grown at 30°C for 21 h followed by a temperature switch to 37°C and incubation for an additional three h to upregulate the presentation of potential antigens. The cultures were pelleted by centrifugation and then inactivated with γ- radiation; temperature-shifted antigens were designated TS. The rF1V fusion protein vaccine construct and the rV protein were diluted in 0.1 M carbonate buffer, pH 9.5, to a concentration of 2 μg / mL, while inactivated Y. pestis TS CO92 or TS C12 whole cells were plated at a concentration of 10 μg / mL on 96-well Immulon 2HB plates (Thermo Fisher, Grand Island, NY, USA). Plates were stored at 4°C overnight, then washed and blocked, and samples were processed as previously described (Biryukov, et al.2021). Two-fold dilutions of the serum were made in triplicate and the results are reported as the geometric mean (GM) and geometric standard error (GSE) of thereciprocal of the highest dilution giving a mean OD of at least 0.1 ± 1 SD at 450 nm with a reference filter (570 nm). Samples with an antibody titer of < 50 were considered negative. 1.1.8. Cellular immune responses

[0109] Four weeks following vaccination, purified splenocytes were prepared based on a previously published protocol (Amemiya et al. 2006, Cote, et al. 2021). Briefly, spleens were excised from mice (n = 5 mice per group), weighed, and disaggregated in RPMI 1640 medium (Thermo Fisher, Grand Island, NY). Red cells in the spleen homogenate were lysed with Ammonium-Chloride-Potassium (ACK) Lysing Buffer (BioWhittaker, Walkersville, MD) after the extract was diluted with RPMI 1640 medium and cells pelleted by centrifugation at 335 x g (1,200 rpm) for 10 min. Splenocytes were then re-suspended in CTL-Medium (Cellular Technology Limited, Cleveland, OH) supplemented with 1% L-glutamine and the cells counted. Purified splenocytes were re-stimulated for ~48 h in the presence of rF1V (25 μg / mL) or irradiated Y. pestis TS C12 (5 μg / mL) in complete medium containing 10% heat-inactivated fetal calf serum (Thermo-Fisher), 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 100 U / mL penicillin, 100 μg / mL streptomycin, and 50 μM 2-mercaptoethanol. A solution of phorbol 12-myristate 13- acetate (PMA; 100 ng / mL) and ionomycin (0.5 μg / mL) was used as the positive control stimulant and resulted in uniformly strong signals (data not shown). Following restimulation, the plates were centrifuged at 1,200 × g for 10 min at RT and the cytokine expression in the supernatants was evaluated by Luminex Mag Pix 36-plex mouse panel per manufacturer directions (Thermo Fisher Scientific, Grand Island, NY, USA).

[0110] In addition, three days post challenge spleen and lung homogenates were prepared by removing and homogenizing the whole organs in KPhos using disposable PRECISION™ homogenizers (Covidien, Dublin, Republic of Ireland). Samples were irradiated with approximately 21 kGy of gamma-radiation and confirmed sterile by testing 10% of the sample before use. Aliquots of the homogenates were saved for cytokine / chemokine determination and stored at -70°C. Cytokine and chemokine levels were assayed in spleen and lung homogenates, as previously described (Trevino, et al.2018). The homogenate samples were thawed, centrifuged at 10,000 x g for 10 min, and the supernatant was then examined for cytokine expression by Luminex Mag Pix 36-plex mouse panel (Thermo Fisher Scientific, Grand Island, NY, USA) as per manufacturer directions. The levels (pg / mL) were measured, and heat maps were generated toreport the fold increase [vaccine group (pg / mL) / PBS control group (pg / mL)] in cytokine or chemokine levels.

[0111] ELISpot assays were performed to measure interferon gamma (IFN-γ) expression. Purified splenocytes, four weeks after vaccination, were seeded in the presence of rF1V, TS C12, medium alone, or PMA / ionomycin. Briefly, 96-well plates were coated overnight at 4°C with 80 µl / well capture anti-mouse IFN-γ monoclonal antibody. Plates were washed one time with 1x PBS. Recombinant F1V (25 μg / mL) or TS C12 (5 µg / mL) were re-suspended in CTL-Medium with 1% L-Glutamine and 100 µl was added to each well. The plates were incubated at 37°C, 9% CO2for 15 min. Splenocytes were resuspended in CTL-Medium with 1% L-Glutamine and seeded at 8x105cells per well for rF1V and 8x104cells per well for TS C12 stimulations. Plates were incubated for 24 h at 37°C and 9% CO2 and splenocytes were removed and plates were washed twice with PBS alone and then twice with PBS and 0.05 % Tween. Eighty μL per well of biotinylated detection anti-mouse IFN-γ monoclonal antibody was added. After two h incubation at room temperature, plates were washed three times with PBS and 0.05 % Tween. Eighty μL of Strep-AP antibody solution was added to the wells, and the plates were incubated for 30 min at room temperature. Development reagents were added and incubated for 15 min at room temperature according to manufacturer recommendations. The colorimetric reaction was stopped by washing the plates three times with distilled water and air drying overnight. Spots were scanned and analyzed using an automated ELISpot reader (CTL-Immunospot S6 Analyzer, CTL, Germany). The splenocyte response was assessed as spot forming cells (SFC), adjusted to 106cells per well, which was automatically calculated by the ImmunoSpot® Software for each stimulation condition and the medium only control. 1.1.9. Statistical analyses

[0112] Statistical analyses were performed using SAS version 9.4, except as indicated. Survival curves of the vaccinated and control mice were estimated with the Kaplan-Meier method using Graph Pad Prism 9.0 and were compared statistically using the log-rank test. Significant differences in survival rates at days 7 and 21 after virulent challenge were determined using the Fisher Exact test. The time-to-mortality (TTM) values were expressed as the median and interquartile range and compared with the log-rank test. The runs of aerosol-challenged mice were analyzed separately or combined, as indicated. The viable counts of Y. pestis from organ and bloodsamples were compared using the Wilcoxon rank sum test for pairwise comparisons test. The CFU were log-transformed and mean results summarized as GM and GSD values, using the LOD / SQRT(2) to replace the values with no recoverable CFU.

[0113] For the ELISA antibody titers, determined as described above, Cytokine concentrations determined in Luminex assays, ELISpot assays, pairwise treatment groups were compared by Wilcoxon rank sum test. The median, Q1, Q3, GM, and GSE were reported.

