Introduction:Yersinia pestis is the etiologic agent of plague, and the disease is categorized into several forms, including bubonic, septicemic, and pneumonic. Plague meningitis is a rare but severe complication and estimated to occur in 6-11% of documented cases. It is most frequently observed in bubonic plague patients under 15 years old that receive inadequate or no antibiotic treatment. To date, there are no reports describing plague meningitis in laboratory animal models of pneumonic plague. Methods:Therefore, we sought to use the BALB/c mouse pneumonic plague model to investigate central nervous system (CNS) involvement after exposure to aerosolized Y. pestis. We used a multifaceted approach analyzing bacterial burden, histopathological analyses, transcriptomic data, and cytokine expression in mice exposed to aerosolized Y. pestis CO92 collected at intervals post-exposure for 3 days. Results:Y. pestis was detected in brain homogenates as early as 2 days post challenge. CNS involvement is further supported by increased pro-inflammatory cytokine expression in the brain homogenates including IL-6. Histopathological analyses conducted in mice and confirmed in non-human primate tissue sections did not demonstrate meningitis but rather indicated that the bacteria remain within the blood vessels of the cerebellum, cerebrum, and nasal turbinates. However, transcriptomic data targeting mouse neuroinflammatory responses indicated alterations in several transcriptional signatures of gene sets, including those that regulate astrocyte, oligodendrocyte, and microglial cell functions. Discussion:While Y. pestis does not appear to breach the blood vessels resulting in meningitis in our acute models of pneumonic plague, we found evidence of a neuroinflammatory response within the brain homogenates of infected mice. We also compared this mouse model of pneumonic plague to a mouse model of inhalational melioidosis, a known neuroinvasive disease caused by Burkholderia pseudomallei. The establishment of a murine model of plague-induced neuroinflammation described herein will contribute to the refinement of animal models, development of medical countermeasures for neurological infections or neurological impacts associated with systemic infection, and improvement of diagnostic strategies for Y. pestis.

Encephalomyelitis is uncommon but is associated with Burkholderia pseudomallei strains whose bimA gene, encoding an actin-based motility protein, resembles bimA from Burkholderia mallei (designated bimABm). We previously characterized the virulence of B. pseudomallei wild-type ATS2021 (bimABm). In a mouse model of ATS2021 aerosol exposure, lesions and inflammatory cytokines in the brain were seen early post-exposure and reached the central nervous system (CNS) through the olfactory nerve. In the current study, the bimA allele in the wild-type ATS2021 was replaced with the bimABp, and virulence was assessed by estimations of LD50, histopathological analyses, and bacterial dissemination. ATS2021 bimABp reached the CNS but was significantly attenuated, with higher LD50 estimates, altered bacterial dissemination, and reduced neuroinflammation. The immune response in lungs, spleens, and brains from mice exposed to aerosols indicated either strain induced shared and divergent immune patterns. Finally, the evaluation of the host response in brains via NanoString transcriptomic platform analyses suggested that the wild-type ATS2021 induced substantial changes in astrocyte and microglial activation, oligodendrocyte function, and overall inflammatory signaling, compared to the mutant bimABp bacteria. These results support a direct role of the bimABm gene product for bacterial dissemination after inhalation.

Melioidosis, caused by Burkholderia pseudomallei, is an emerging disease in the United States and was declared endemic to the Gulf Coast region in 2022. Melioidosis has been sporadically reported in the American continents, the Caribbean, and more recently in Africa. We conducted lethal dose analyses in BALB/c and C57BL/6 mice exposed to small particle aerosols of B. pseudomallei strains from the Western Hemisphere and Africa. Those isolates exhibit a variety of virulence patterns, including rapidly-lethal disease and delayed onset of fatal disease. We found that the isolates we tested grew similarly in culture but displayed major differences in biofilm formation. Our data contribute to the growing knowledge of geographically distinct B. pseudomallei isolates and aid in ensuring that medical countermeasures in development are effective against a diverse collection of bacterial strains.