[0114] The potential synergistic effects of vaccine and antibiotic on protection were analyzed using the Bliss synergy (Demidenko and Miller 2019). Synergy score values indicated the ratio of median TTM and were based on a log-logistic or log-normal parametric survival model. 1.2. The live attenuated vaccines protected mice against bubonic plague but not against pneumonic plague after challenge with Y. pestis C12

[0115] Previously the efficacy of live attenuated vaccine strains of Y. pestis was demonstrated for protection of mice against bubonic and pneumonic plague after exposure to the virulent encapsulated Y. pestis CO92 strain (Cote, et al.2021). Here it is described that the same strategies could not similarly protect BALB / c mice against plague in animals exposed to Y. pestis strain C12, a nonencapsulated derivative of CO92. As demonstrated in Figure 1A, protection was afforded to mice vaccinated with a single dose of Y. pestis mutant CO92 ∆yscN, C12 ∆yscN, or a combination of both ∆yscN vaccine strains (Combo LAV), against a SC challenge with approximately 214 LD50s of Y. pestis C12 (40%, 60%, or 70%, respectively); these survival rates were greater or nearly greater than the mean rate of the control mice, with p = 0.087, p = 0.011, and p = 0.003, respectively. Nevertheless, the vaccine-mediated protection was less robust than that reported previously for mice challenged with the wild-type Y. pestis CO92 strain, against which some of the live vaccines protected completely (Cote, et al.2021). Not surprisingly, these single dose strategies offered no protection to mice after exposure to aerosolized Y. pestis C12 with only slight, statistically insignificant differences in mean time-to-mortality (TTM) (Figure 1B).

[0116] As demonstrated in Figure 2, two doses of the LAV (prime-boost) given on days 0 and 23 yielded only a modest increase in protection against C12 challenge compared to the single dose cohorts (Figure 1A). In the bubonic model, the protection afforded after a booster vaccination byCO92 ∆yscN and C12 ∆yscN was increased 20% (to 60% and 80%, respectively), and the extent of protection by the Combo LAV vaccine was unchanged (70%, Figure 2A); the mean survival rates were statistically greater than the mean survival rate of the control mice, p = 0.057 to p = 0.006). The two-dose homologous vaccination schemes with our live attenuated ∆yscN strains did not improve survival after exposure to aerosolized Y. pestis C12 (Figure 2B), but it slightly extended the TTMs at days 7 and 21 after challenge (p = 0.022 to p < 0.0001) as compared to the controls. 1.3. Heterologous vaccination strategies typically protected better than live attenuated vaccines alone against a Y. pestis C12 challenge

[0117] The LAV strains are clearly immunogenic as determined by humoral and cell-mediated immune responses that protect against Y. pestis CO92 (Cote, et al. 2021), and these immune responses protect mice against bubonic, but not pneumonic, plague caused by Y. pestis C12. Capsule producing LAV candidate strains produce a robust anti-F1 humoral immune response, but due to possible immune interference the anti-F1 responses may diminish the induction of a more diverse immunity against other protective antigens (Cote, et al.2021). To enhance the availability of other protective antigens, but still retain the F1 mediated protection, approximately equal parts CO92 ∆yscN and C12 ∆yscN vaccine mix (Combo LAV) was selected for further evaluation in heterologous vaccine strategies to improve the protection against Y. pestis C12 infection.

[0118] In these experiments, mice were primed with a dose of the Combo LAV. The mice were then boosted (28 days later) with either the same LAV given in the first injection or with a subunit vaccine, rF1V fusion protein or rV protein [groups 2-4, 7, 8] (Figure 3). The impact of further adjuvanting the recombinant protein boost with the addition of oligonucleotide CpG2006 was also tested. Mice which received any of the four heterologous vaccines [groups 3, 4, 7,8] were highly protected against bubonic disease when challenged four weeks after the boost vaccination, as illustrated in Figure 3A. Vaccination with the Combo LAV followed by rF1V + CpG, or rV + / - CpG, resulted in 100% survival [groups 4, 7, 8], p < 0.001 versus KPhos control; and 90% survival was observed in groups given either the Combo LAV first and then rF1V without CpG [group 3] or with two doses of the Combo LAV vaccine [group 2], p = 0.0001. Two groups receiving KPhos and CpG alone plus the Combo LAV, as either the prime or boost doses, resulted in 80% survival after SC challenge [groups 6 and 9], p = 0.0007 vs. the control, respectively. Thus, in the bubonicmodel, the heterologous vaccine combinations were generally more protective against Y. pestis C12 than the homologous vaccine schemes.

[0119] Furthermore, heterologous approaches significantly improved the clinical outcome in mice exposed to aerosolized Y. pestis C12 (Figure 3B). The best survival against aerosolized Y. pestis C12 challenge was observed in mice vaccinated with the heterologous combinations of Combo LAV (prime) followed by a boost of rF1V with CpG (Figure 3B). The survival rates of these mice at both early (day 7) and final timepoints post-challenge [group 4] were significantly better (90%) than those of four of the other five groups (p values ranged from p = 0.0001 to p = 0.048). Although 50% of the group 3 (Combo LAV and rF1V) survived, this survival rate was not significantly different from that of group 4 (p = 0.141). However, the TTM of the latter was longer than that of mice given the Combo LAV and rF1V without CpG (p = 0.048). All five vaccinated groups survived slightly but significantly longer than did the KPhos controls (p < 0.041), with mean TTMs for non-survivors of 3.5 days (controls), and 4.0 to 5.4 days for the vaccinated groups. Thus, the immunostimulant CpG enhanced protection afforded by the heterologous Combo LAV and rF1V vaccine.

[0120] To further support these findings, mice were vaccinated again with the homologous or heterologous prime / boost scheme but were challenged with a mean inhaled dose of Y. pestis C12 approximately two-folds greater than the dose in the Figure 3B study, i.e., 1.65 x 106CFU (22 LD50s). As shown in Figure 3C, the heterologous vaccines outperformed two doses of the Combo LAV. Whereas the latter provided no protection, the groups vaccinated with the LAV followed by rF1V, with or without CpG, protected 50% or 40%, respectively. The survival rate of the group receiving the heterologous vaccine with CpG was significantly greater than that of the control and Combo LAV x2 groups at day 7 and day 21 after challenge (p = 0.0325); however, these survival rate comparisons to the control and Combo LAV x2 groups were not statistically different for the mice receiving the heterologous vaccine without CpG. The TTMs of the two heterologous vaccination groups were greater than those of the KPhos control and LAV x2 groups at both day 7 and day 21 post-challenge (p < 0.0423). Overall, vaccination with heterologous combinations of LAVs and subunit protein vaccines were more strongly protective than homologous vaccine combinations of LAVs or subunit, especially against aerosol challenge (Figures 3B and 3C). Importantly, vaccination with the recombinant protein alone was shown to be less effective againstsimilar challenge doses of C12 compared to mice challenged with Y. pestis CO92 (Biryukov, et al. 2021).

[0121] Tissue samples and blood were collected from cohorts of mice in the study described in Figure 3B to assess bacterial load and antibody titers on day three after the aerosol challenge and results are shown in Tables 1 and 2. Although there were large variations in organ and blood bacterial loads between mice in some groups, statistical analyses revealed significant vaccine- related differences. For the group vaccinated twice with the Combo LAV, there were no significant reductions in bacterial burden compared to the KPhos control mice in the lung, spleen, and blood. In contrast, mice vaccinated with the heterologous Combo LAV / rF1V + CpG regimen had a reduced burden in the lungs (GM of 274 CFU / g), and the bacterial load was significantly reduced compared to the other five groups (p = 0.026 to 0.0022). In addition, a GM of 123 CFU / g was isolated from the spleen samples of mice vaccinated with Combo LAV / rF1V + CpG, with four of six mice having no detectable CFU, and blood samples yielded a GM of 237 CFU / ml (Table 1); no CFU were recovered from the blood of 5 / 6 mice vaccinated with Combo LAV / rF1V + CpG, and one mouse had 1.0 x 105CFU / ml.

[0122] For the Combo LAV / rF1V vaccinated mice, the lung CFU were only significantly less than those recovered from the Combo LAV x2-vaccinated mice (p = 0.041); the spleen and blood burdens were reduced, but there were no differences compared to the other five groups. Overall, the three vaccine groups Combo LAV / rF1V, KPhos / rF1V, and KPhos / rF1V + CpG had generally similar bacterial loads. The GM of lung, spleen, and blood CFU of mice with positive cultures were not statistically different in comparisons between these three vaccine groups. However, compared to the Combo LAV x2 burdens, two of these vaccine groups had significant reductions of one to two logs in lung CFU (p = 0.026 for the KPhos / rF1V group and p = 0.041 for the Combo LAV / rF1V vaccinated mice), at least one log lower spleen CFU counts, and no detectable CFU in half of the blood samples from the KPhos / rF1V and Combo LAV / rF1V vaccinated mice. Thus, the bacterial burdens of these three groups were generally intermediate between those mice with the smallest bacterial loads (Combo LAV / rF1V + CpG) and the KPhos controls.

[0123] Antibody titers to four antigens were assessed by ELISA on sera from blood collected three days after challenge. The antigens included rF1V, rV, irradiated TS CO92 whole cells and irradiated TS C12 whole cells, and the results are shown in Table 2. The most elevated antibodytiters overall were those to the rF1V fusion protein or the rV antigen which reiterates the strong immunogenicity of the F1 and V antigens in BALB / c mice. All five vaccines induced anti-rF1V and rV titers that were significantly greater than those of the KPhos control mice, with p < 0.0159 for all groups (Table 2). Mice vaccinated with Combo LAV / rF1V + CpG generated highest anti- rV titers that were 2- and 3-fold higher than Combo LAV / rF1V and KPhos / rF1V + CpG, although these differences did not reach significance. Vaccination with a priming dose of the Combo LAV and a boosterdose of rF1V induced the highest titers to TS CO92 whole cells, whereas two doses of the Combo LAV vaccine induced the highest titers to TS C12 (p = 0.017). Likely, the antibody responses to TS CO92 whole-cell the antigen were largely directed against the F1 capsule; and adding rF1V to the Combo LAV elicited higher titer to the rF1V and TS CO92 whole-cell antigens. The addition of CpG to Combo + rF1V in group 4 greatly increased titers to rF1V compared to the mean titers of group 1 (p = 0.0095) and group 5 (p = 0.0022). However, the addition of CpG was associated with repressed titers when TS CO92 whole cells were used as the capture antigen and did not significantly increase the titers to TS C12 cells. Specifically, the anti-TS CO92 levels in group 4 mice were less than those in group 3 (p = 0.0043), though not group 2 (p = 0.643). The vaccines containing only a single dose of rF1V elicited the lowest titers overall, albeit these were still elevated against the rF1V antigen. Overall, the antibody levels against the TS C12 antigen were much less than those to the rF1V and TS CO92 antigens. This finding might suggest either that surface antigens other than the immune-dominant F1 protein do not elicit strong humoral responses or that the C12 ∆yscN LAV was cleared in the mice before reaching immunostimulatory levels due to the lack of an anti-phagocytic capsule.

[0124] Lungs and spleens were obtained from animals collected on day 3 post-challenge to analyze the cytokine responses. The lung sample data revealed that three vaccine groups had levels of cytokines that were repressed or not significantly different overall relative to the KPhos group levels (heat map Table 3). The Combo LAV / rF1V + CpG and KPhos / rF1V groups had responses that were relatively most repressed. The KPhos / rF1V + CpG group cytokines were also moderately suppressed, whereas the Combo LAV / rF1V group responses were not appreciably different from those of the KPhos group. For example, in the Combo LAV / rF1V + CpG group, the levels of three cytokines were approximately100-fold lower than those of the controls (IL-6, MIP-2α, G-CSF) while seven additional cytokines were approximately 10- to 30-fold lower (Gro- α (CXCL1), LIF, IL-1β, GM-CSF, MIP-1α, IL-1α, and IFN-γ). In contrast, the cytokine levelswere generally significantly elevated in the Combo LAV x2 vaccinated mice. The levels of IL-3, Rantes (CCL5), IL-4, and IFN-γ were from 5.3- to 7.1-fold greater than KPhos controls. These data were inversely associated in part with the vaccine-associated protection against C12 challenge in that the Combo LAV / rF1V + CpG vaccine was the most protective and Combo / LAV x2 the least protective (Figure 3 B and C). The results of the analysis of spleen samples were similar overall to those of the lung samples, but the degree of differences in levels between the vaccine groups was less (Table 4). In contrast to the lung results, IFN-γ, MCP-1, IL-1α, IP-10 and other cytokine levels were repressed in all vaccine groups, and IL-23 (a Th17 cytokine) was uniquely elevated in the Combo LAV / rF1V mice (but not in the Combo LAV / rF1V + CpG group). In aggregate, the spleen data do not appear to be as informative as the lung data in comparing cellular immune responses to vaccination these female BALB / c mice; but they suggest that early post- challenge decreases in bacterial lung burdens of vaccine-protected mice might result in less dissemination to the spleen. 1.4. Extended time between vaccination doses increased overall titers

[0125] In an effort to further characterize heterologous plague vaccination approaches, the impact of extended times between the prime and boost vaccine injections was examined along with varying durations between the prime vaccine and subsequent booster dose(s) in the absence of CpG. The ability of the CO92 pgm- pPst- LAV to protect mice from C12 challenge was also reevaluated because of its significantly different protein expression profile and the fact that it is an excluded non-select agent strain. In this experiment, the vaccines were administered once only or with one or two additional booster dose(s) given at different times after the first dose, as detailed in Figure 4 (table in Figure legend). Specifically, one group received a prime dose only (group 2). Single boosters were administered 21 days after the prime dose (group 3), 46 days after the prime (groups 4 and 9), or 90 days after the prime (groups 5, 7, 8, 10, 11). Double boosters were injected on day 46 and 90 after the prime (groups 6 and 12). Figure 4 illustrates survival rates after aerosol challenge with a dose of 13 LD50s of C12. Two groups which had been primed with an LAV (CO92 pgm- pPst- or CO92 ∆yscN, groups 7 and 11, respectively) and given one booster of rF1V (without CpG) 90 days after the prime dose had the highest survival rates (37.5 %); but survival did not reach significance relative to the KPhos group. A small number of survivors (12.5%) were observed in a heterologous group of mice initially given the CO92 pgm- pPst- LAVand boosted twice, first with CO92 pgm / pPst- at 46 days after the prime and second with rF1V at 90 days after the initial vaccination (group 6). The mice primed with CO92 ∆yscN and boosted on day 46 with the same LAV and on day 90 with rF1V (group 12) had no survivors but had a significantly longer mean TTM than the KPhos controls, with geometric means of 5.0 and 3.8 days, respectively (p = 0.0285). The remaining groups of mice had no survivors and succumbed at the same time post-challenge as the KPhos controls (day 4). Albeit complete protection was not achieved in this study, the results again suggested that heterologous vaccination strategies are superior and that administering one booster injection of rF1V vaccine 3 months after priming with an LAV could be associated with better protection than schedules where the prime and booster doses were spaced more closely in time. Of interest, priming and boosting 90 days later with homologous LAVs (groups 5 and 10) were not protective. Thus, a heterologous LAV / rF1V vaccination scheme appears to be optimal, but further evaluation is needed.