IntroductionEthiqa XR is an extended-release formulation of the potent partial opioid buprenorphine and has been FDA-indexed for mice and other laboratory animal species to relieve pain. Unfortunately, the use of analgesia may produce confounding effects that distort physiological and pathophysiological responses during laboratory animal studies. Since various reports have indicated that Ethiqa XR can affect the inflammatory response in several in vivo models, we sought to evaluate the effects of Ethiqa XR treatment on the immune response to and disease pathogenesis associated with bacterial biothreat agents.MethodsBALB/c and C57BL/6 mouse strains were treated or not with Ethiqa XR before and 48 h after challenge with aerosolized Burkholderia pseudomallei K96243 or Yersinia pestis CO92. Control mice were similarly treated with Ethiqa XR but were not challenged. Mice were euthanized 60-72 hours post-treatment/challenge to harvest blood, serum, lung, brain and spleen for bacterial burden and immunological profiling.ResultsBoth C57BL/6 and BALB/c mouse strains showed higher bacterial dissemination to the spleen 60-72 h after B. pseudomallei challenge when treated with Ethiqa XR. Consistently, we found increased concentration of pro-inflammatory cytokines in the spleen (e.g., IFN-γ and IL-6) in Ethiqa XR-treated mice. Compared to BALB/c, C57BL/6 mice had trends of higher cytokine dysregulation not only in the spleens, but also in the lungs and brains. In contrast, we found less profound differences in either mouse strain treated with Ethiqa XR and challenged with Y. pestis. In the absence of bacterial challenge, both BALB/c and C57BL/6 mouse strains treated with Ethiqa XR had an overall higher concentration of pro-inflammatory cytokines in the lungs and spleens. However, C57BL/6 mice showed a higher dysregulation of the cytokine profile in both lungs and spleens. We also found decreased macrophage activity (iNOS concentration) in the spleens of BALB/c mice and decreased neutrophil activity (MPO concentration) in the lung of C57BL/6 mice treated with Ethiqa XR. DiscussionThese results suggest that the effect of Ethiqa XR on the immune response and disease pathogenesis increases the complexity of data interpretation. Thus, prior to providing analgesia to laboratory animals, bridging studies and cost benefit analyses must be considered to avoid misinterpretation of immunological data collected during the development, testing, and evaluation of medical countermeasures.

ABSTRACT Bacillus anthracis is a gram-positive, spore-forming bacterium and the etiological agent of anthrax. As a Tier 1 Select Agent, the pathogen can be isolated from the natural environment, and the endospore is a significant biological threat that can be produced in large quantities, stored, and disseminated by aerosolization. The intentional release of B. anthracis spores has the potential for mass casualties and is a serious threat to public and military health. Fluoroquinolones and tetracyclines are common antibiotics used for post-exposure prophylaxis and treatment in the United States (U.S.), whereas penicillin is the drug of choice throughout the rest of the world. However, these commonly used antibiotics may be contraindicated or suboptimal in a public health crisis. Thus, there is a need for novel medical countermeasures to be developed and tested against B. anthracis . Sulopenem is a broad-spectrum, orally bioavailable penem antibiotic recently approved in the U.S. for the treatment of uncomplicated urinary tract infections in women with limited therapeutic options. Here, we demonstrate that sulopenem has potent in vitro activity against both wild-type virulent B. anthracis strains, as well as biosafety level 2 surrogate strains resistant to current standards of care. Using human-equivalent dosing regimens, we show that sulopenem is highly protective in both the mouse and rabbit inhalational anthrax models. Together, these results support continued evaluation of this oral penem as a countermeasure for inhalational anthrax.

Burkholderia pseudomallei is a facultative intracellular bacterium found in soil, which causes melioidosis, a disease with diverse symptomatology. B. pseudomallei is an emerging threat in the United States based on recent environmental samples and case reports. Acute infection is 10%-40% fatal depending on treatment conditions. No vaccines for B. pseudomallei have been approved for human use, although several are under development, mostly targeting the antigens Hcp1 (hemolysin-coregulated protein 1) and CPS (capsular polysaccharide). For development of new vaccines, DNA compares favorably to other platforms in storage stability, low cost, and ease of design. Needle-free jet injection has been effective in immunizing against several infections in laboratory animals; the delivery devices are simple to use and have been FDA 510k cleared for human use. Herein, we developed a DNA vaccine targeting Hcp1 (pWRG/Hcp1) and delivered it to rabbits and mice by jet injection using a PharmaJet Stratis and a prototype adjustable-dose PharmaJet Tropis, respectively. The Hcp1 DNA vaccine was unadjuvanted and not combined with any other B. pseudomallei antigens. Immunization was followed by assessment of serum antibodies and cellular immunity against Hcp1 protein. Rabbits and mice showed induction of anti-Hcp1 antibodies after as few as two doses of pWRG/Hcp1, and splenocytes responsive to restimulation with Hcp1 protein were also detected after two doses. These results demonstrate the feasibility of inducing immunity against Hcp1 of B. pseudomallei using DNA alone. These results also serve as a proof-of-concept for immunizing mice with a PharmaJet device previously only used for larger animals.