[0126] Antibody titers to the four antigens were assessed by ELISA on sera collected from a cohort of mice one day before aerosol challenge. The antigens included rF1V, rV, Y. pestis TS CO92, and Y. pestis TS C12. In agreement with the protection data, the two groups (7 and 11) with the greatest survival rates had the highest titers to rF1V and CO92 whole cells but not to C12 whole cells (Table 5), similar to the responses to C12 described in Table 2, and the differences were confirmed statistically. Importantly, groups 7 and 11 did have some of the highest anti-rV titers in this study. These two groups had been primed with either the encapsulated CO92 pgm- pPst- or CO92 ∆yscN LAV and boosted once with rF1V 90 days later. Similarly, mice in groups 6 and 12, which had received prime and boost doses at day 46 of a CO92 LAV (pgm- pPst- or ∆yscN) and a second boost at day 90 with rF1V, and which were partially protected or had an extended TTM, exhibited the second highest antibody responses to rF1V. However, this partial protection was not clearly associated with humoral responses to either the F1+or F1- whole cell antigens (Table 5). The most elevated anti-TS C12 levels were observed in mice given two homologous doses of a LAV or heterologous prime and boost doses of the LAVs 90 days apart, i.e., groups 5, 8, and 10 in Table 5, although the GM anti-TS C12 titers of all 11 vaccine groups were significantly elevated compared to the KPhos group (p = 0.008 to 0.029). The GM Y. pestis TS C12 antibody titer of group 8 (320,000) was significantly greater than that of vaccine groups 2, 3, 6, 7, 9, 11, and 12 (p = 0.008 to 0.04,). Apart from group 2 (which received one prime dose only), these groups had received a final dose on day 90 of rF1V. The presence of rF1V in thevaccines appears to mostly blunt the humoral response to Y. pestis TS C12. Generally, the humoral responses to Y. pestis TS C12 were not associated with protection.

[0127] To assess cellular immune responses, spleens were collected from animals one day before aerosol challenge for ELISpot and Luminex restimulation assays. ELISpot assays for IFN-γ were performed using unstimulated splenocytes or splenocytes stimulated with rF1V or Y. pestis TS C12. The unstimulated cells from the vaccine groups produced < 15 IFN-γ producing cells (SFC) per 106splenocytes (Figure 10); note the differences in Y axis ranges of panels A – B of Figure 5. In contrast, splenocytes from all the vaccinated mice stimulated with rF1V secreted much higher levels than did the KPhos controls (Figure 5A), p = 0.0034 by pairwise comparison analyses. The greatest levels of IFN-γ were produced by rF1V-stimulated cells from mouse groups 6, 7, 11, and 12 (p < 0.0001). As illustrated for the humoral immune responses, these results again paralleled the differential protective efficacies afforded by the vaccines. Interestingly, as depicted in Figure 5B, Y. pestis TS C12 whole cells elicited IFN-γ maximally from groups 9 – 12 (p < 0.0001 compared to KPhos controls), all of which had been primed with the LAV CO92 ∆yscN and not with the CO92 pgm- pPst- vaccine. It was hypothesized that the difference in IFN-γ production may be associated with the expression of V by the CO92 pgm- pPst LAV, as V is linked to immune- suppression. Additionally, the altered secretion of the other Yops may also have contributed to the different immune responses observed. The numbers of SFCs from splenocytes of these four groups (Figure 5B) were greater than those produced after stimulation with rF1V (Figure 5A). IFN-γ expression by Y. pestis TS C12-stimulated cells was maximal for groups 11 and 12 (but not statistically different from groups 9 and 10; p > 0.17); both groups 11 and 12 had been primed with CO92 ∆yscN and boosted on day 90 with rF1V. These results contrast with the humoral responses to Y. pestis TS C12, where inclusion of rF1V reduced antibody titers. In addition, this may reflect a greater role for cell-mediated immunity in protection associated with the ∆yscN LAVs relative to the CO92 pgm- pPst- vaccine.

[0128] Cytokine production by stimulated splenocytes from vaccinated groups was analyzed with Luminex bead-based immunoassay. The levels of cytokines produced by splenocytes stimulated with rF1V of the vaccinated groups relative to the KPhos group are illustrated in Table 6. Relative to KPhos, all CO92 ∆yscN vaccinated groups promoted a strong cytokine response, with most notable up-regulation of MIP-1α, IL-2, MCP-3, MIP-1β, IL-3, IL-17A, MIP-2α, and RANTES.Addition of rF1V to CO92 ∆yscN vaccinated mice drastically enhanced overall cytokine response, while addition of rF1V to CO92 pgm- pPst- vaccinated groups only moderately and selectively enhance some cytokines, notably , MIP-1α, IL-2, MIP-1β, IL-4, and IL-6.

[0129] Relative cytokine production by splenocytes stimulated with Y. pestis TS C12 showed an overall increased cytokine response for six groups (groups 2 – 7), all of which had received the CO92 pgm- pPst- LAV with or without rF1V. This increased response was most pronounced for mice vaccinated with a double dose of CO92 pgm- pPst- 45 days apart with or without F1V (groups 4 and 6) (Table 7). Substantially greater responses were observed for five groups (groups 8 – 12), all of which were CO92 ∆yscN vaccinees (Table 7). These results agreed in part with the humoral antibody and ELISpot data and suggest that the elevated antibody responses to rF1V and Y. pestis TS CO92, and the heighted Y. pestis TS C12-stimulated cellular immune responses following CO92 ∆yscN vaccination, may be generally associated with protection. However, the associations between the cell-mediated immune responses and survival rates are not readily apparent, likely due to the low vaccine-mediated protection against the high challenge dose of Y. pestis C12 delivered in this study. 1.5. Live attenuated Y. pestis vaccines could be an important component of layered defense strategies against plague

[0130] Following possible exposure to Y. pestis, the time between infection, and manifestation of lethal disease is brief. During that time, an individual needs to be properly diagnosed in order to receive the appropriate therapy, hence any effort to prolong the window of opportunity for therapeutic intervention can dramatically improve disease outcome. The protection afforded BALB / c mice against aerosol challenge with Y. pestis C12 by a suboptimal vaccination and delayed post-challenge antibiotic treatment strategy was examined. The mice were either vaccinated twice with mutant CO92 ∆yscN (21 days apart) or administered buffer alone, and then challenged 4 weeks after the second vaccine dose with a lethal aerosol dose of Y. pestis C12. They were subsequently treated with 20 mg streptomycin / kg (or buffer alone) beginning 48 or 60 h after challenge, and the effects on survival determined, as shown in Figure 6A and Figure 6B.