Introduction:Yersinia pestis is the etiological agent of plague, a disease that remains a concern as demonstrated by recent outbreaks in Madagascar. Infection with Y. pestis results in a rapidly progressing illness that can only be successfully treated with antibiotics given shortly after symptom onset. Live attenuated or whole cell inactivated vaccines confer protection against bubonic plague, but pneumonic plague has been more difficult to prevent. Novel effective subunit vaccine formulations may circumvent some of these shortfalls. Here, we compare the immunogenicity generated by an advanced subunit vaccine (F1V fusion protein) and a nanolipoprotein particle (NLP)-based vaccine. Methods:The NLP, a high-density lipoprotein mimetic, provides a nanoscale delivery platform for recombinant Y. pestis antigens LcrV (V) and F1. BALB/c mice were immunized via subcutaneous injection twice, three or four weeks apart. Four weeks later, splenocytes and sera were collected for immune profiling, and mice were challenged with aerosolized Y. pestis CO92. Results:Both formulations induced a strong IgG response against the F1 and V proteins, along with a robust memory B cell response and a balanced cell-mediated immune response as evidenced by both Th1- and Th2-related cytokines. The NLP-based vaccine induced a stronger cytokine response against F1, V, and F1V proteins relative to the F1V vaccine. As with F1V, the inclusion of Alhydrogel (Alu) in NLP vaccine formulations was critical for enhanced immunogenicity and protective efficacy. Mice that received two doses of F1:V:NLP + Alu and CpG were completely protected from a challenge with approximately eight median lethal doses of aerosolized Y. pestis CO92 and this protection confirmed the well-documented synergy between the F1 and V antigens in context of pneumonic plague. The NLPs have defined regions of polarity that facilitates the incorporation of a wide range of adjuvants and antigens with distinct physicochemical properties and are an excellent candidate platform for the development of multi-antigen vaccines.

Introduction:Melioidosis is a major cause of disease and mortality in endemic tropical regions, and the etiologic agent, Burkholderia pseudomallei, is being isolated increasingly from an expanded range of environmental and clinical sources in locations including the United States. The disease can have multi-faceted clinical presentations and requires a complex and protracted treatment regimen which is confounded by resistance of this microbe to numerous antibiotics. Thus, prophylactic countermeasures are needed; however, a vaccine has yet to be licensed for human use. Since B. pseudomallei is classified as a Tier 1 select agent, the development of a safe and effective vaccine is both a military and public health need. Our laboratories have focused on the development of vaccines composed of live attenuated strains and defined subunit antigens. Methods:In the current study, we evaluated homologous and heterologous combinations of candidate subunits and live vaccines in a murine aerosol model of melioidosis to determine the effects of vaccine composition and delivery scheme on protection in conjunction with immune responses and bacterial clearance. Results:Both strategies provided significant protection against lethal aerosol challenges, and the accumulated data support that a heterologous vaccination strategy employing capsular polysaccharide conjugate and Hcp1 subunits and a live but highly safe capsular polysaccharide-producing surrogate strain of B. thailandensis is an effective and potentially agile prophylactic strategy.

Francisella tularensis is the etiological agent of the potentially fatal disease tularemia. F. tularensis is sensitive to several antibiotic classes; however, the ability to derive antibiotic resistant Francisella strains has been well documented. Therefore, there is a need for new antimicrobials to be developed which requires animal models suitable to recapitulate critical inpatient therapy. We performed a proof-of-concept study utilizing intravenous catheterized rats which had been challenged with the Schu S4 strain of F. tularensis. Using gentamicin and levofloxacin as representative antibiotics, we first determined the pharmacokinetics from intravenous infusions of these drugs in rats to establish human-equivalent doses. Next, catheterized Fischer rats were aerosol challenged with F. tularensis. Three days after challenge, the rats received gentamicin or levofloxacin via intravenous infusion. Antibiotic or saline (vehicle control) was delivered twice daily for 14 days. All challenged rats provided antibiotic treatment survived to the end of the study. In contrast, the rats provided only saline succumbed or were euthanized in accordance with early endpoint euthanasia criteria by day 7 post-challenge. This study was purposely performed in two iterations to allow technical optimization. In the first iteration, catheter patency pre- and post-exposure was difficult to maintain leading to adverse clotting events and the loss of six rats. In the second iteration, animals were provided a constant rate infusion via syringe pumps with saline which improved catheter patency. However, two rats were still lost. Overall, we demonstrate the ability of delivering an intravenous humanized antibiotic treatment in the preferred small animal model of tularemia which can be used as a basis to test future therapeutics.

Bacillus anthracis is the etiological agent of anthrax and is classified as a Tier 1 biothreat pathogen. The fluoroquinolone ciprofloxacin is a preferred prophylactic drug for potential anthrax infections and acts by stabilizing DNA strand breaks formed by the bacterial type II topoisomerases, gyrase and topoisomerase IV. Unfortunately, widespread fluoroquinolone usage has increased levels of resistance in common bacterial pathogens, raising concern that resistant B. anthracis strains could be misused. Therefore, there is great interest in developing new classes of antibacterials that are efficacious against both wild-type and fluoroquinolone-resistant B. anthracis infections. Previous studies have demonstrated that gepotidacin, a triazaacenaphthylene antibacterial that targets gyrase and topoisomerase IV, displays potent activity against B. anthracis and was efficacious in a rabbit inhalation anthrax model. Given these promising results, we evaluated the activity of OSUAB-0284, a Novel Bacterial Topoisomerase Inhibitor (NBTI) that shares a general pharmacophore with gepotidacin, against B. anthracis. OSUAB-0284 displayed activity against B. anthracis that was comparable to or better than gepotidacin. Both compounds displayed activity against fluoroquinolone-resistant cells. Gepotidacin and OSUAB-0284 increased levels of gyrase- and topoisomerase IV-mediated DNA single-stranded breaks and inhibited the overall catalytic activity of the two enzymes. Both compounds were also more potent than ciprofloxacin against wild-type gyrase and topoisomerase IV and maintained activity against fluoroquinolone-resistant enzymes. Finally, OSUAB-0284 displayed efficacy in a mouse model of inhalation anthrax. These results provide mechanistic underpinnings supporting the use of gepotidacin and OSUAB-0284 against B. anthracis and suggest that they may be potential candidates for the treatment of anthrax.

The development of medical countermeasures against pathogens of biodefense concern remains critical to protecting military and public health. This review compares data detailing antibacterial activity and efficacy for a selection of antibiotics evaluated against potential bacterial biothreat pathogens. The human safety and tolerability of these formulations were also considered. This review includes finafloxacin, levofloxacin, delafloxacin, omadacycline, gepotidacin, tebipenem and sulopenem. The selection criteria of these antibiotics were 1) the availability of an oral formulation, 2) the regulatory status (licensed by a regulatory authority or in an advanced stage of development) and 3) the availability of publicly available information on the biodefence pathogens of concern. We hope to highlight approved or advanced clinical candidates that have significant and unique potential in the biodefense space which may be deployed to protect both the public and warfighter against these bacterial infections.