[0131] The vaccination had no effect on the survival of mice after challenge with Y. pestis C12, while the administration of the antibiotic 48 h following challenge was associated with 90% and 100% of the treated (only) and vaccinated, treated mice, respectively (Figure 6A). However,increasing the time between challenge and antibiotic treatment to 60 h revealed the enhanced effect on survival of the layered approach.

[0132] Whereas all the mice treated with 20 mg / kg streptomycin alone or vaccinated alone succumbed, 80% of the vaccinated and antibiotic treated mice survived (p = 0.0007 versus treated and p = 0.0011 versus vaccinated alone); the median TTM of the treated group was extended compared to the unvaccinated and vaccinated control groups (Figure 6B), i.e., 7.0 days versus 5.0 days (p = 0.0044) or versus 5.5 days (p = 0.044), respectively. The benefits accrued from this vaccination-treatment scheme was further illustrated in Figure 6C.

[0133] To improve the survival of unvaccinated mice treated with antibiotic at a delayed time (60 h vs 48 h) after challenge, animals were given a higher dose of streptomycin. A total of 20% of mice receiving 40 mg / kg streptomycin 60 h after challenge survived. In contrast, 70% of the CO92 ∆yscN -vaccinated and antibiotic treated mice survived, an increase that approached significance at days 7 and 21 (p = 0.070). All of the mice in the two control groups succumbed by day 6, p = 0.0044 versus CO92 ∆yscN-vaccinated and KPhos control groups compared to vaccinated and antibiotic treated mice (Figure 6C).

[0134] The C12 ∆yscN vaccine was evaluated similarly in the context of antibiotic treatment (Figure 7). Vaccination and treatment with 20 mg / kg streptomycin 60 h post-challenge with Y. pestis C12 elicited significantly better protection against aerosol infection. In addition, the Y. pestis C12 challenge dose was higher in this experiment (1.67 x 106CFU, 21 LD50s) than that used in the Figure 6 study (GM 3.62 x 105CFU, 5 LD50s). Half of the vaccinated and antibiotic treated mice survived and none of the vaccinated only or antibiotic treated only controls survived (p = 0.0325 for survival at days 7 and 21 after challenge). This layered therapy revealed that the combinations of vaccination and post-exposure antibiotic treatment were synergistic in the challenge experiments shown in Figure 6B (synergy score 4.84, p = 0.0013), Figure 6C (synergy score 2.66, p = 0.040), and Figure 7 (synergy score 3.16, p = 0.009). These synergistic effects allowed the treatment to be delayed after exposure to aerosolized Y. pestis and may facilitate a sparing effect on the amount of antibiotic required. TABLESTable 1. The bacterial burden of mice collected three days after challenge with Y. pestis C12Table 2. Total IgG antibody response against rF1V, Y. pestis CO92 or Y. pestis C12Example 2. Burkholderia pseudomallei and melioidosis 2.1. Materials and methods 2.1.1. Mouse challenge models

[0135] C57BL / 6 female mice (approximately 7–9 weeks at time of vaccination) were purchased from Charles River (Frederick, MD, United States). In this study only female mice were used. It has been previously shown that female and male mice are similar regarding disease course but females are preferred for long course studies due to aggression patterns and self-injury in male mice that often require early euthanasia intervention (Klimko et al., 2020). For challenge studies, mice were exposed to aerosolized B. pseudomallei K96243 on day 32 (±3) post-last vaccination, as described previously (Bearss et al., 2017; Trevino et al., 2018). Except as identified in footnotes included in tables, all groups of mice had N = 10. Briefly, B. pseudomallei K96243 was grown overnight in 4% glycerol (Sigma Aldrich, St. Louis, MO, United States), 1% tryptone (Difco, Becton Dickinson, Sparks, MD, United States), and 5% NaCl (Sigma Aldrich) broth (GTB) at 37°C and shaking at 200 rpm. Cultures were harvested by centrifugation and resuspended in fresh GTB medium prior to aerosolization. During the aerosolization procedures an all-glass impinger (AGI) was used to sample aerosolized material so that approximations of inhaled doses could be determined. The challenge doses for each cohort are described in the results. Early endpoint euthanasia was employed in accordance with previously approved intervention criteria. Research was conducted under an IACUC approved protocol in compliance with the Animal Welfare Act, PHS Policy, and other Federal statutes and regulations relating to animals and experiments involving animals. The facility where this research was conducted is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 2011.

[0136] At the end of study, surviving mice were euthanized and examined for evidence of detectable pyogranulomas during gross necropsy. After euthanasia, the lungs and spleens were removed, weighed, and homogenized for colony forming unit (CFU) determination to approximate bacterial load present in each organ. Homogenates were plated on sheep blood agar plates (Remel ThermoFisher, Rockville, MD, United States) and incubated at 37°C for 48 h. The limit ofdetection for each organ was determined to be approximately 5 CFU per organ based upon the homogenization and plating protocol used. 2.1.2. Vaccination strategies

[0137] Vaccinations were carried out as previously described (Burtnick et al., 2012, 2018; Amemiya et al., 2019; Biryukov et al., 2022). Briefly, mice receiving the live attenuated vaccine 668 ΔilvI were vaccinated on day 0 and day 21 or 24 with a target dose of approximately 1.0 × 107CFU delivered in 200 μl injections subcutaneously. Mice described in Table 1 received a prime vaccine dose of approximately 8.6 × 106CFU on day 0 and then a booster dose of approximately 9.8 × 106CFU on day 21. Mice described in Tables 9 & 10 received a prime vaccine dose of 8.0 × 106CFU on day 0 and then a booster dose of approximately 1.4 × 106CFU on day 24.

[0138] Mice receiving subunit vaccines were vaccinated three times (day 0, day 21, and day 35 for the experiment listed in Table 8 and day 0, day 21, and day 38 for the experiments listed in Tables 9 & 10). Each dose of the subunit vaccine included 0.5 μg of the recombinant proteins listed: enzymatically inactive AhpC; (Schmidt et al., 2022) and / or tagless Hcp1;, 0.25 μg of the CPS conjugated to CRM197 (conjugate) (Fina Biosolutions, Rockville, MD, United States); 250 μg of Alhydrogel (Invivogen, San Diego, CA, United States), and 10 μg of CpG ODN 2006 (Invivogen). The subunit vaccines were also delivered in 200 μl doses, but with 100 μl being delivered to each hind flank. In general, the mice responded well to all vaccines administered; however, in some cases significant reactogenicity (most often associated with the administration of the vaccine formulation containing AhpC) resulted in several mice being removed from the study before exposure to aerosolized B. pseudomallei. The experimental groups that had mice removed from study are noted as footnotes in the data tables. Submandibular blood collections were taken approximately 1 week prior to challenge to assess the immune response in the mice prior to exposure to aerosolized B. pseudomallei. 2.1.3. Antibiotic regimens