Abstract Background Sulopenem (SUL) is a penem β-lactam antibiotic in development for the treatment of resistant bacterial infections. It is available as intravenous and oral prodrug formulations, and its activity aligns with the most urgent drug-resistant antimicrobial threats defined by the CDC. SUL possesses potent activity against Enterobacterales species that encode ESBLs or AmpC-type β-lactamases that confer resistance to 3rd generation cephalosporins. SUL has also demonstrated potent in vitro activity against numerous biothreat pathogens, including Bacillus anthracis at concentrations likely to be achieved after oral dosing in humans. B. anthracis is a CDC Tier 1 Select Agent, and remains of considerable concern for biodefense. The potential for mass casualties resulting from a biothreat incident combined with the risk of antibiotic resistance necessitates development of novel medical countermeasures (MCM) for B. anthracis. Orally administered SUL was previously shown to be protective in the murine model of inhalational anthrax and provided proof-of-concept to advance the drug into the well-established rabbit model. Methods Female New Zealand White rabbits were challenged with a lethal dose of B. anthracis Ames spores via the inhalational route. Post-exposure prophylaxis (PEP) was initiated at 24 ±1 h postchallenge with cohorts (N=6) of animals receiving vehicle (saline, SC), ciprofloxacin (CIP) 10 mg/kg, SC or SUL 10 mg/kg, SC. This dosing regimen was informed by pharmacokinetic models to establish a human-equivalent dose. Treatment was continued BID for 14 days. Clinical progression and survival were assessed up to 30 days postchallenge. Results The untreated saline control group demonstrated 100% lethality with a median time to death of 48 h. Both the SUL and CIP groups demonstrated 100% survival through study termination on day 30. Conclusion As a MCM for inhalational anthrax, SUL would provide considerable advantages as an addition to the slate of current standards of care. Combined with positive efficacy results in two preclinical models of inhalational anthrax and advantages offered by an orally available penem antibiotic, SUL remains a promising candidate for advanced development for treating B. anthracis. Disclosures Sailaja Puttagunta, MD, Iterum Therapeutics: Employee|Iterum Therapeutics: Employee|Iterum Therapeutics: Stocks/Bonds (Public Company)|Iterum Therapeutics: Stocks/Bonds (Public Company) Steven I. Aronin, MD, Iterum Therapeutics: Employee|Iterum Therapeutics: Employee|Iterum Therapeutics: Stocks/Bonds (Public Company)|Iterum Therapeutics: Stocks/Bonds (Public Company)

Francisella tularensis is one of the several biothreat agents for which a licensed vaccine is needed. To ensure vaccine protection is achieved across a range of virulent F. tularensis strains, we assembled and characterized a panel of F. tularensis isolates to be utilized as challenge strains. A promising tularemia vaccine candidate is rLVS ΔcapB/iglABC (rLVS), in which the vector is the LVS strain with a deletion in the capB gene and which additionally expresses a fusion protein comprising immunodominant epitopes of proteins IglA, IglB, and IglC. Fischer rats were immunized subcutaneously 1-3 times at 3-week intervals with rLVS at various doses. The rats were exposed to a high dose of aerosolized Type A strain Schu S4 (FRAN244), a Type B strain (FRAN255), or a tick derived Type A strain (FRAN254) and monitored for survival. All rLVS vaccination regimens including a single dose of 107 CFU rLVS provided 100% protection against both Type A strains. Against the Type B strain, two doses of 107 CFU rLVS provided 100% protection, and a single dose of 107 CFU provided 87.5% protection. In contrast, all unvaccinated rats succumbed to aerosol challenge with all of the F. tularensis strains. A robust Th1-biased antibody response was induced in all vaccinated rats against all F. tularensis strains. These results demonstrate that rLVS ΔcapB/iglABC provides potent protection against inhalational challenge with either Type A or Type B F. tularensis strains and should be considered for further analysis as a future tularemia vaccine.

Abstract Yersinia pestis (Yp) is the etiological agent of plague, a disease that remains a concern as demonstrated by recent outbreaks in Madagascar. Infection with Yp results in a rapidly progressing illness that can only be treated with antibiotics. Live attenuated or whole-cell inactivated vaccines confer short-lived protection against bubonic but not pneumonic plague. Subunit vaccine formulations may circumvent some of these shortfalls. Here we compare the immunogenicity generated by the most advanced subunit vaccine (rF1V) against a nanolipoprotein particle (NLP)-based vaccine. BALB/c mice were immunized twice, four weeks apart. Four weeks later, splenocytes and sera were collected for immune profiling and mice were aerosol challenged with Yp CO92. Both formulations induced a strong IgG response against F1 and V proteins, along with a robust memory B cell response and cell-mediated response with Th1- and Th2-related cytokines. NLP-based vaccine induced a stronger cytokine response against F1, V, and F1V proteins relative to the rF1V vaccine. As with rF1V, the inclusion of Alhydrogel in NLP vaccine formulations was critical for enhanced immunogenicity and protective efficacy. Addition of Francisella tularensis antigens to the Yp NLP vaccine platform did not impair vaccine efficacy, based on protection and antibody response. The modularity and lipid bilayer structure of NLPs allows for incorporation of many protective antigens with variable ratios against multiple pathogens.

BackgroundYersinia pestis is the etiological agent of plague, which can manifest as bubonic, septicemic, and/or pneumonic disease. Plague is a severe and rapidly progressing illness that can only be successfully treated with antibiotics initiated early after infection. There are no FDA-approved vaccines for plague, and some vaccine candidates may be less effective against pneumonic plague than bubonic plague. Y. pestis is not known to impact males and females differently in mechanisms of pathogenesis or severity of infection. However, one previous study reported sex-biased vaccine effectiveness after intranasal Y. pestis challenge. As part of developing a safe and effective vaccine, it is essential that potential sex differences are characterized. MethodsIn this study we evaluated novel vaccines in male and female BALB/c mice using a heterologous prime-boost approach and monitored survival, bacterial load in organs, and immunological correlates. Our vaccine strategy consisted of two subcutaneous immunizations, followed by challenge with aerosolized virulent nonencapsulated Y. pestis. Mice were immunized with a combination of live Y. pestis pgm- pPst-Δcaf1, live Y. pestis pgm- pPst-Δcaf1/ΔyopD, or recombinant F1-V (rF1-V) combined with adjuvants. ResultsThe most effective vaccine regimen was initial priming with rF1-V, followed by boost with either of the live attenuated strains. However, this and other strategies were more protective in female mice. Males had higher bacterial burden and differing patterns of cytokine expression and serum antibody titers. Male mice did not demonstrate synergy between vaccination and antibiotic treatment as repeatedly observed in female mice.ConclusionsThis study provides new knowledge about heterologous vaccine strategies, sex differences in plague-vaccine efficacy, and the immunological factors that differ between male and female mice.

Abstract Pneumonic plague, caused by the bacterium Yersinia pestis, is a severe and rapidly progressing illness transmitted via aerosol. There are no FDA-approved vaccines, and candidate vaccines may be less effective against pneumonic than bubonic plague. Y. pestis is not known to impact males and females differently in mechanism of pathogenesis or severity. However, a recent study reported sex-biased vaccine effectiveness after intranasal challenge. In this study we observed that when male mice were given a vaccine that is protective in females challenged with aerosolized virulent Y. pestis C12, males were not protected. We then gave male and female BALB/c mice a recently developed heterologous vaccine strategy and monitored survival, bacterial burden (CFU), and immunological factors before and after aerosol challenge. This strategy consisted of two subcutaneous injections: one of Y. pestis live attenuated vaccine (LAV) strain pgm-pPst-Δcaf1 or pgm-pPst-ΔyopD/Δcaf1 and one of rF1-V protein subunit with adjuvants CpG and Alhydrogel. When primed with rF1-V followed by pgm-pPst-Δcaf1 LAV boost, 100% of females and 50% of males survived challenge. This sex difference was observed to a lesser extent with three other vaccines. Males had higher CFU titers and differing patterns of serum antibodies, lung cytokines, and splenic T cells. These data provide new knowledge about sex differences in vaccine efficacy and immunological factors that differ between males and females.

Abstract Yersinia pestis (Yp) is a security concern since it can be aerosolized and cause pneumonic plague, a rapidly progressing illness that is fatal if not treated promptly with antibiotics. There are no FDA-approved vaccines, and candidate vaccines are less effective against pneumonic than bubonic plague. The major protective vaccine antigen has been the F1 capsule protein; protection against nonencapsulated 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, Δcaf1 and ΔyopD/Δcaf1, lack F1 and may be more effective at inducing immunity against nonencapsulated strains like C12. Female BALB/c mice were immunized with a heterologous prime-boost strategy combining one subcutaneous injection of novel LAV and one of recombinant F1-V (rF1-V) subunit, then challenged with aerosolized virulent Yp C12 and monitored for survival, bacterial load, and immunological factors. Priming with rF1-V and boosting with LAV was more protective than the reverse strategy. Protection was accompanied by high antibody titers against rF1-V and rV. rF1-V prime and LAV boost also led to a higher IgG2a/IgG1 antibody ratio than the reverse, suggesting a balanced Th1/Th2 response. These data support rF1-V prime and pgm-pPst-Δcaf1 LAV boost as a promising strategy for a safe and effective vaccine against both encapsulated and nonencapsulated Yp.