[0139] The antibiotic chosen for this study was co-trimoxazole (Teva Pharmaceuticals USA Inc., North Wales, PA, United States) diluted to achieve a dose of approximately 100 mg / kg of sulfamethoxazole USP and 20 mg / kg of trimethoprim USP. The co-trimoxazole was diluted in 5%dextrose in water. Control animals (no antibiotics) received 5% dextrose in water alone. Antibiotics were delivered via intraperitoneal injections every 12 h for either 7 or 21 days as described in the data tables. 2.1.4. Enzyme-linked immunosorbent assay analyses

[0140] Immunoglobulin G (IgG) titers in vaccinated mice were determined by ELISA as described by Trevino et al. (2018). The capture reagents included capsule CPS and whole-cell radiation- inactivated B. pseudomallei K96243 cells (BpK). The antibody titer results obtained from the pooled sera samples are reported as the geometric mean (GM) and geometric standard error (GSE) of the reciprocal of the highest dilution giving a mean OD of at least 0.100 ± 1 SD at 450 nm with a reference filter (570 nm). The limit of detection was a reciprocal titer of 50 and samples with an antibody titer of <50 were considered negative. The labeled secondary antibody used in the ELISAs was goat anti-mouse IgG obtained from Southern Biotechnology Associates, Inc. (Birmingham, AL, United States). 2.1.5. Statistics

[0141] The survival rates at selected time points were compared by Fisher exact test and the times to morbidity (TTM) were analyzed by Log-rank test. Where feasible, the potential synergy between antibiotic and vaccine was analyzed by forming a test of interaction in a log-logistic accelerated failure time model. The synergy score is the fold increase in survival time associated with vaccination in the antibiotic treated animals, divided by the fold increase in survival time associated with vaccination in the absence of antibiotic treatment. A Wald test was used to compare the synergy score to 1. Analysis was implemented in SAS version 9.4 (SAS Institute Inc., Carry, NC, United States). 2.2. Extended co-trimoxazole regimen initiated early after exposure to aerosolized Burkholderia pseudomallei results in significant protection of C57BL / 6 mice

[0142] As shown in Table 8, when 21 days of co-trimoxazole is initiated within 45 h or less after naïve mice are exposed to aerosolized B. pseudomallei (approximately 7 LD50 equivalents) the survival rates 82 days post-exposure to B. pseudomallei are 90% or greater. Mice that received the co-trimoxazole starting within 21 h demonstrated 100% sterile immunity and if the co-trimoxazole was initiated at 45 h post-exposure, 80% of the mice were determined to be free of B. pseudomallei(as determined by culturing of lung, spleen, and blood). If treatment was delayed to approximately 69 h post-exposure to B. pseudomallei, the survival rate was reduced to 30% and sterility was observed in two out of three surviving mice.

[0143] As previously discussed, several current promising B. pseudomallei vaccine candidates have been characterized. These vaccines include protein subunit conjugate vaccines and live attenuated vaccines, and they result in various antibody titers and levels of protection after exposure to aerosolized B. pseudomallei. The protein subunit vaccine consisting of AhpC, Hcp1, CPS conjugated to CRM197, Alhydrogel, and CpG was first examined as well as the live attenuated vaccine strain 668 ΔilvI. In this experiment, the protein subunit vaccine, and the vaccine strain 668 ΔilvI resulted in 60% and 20% protection at the end of study, respectively (Table 8). However, when the vaccinated mice were also treated with co-trimoxazole after exposure to aerosolized B. pseudomallei the survival rates were 90% or greater and all surviving mice were determined to be free from infection with B. pseudomallei. The layering of the medical countermeasures resulted in statistically significant improvement in the survival rates and time to morbidity (TTM) compared with vaccinated mice with no post-exposure antibiotic treatment (Table 8). While promising, this early initiation of treatment coupled with an extended antibiotic dosing schedule resulted in data that did not demonstrate significant synergy between the vaccines and antibiotic regimen because co-trimoxazole alone rescued most of the non-vaccinated mice and only three survivors had detectable bacteria in the lungs. 2.3. Vaccinated mice are protected with shorter duration or delayed courses of co- trimoxazole

[0144] To more stringently evaluate the extent of the protection co-trimoxazole provided vaccinated mice, two additional experiments were performed. As described in Table 9 and Figure 8, naïve or vaccinated mice were exposed to approximately 6 LD50equivalents of aerosolized B. pseudomallei K96243 followed by a shortened 7 day, rather than a 21-day, time course of antibiotics post-challenge. In this experiment, results were similar to those obtained from the cohort that received co-trimoxazole for 21 days (Table 8), with the exception being that the shortened time course offered an opportunity to demonstrate statistically significant synergy between the vaccines and antibiotic therapy. Among animals not treated by co-trimoxazole, modeling of survival times found an estimated 5.7-fold and 3.7-fold increase in survival times inmice immunized with AhpC + Hcp1 + conjugate or 668 ΔilvI, respectively. By comparison, treatment with co-trimoxazole at 69 h gave improvements in survival times of 40.4- and 26.8-fold in these two immunized groups. The increase in the relative effect of these vaccines was statistically significant (P < 0.01 by Wald test) in each case and gives evidence of a synergistic effect. The high levels of survival did not permit similar quantification of the synergy in each immunization group, respectively. However, vaccinated mice that received the antibiotics in this delayed regimen demonstrated 80% or greater survival rates and only a few mice retained low levels of B. pseudomallei in the lungs and spleens.

[0145] A third experiment, summarized in Table 10 and Figure 9, evaluated naïve or vaccinated mice that were exposed to approximately 4 LD50equivalents of aerosolized B. pseudomallei K96243 and were then treated with a 21-day antibiotic regimen with a delayed initiation time (approximately 93 or 117 h post-challenge). With these infection and treatment parameters, none of the naïve mice survived the challenge (all succumbed or were euthanized within 21 days). Importantly, in this experiment, 50% of the non-vaccinated mice succumbed or were euthanized prior to the initiation of co-trimoxazole at 93 h post-exposure to B. pseudomallei and 100% of the non-vaccinated mice succumbed or were euthanized prior to the initiation of co-trimoxazole at 117 h post-exposure to B. pseudomallei. Vaccinated mice that received the antibiotics in this delayed regimen demonstrated 90% or greater survival rates and only a few mice retained low levels of B. pseudomallei in the lungs and spleens.Table 8. Vaccinated or naïve C57BL / 6 mice are similarly protected after receiving 21 days of co- trimoxazole initiated within 45 h after exposure to aerosolized B. pseudomallei. Vaccinated mice were more likely to survive than naive mice when co-trimoxazole was initiated 69 h post-exposure.Table 9. Vaccinated or naïve C57BL / 6 mice are similarly protected after receiving 7 days of co- trimoxazole initiated within 45 h after exposure to aerosolized B. pseudomallei. Only vaccinated mice survived when co-trimoxazole was initiated 69 h post-exposure.Example 3

[0146] The F1 capsule protein (encoded by caf1) is the major protective vaccine antigen ; however, protection against nonencapsulated (F1-) strains has been elusive. We constructed two novel live attenuated vaccine (LAV) strains on the pgm-pPst- background, which is excluded from the CDC select agent list due to established safety. These strains, pgm-, pPst-, Δcaf1 (nonencapsulated) and pgm-, pPst-, ΔyopD / Δcaf1 (nonencapsulated and impaired type III secretion system), both lack F1 and may be more effective at inducing immunity against nonencapsulated strains like Y. pestis C12.