In the United States in 2021, an outbreak of 4 cases of Burkholderia pseudomallei, the etiologic agent of melioidosis and a Tier One Select Agent (potential for deliberate misuse and subsequent harm), resulted in 2 deaths. The causative strain, B. pseudomallei ATS2021, was unintentionally imported into the United States in an aromatherapy spray manufactured in India. We established that ATS2021 represents a virulent strain of B. pseudomallei capable of robust formation of biofilm at physiologic temperatures that may contribute to virulence. By using mouse melioidosis models, we determined median lethal dose estimates and analyzed the bacteriologic and histopathologic characteristics of the organism, particularly the potential neurologic pathogenesis that is probably associated with the bimABm allele identified in B. pseudomallei strain ATS2021. Our data, combined with previous case reports and the identification of endemic B. pseudomallei strains in Mississippi, support the concept that melioidosis is emerging in the United States.

Yersinia pestis is a gram-negative bacterium that causes plague in animals and humans. Depending on the route of disease transmission, the bacterium can cause an acute, often fatal disease that has a narrow window for treatment with antibiotics. Additionally, antibiotic resistant strains have been identified, emphasizing the need for novel treatments. Antibody therapy is an appealing option that can direct the immune system to clear bacterial infections. Advances in biotechnology have made both engineering and producing antibodies easier and more affordable. In this study, two screening assays were optimized to evaluate the ability of antibodies to promote phagocytosis of Y. pestis by macrophages and to induce a cytokine signature in vitro that may be predictive of protection in vivo. We evaluated a panel of 21 mouse monoclonal antibodies targeting either the anti-phagocytic capsule F1 protein or the LcrV antigen, which is part of the type 3 secretion system that facilitates translocation of virulence factors into the host cell, using two functional assays. Anti-F1 and anti-LcrV monoclonal antibodies both increased bacterial uptake by macrophages, with greater uptake observed in the presence of antibodies that were protective in the mouse pneumonic plague model. In addition, the protective anti-F1 and anti-LcrV antibodies produced unique cytokine signatures that were also associated with in vivo protection. These antibody-dependent characteristics from in vitro functional assays will be useful in down-selecting efficacious novel antibodies that can be used for treatment of plague.

OBJECTIVES To evaluate the in vitro activity and in vivo efficacy of delafloxacin against Bacillus anthracis, the causative agent of anthrax. METHODS MICs were obtained according to CLSI guidelines for 30 virulent isolates and 14 attenuated antibiotic-resistant strains. For the in vivo efficacy study, mice were administered delafloxacin (30-62.5 mg/kg) subcutaneously, or ciprofloxacin (30 mg/kg) intraperitoneally beginning at either 24 or 48 ± 1 h post-challenge (post-exposure prophylaxis) and continued every 12 h for 14 days with study termination on day 30. The mean inhaled dose in the study was approximately 103 × LD50 equivalents, and the range was 87-120 × LD50. RESULTS Delafloxacin (MIC90 = 0.004 mg/L) was 16-fold more potent than ciprofloxacin (MIC90 = 0.06 mg/L) against a 30-strain set of virulent B. anthracis. Against a panel of attenuated antibiotic-resistant strains, delafloxacin demonstrated potency ≥128-fold over that observed with ciprofloxacin. When evaluated in vivo, mice treated with all delafloxacin doses tested at 24 h post-challenge demonstrated equivalent survival compared with mice treated with the positive control ciprofloxacin. Because of the high challenge dose of spores, mice treated at 48 h showed rapid and high mortality in all groups including the positive control. Surviving animals in all delafloxacin- and ciprofloxacin-treated groups (24 and 48 h) showed complete splenic clearance of infection and <2.2 × 103 cfu/g lung tissue. CONCLUSIONS Given the high bar set by the 100 × LD50 challenge dose in this study, the results from delafloxacin treatment are promising for the treatment of inhaled anthrax.