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Claims

CLAIMS What is claimed is 1. A method of prophylactic or therapeutic treatment of bubonic- and / or pneumonic plague caused by Yersinia pestis infection in a subject; the method comprising steps of: i. before infection, optionally at least about 28 (±3) days before infection, administering a therapeutically effective amount of a vaccine composition to the subject comprising at least one live attenuated vaccine (LAV) selected from Y. pestis mutant C12 ∆yscN, CO92 ∆yscN, or CO92 pgm- pPst-, in particular combination of C12 ∆yscN and CO92 ∆yscN, or CO92 pgm- pPst-, and ii. before 48-60 hours after infection, administering to the subject an antibiotic treatment to an effective amount of an antibiotic or antibiotics.

2. A method of prophylactic or therapeutic treatment of bubonic- and / or pneumonic plague caused by Yersinia pestis infection in a subject; the method comprising steps of, i. administering to the subject a therapeutically effective amount of a first vaccine composition comprising a recombinant protein subunit of rF1V or rV with or without CpG, combination of rF1V and rV with or without CpG, or at least one LAV selected from Y. pestis mutant C12 ∆yscN, CO92 ∆yscN, or CO92 pgm- pPst-, in particular combination of CO92 ∆yscN and C12 ∆yscN, or CO92 pgm- pPst-, ii. between 20-90 days, in particular between 20-46 days, more in particular between 20- 28 days, after the first vaccination, and before infection, optionally at least about 28 (±3) days before infection, administering to the subject a therapeutically effective amount of a second (a first booster) vaccine composition comprising CpG, a recombinant protein subunit of rF1V or rV with or without CpG, combination of rF1V and rV with or without CpG, and / or at least one LAV selected from Y. pestis mutant C12 ∆yscN, CO92 ∆yscN, or CO92 pgm- pPst-, wherein if the first vaccine composition comprises only subunit(s) of rF1V and / or rV, the second vaccine composition comprises at least one LAV selected from Y. pestis mutant C12 ∆yscN, CO92 ∆yscN, or CO92 pgm- pPst-, and, optionally,iii. before 48-60 hours after infection, administering to the subject an antibiotic treatment with an effective amount of an antibiotic or antibiotics.

3. A method of prophylactic or therapeutic treatment of bubonic and / or pneumonic plague caused by Yersinia pestis infection in a subject; the method comprising steps of, i. Administering to the subject a therapeutically effective amount of a first vaccine composition comprising a recombinant protein subunit of rF1V or rV with or without CpG, combination of rF1V and rV with or without CpG, and / or at least one LAV selected from Y. pestis mutant C12 ∆yscN, CO92 ∆yscN, or CO92 pgm- pPst-, in particular combination of CO92 ∆yscN and C12 ∆yscN, or CO92 pgm- pPst-, ii. between 20-90 days, in particular between 20-46 days, more in particular between 20- 28 days, after the first vaccination, administering to the subject a therapeutically effective amount of a second (a first booster) vaccine composition comprising CpG, a recombinant protein subunit of rF1V or rV with or without CpG, combination of rF1V and rV with or without CpG, or at least one LAV selected from Y. pestis mutant C12 ∆yscN, CO92 ∆yscN, or CO92 pgm- pPst-, iii. between 20-90 days, in particular between 20-46 days, more in particular between 20- 28 days, after the second vaccination, and before infection, optionally at least about 28 (±3) days before infection, administering to the subject a therapeutically effective amount of a third (a second booster) vaccine composition comprising CpG, a recombinant protein subunit of rF1V or rV with or without CpG, combination of rF1V and rV with or without CpG, or at least one LAV selected from Y. pestis mutant C12 ∆yscN, CO92 ∆yscN, or CO92 pgm- pPst-, wherein if the first and second vaccine compositions do not comprise a LAV or LAVs, the third composition comprises at least one LAV selected from Y. pestis mutant C12 ∆yscN, CO92 ∆yscN, or CO92 pgm- pPst-, and, optionally, iv. before 48-60 hours after infection, administering to the subject an antibiotic treatment with an effective amount of an antibiotic or antibiotics.

4. The method of any one of the preceding claims, wherein the antibiotic is an aminoglycoside antibiotic or combination thereof, in particular streptomycin.

5. The method of any one of the preceding claims, wherein a dose of streptomycin is 10-100 mg / kg / dose, in particular 20-40 mg / kg / dose, and wherein the streptomycin is administered through intramuscular or intravenous injection every 6 hours for 5-30 days.

6. The method of any one of claims 1-3, wherein a single dose volume of the vaccine composition is 0.1-0.5 mL, and wherein the vaccine composition is administered through subcutaneous, intradermal or intramuscular route.

7. The method of any one of claims 1-3, wherein a therapeutically effective amount of a LAV or a total amount of LAVs in the vaccine composition is between 0.1 - 10.0 × 106-7CFU per kg body weight.

8. The method of any one of claims 1-3, wherein a therapeutically effective amount of a recombinant protein subunit rF1V or rV, or combination of both subunits in the vaccine composition is between 0.1-10 µg per kg body weight.

9. The method of claim 2 or 3, wherein a content of CpG is between 0.1-30 µg per kg body weight.

10. The method of claim 2 or 3, wherein the recombinant protein subunit(s), when administered without a LAV, is administered in a buffer solution, in particular potassium phosphate (10 mM, pH 7.3–7.4) with an adjuvant comprising an aluminum hydroxide wet gel suspension, wherein a content of the aluminum hydroxide wet gel suspension is between 1-500 µg per kg body weight.

11. The method of claim 2 or 3, wherein the first vaccine composition comprises the combination of CO92 ∆yscN and C12 ∆yscN, or CO92 pgm- pPst-, and the second vaccine composition comprises rF1V or rV with or without CpG.

12. A method of prophylactic or therapeutic treatment of melioidosis caused by Burkholderia pseudomallei infection in a subject; the method comprising steps of: i. before infection, optionally at least about 32 (±3) days before infection, administering to the subject a therapeutically effective amount of a live attenuated vaccine (LAV) composition of B. pseudomallei, 668 ΔilvI two times, optionally on day 0 and day 21-24, andii. before 117 hours, in particular 69 hours, in more particular 45 hours, after infection, administering to the subject an antibiotic treatment with an effective amount of an antibiotic or antibiotics.

13. A method of prophylactic or therapeutic treatment of melioidosis caused by Burkholderia pseudomallei infection in a subject; the method comprising steps of: i. before infection, optionally at least about 32 (±3) days before infection, administering to the subject a therapeutically effective amount of recombinant protein subunit vaccine composition comprising AhpC and / or Hcp1 three times, optionally on day 0, day 21-24, and day 35-38; ii. before 117 hours, in particular 69 hours, in more particular 45 hours, after infection, administering to the subject an antibiotic treatment with an effective amount of an antibiotic or antibiotics.

14. A method of prophylactic or therapeutic treatment of melioidosis caused by Burkholderia pseudomallei infection in a subject; the method comprising steps of; before infection, optionally at least about 32 (±3) days before infection, i. administering to the subject a first therapeutically effective amount of a live attenuated vaccine (LAV) composition of B. pseudomallei, 668 ΔilvI or recombinant protein subunit vaccine composition comprising AhpC and / or Hcp1, ii. administering to the subject a second therapeutically effective amount of a live attenuated vaccine (LAV) of B. pseudomallei, 668 ΔilvI or recombinant protein subunit vaccine composition comprising AhpC and / or Hcp1, before 90 days optionally before 60 days, more optionally before 30 days after the first vaccination, wherein the second vaccine composition comprises a LAV or protein subunit(s) not included in the first vaccine composition, , and iii. before 117 hours, in particular 69 hours, in more particular 45 hours, after infection, administering to the subject an antibiotic treatment with an effective amount of an antibiotic or antibiotics.

15. The method of claim 13 or 14, wherein the recombinant protein subunit vaccine composition can be administered with adjuvant, which comprises capsular polysaccharides (CPSs) conjugated to Cross-Reactive-Material-197 (CRM197, a non-toxic mutant of diphtheriatoxin as a carrier protein), CpG oligodeoxynucleotides, and / or an aluminum hydroxide wet gel suspension, wherein the content of the CPS conjugated to CRM197 is 0.1-10 μg per kg body weight, the content of CpG ODN is 0.1-20 μg per kg body weight and the content of the aluminum hydroxide wet gel suspension is 0.1-500 μg per kg body weight.

16. The method of any one of claims 12-14, wherein the antibiotic is co-trimoxazole (trimethoprim / sulfamethoxazole).

17. The method of any one of claims 12-14, wherein a dose of co-trimoxazole is trimethoprim 160 mg and sulfamethoxazole 800 mg, and wherein the co-trimoxazole is administered through intramuscular injection or oral tablet every 12 hours for 7-21 days.

18. The method of any one of claims 12-14, wherein a single dose volume of the vaccine composition is 0.1-0.5 mL, and wherein the vaccine composition is administered through subcutaneous, intradermal or intramuscular route.

19. The method of claim 12, wherein a therapeutically effective amount of LAV in the first vaccine composition is between between 0.1 - 10.0 × 106-7CFU per kg body weight, and a therapeutically effective amount of LAV in the second vaccine composition is between between 0.1 - 10.0 × 106-7CFU per kg body weight.

20. The method of claim 14, wherein a therapeutically effective amount of LAV in the vaccine composition is between 0.1-10 µg per kg body weight.

21. The method of claim 13 or 14, wherein a therapeutically effective amount of a recombinant protein subunit AhpC and / or Hcp1, or combination of both subunits in the vaccine compositions is between0.1-10 µg per kg body weight.

22. A composition comprising a recombinant protein subunit of rF1V or rV with or without CpG, combination of rF1V and rV with or without CpG, and / or at least one LAV selected from Y. pestis mutant C12 ∆yscN, CO92 ∆yscN, or CO92 pgm- pPst-, in particular combination of CO92 ∆yscN and C12 ∆yscN, or CO92 pgm- pPst-.

23. The composition of claim 22, wherein the composition comprises at least one LAV selected from Y. pestis mutant C12 ∆yscN, CO92 ∆yscN, or CO92 pgm- pPst-, in particular combination of CO92 ∆yscN and C12 ∆yscN, or CO92 pgm- pPst-.

24. A composition comprising at least one LAV strain selected from pgm-, pPst-, Δcaf1 or pgm- , pPst-, ΔyopD / Δcaf1.

25. A method of prophylactic or therapeutic treatment of bubonic- and / or pneumonic plague caused by Yersinia pestis infection in a subject; the method comprising steps of, i. administering to the subject a therapeutically effective amount of a first vaccine composition comprising a recombinant protein subunit of rF1V or rV with or without CpG, combination of rF1V and rV with or without CpG, or at least one LAV selected from Y. pestis mutant pgm-, pPst-, Δcaf1 and / or pgm-, pPst-, ΔyopD / Δcaf1, ii. between 20-90 days, in particular between 20-46 days, more in particular between 20- 28 days, after the first vaccination, and before infection, optionally at least about 28 (±3) days before infection, administering to the subject a therapeutically effective amount of a second (a first booster) vaccine composition comprising CpG, a recombinant protein subunit of rF1V or rV with or without CpG, combination of rF1V and rV with or without CpG, and / or at least one LAV selected from Y. pestis mutant pgm-, pPst-, Δcaf1 and / or pgm-, pPst-, ΔyopD / Δcaf1, wherein if the first vaccine composition comprises only subunit(s) of rF1V and / or rV, the second vaccine composition comprises at least one LAV selected from Y. pestis mutant pgm-, pPst-, Δcaf1 and / or pgm-, pPst-, ΔyopD / Δcaf1, and, optionally, iii. before 48-60 hours after infection, administering to the subject an antibiotic treatment with an effective amount of an antibiotic or antibiotics.

26. A method of prophylactic or therapeutic treatment of bubonic and / or pneumonic plague caused by Yersinia pestis infection in a subject; the method comprising steps of, i. Administering to the subject a therapeutically effective amount of a first vaccine composition comprising a recombinant protein subunit of rF1V or rV with or without CpG, combination of rF1V and rV with or without CpG, and / or at least one LAV selected from Y. pestis mutant pgm-, pPst-, Δcaf1 and / or pgm-, pPst-, ΔyopD / Δcaf1, ii. between 20-90 days, in particular between 20-46 days, more in particular between 20- 28 days, after the first vaccination, administering to the subject a therapeutically effective amount of a second (a first booster) vaccine composition comprising CpG, a recombinant protein subunit of rF1V or rV with or without CpG, combination of rF1V and rV with or without CpG, or atleast one LAV selected from Y. pestis mutant pgm-, pPst-, Δcaf1 and / or pgm-, pPst- ,ΔyopD / Δcaf1, iii. between 20-90 days, in particular between 20-46 days, more in particular between 20- 28 days, after the second vaccination, and before infection, optionally at least about 28 (±3) days before infection, administering to the subject a therapeutically effective amount of a third (a second booster) vaccine composition comprising CpG, a recombinant protein subunit of rF1V or rV with or without CpG, combination of rF1V and rV with or without CpG, or at least one LAV selected from Y. pestis pgm-, pPst-, Δcaf1 and / or pgm-, pPst-, ΔyopD / Δcaf1, wherein if the first and second vaccine compositions do not comprise a LAV or LAVs, the third composition comprises at least one LAV selected from Y. pestis mutant pgm- , pPst-, Δcaf1 and / or pgm-, pPst-, ΔyopD / Δcaf1, and, optionally, iv. before 48-60 hours after infection, administering to the subject an antibiotic treatment with an effective amount of an antibiotic or antibiotics.

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