Multistate Outbreak of Melioidosis Associated with Imported Aromatherapy Spray.

Melioidosis, caused by the bacterium Burkholderia pseudomallei, is an uncommon infection that is typically associated with exposure to soil and water in tropical and subtropical environments. It is rarely diagnosed in the continental United States. Patients with melioidosis in the United States commonly report travel to regions where melioidosis is endemic. We report a cluster of four non-travel-associated cases of melioidosis in Georgia, Kansas, Minnesota, and Texas. These cases were caused by the same strain of B. pseudomallei that was linked to an aromatherapy spray product imported from a melioidosis-endemic area.

Virulence of Burkholderia pseudomallei ATS2021 Unintentionally Imported to United States in Aromatherapy Spray.

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.

COVID-19 Exacerbated Long-standing Challenges for the Home Care Workforce.

The purpose of this study was to understand staffing challenges faced by home care (including home health) agencies due to the COVID-19 pandemic and the policies and practices put into place by the federal government, state governments, and home care agencies themselves to mitigate these challenges. This study included a review of federal and state policy changes enacted in reaction to the pandemic from March through December 2020, a review of home care agency practices described in media reports, peer-reviewed literature, and gray literature focused on responses to workforce challenges encountered during the pandemic, and interviews with a variety of stakeholders. Some of the challenges encountered were entirely new and resulted directly from the pandemic. In other cases, the pandemic worsened long-standing challenges in the industry. States and the federal government addressed some of these issues through changes to policies, regulations, and guidance. Home care agencies also responded with changes to their own policies and practices.

Point-of-Care Antigen Test for SARS-CoV-2 in Asymptomatic College Students.

We used the BinaxNOW COVID-19 Ag Card to screen 1,540 asymptomatic college students for severe acute respiratory syndrome coronavirus 2 in a low-prevalence setting. Compared with reverse transcription PCR, BinaxNOW showed 20% overall sensitivity; among participants with culturable virus, sensitivity was 60%. BinaxNOW provides point-of-care screening but misses many infections.

Combinations of early generation antibiotics and antimicrobial peptides are effective against a broad spectrum of bacterial biothreat agents.

The misuse of infectious disease pathogens as agents of deliberate attack on civilians and military personnel is a serious national security concern, which is exacerbated by the emergence of natural or genetically engineered multidrug resistant strains. In this study, the therapeutic potential of combinations of an antibiotic and a broad-spectrum antimicrobial peptide (AMP) was evaluated against five bacterial biothreats, the etiologic agents of glanders (Burkholderia mallei), melioidosis (Burkholderia pseudomallei), plague (Yersinia pestis), tularemia (Francisella tularensis), and anthrax (Bacillus anthracis). The therapeutics included licensed early generation antibiotics which are now rarely used. Three antibiotics and one 24- amino acid AMP were selected based on MIC assay data. Combinations of the AMP and tigecycline, minocycline, or novobiocin were screened for synergistic activity by checkerboard MIC assay. The combinations each enhanced the susceptibility of several strains. The tetracycline-peptide combinations increased the sensitivities of Y. pestis, F. tularensis, B. anthracis and B. pseudomallei, and the novobiocin-AMP combination augmented the sensitivity of all five. In time-kill assays, down-selected combinations of the peptide and minocycline or tigecycline enhanced killing of B. anthracis, Y. pestis, F. tularensis, and Burkholderia mallei but not B. pseudomallei. The novobiocin-AMP pair significantly reduced viability of all strains except B. mallei, which was very sensitive to the antibiotic alone. The results suggested that antibiotic-AMP combinations are useful tools for combating diverse pathogens. Future studies employing cell culture and animal models will utilize virulent strains of the agents to investigate the in vivo availability, host cytotoxicity, and protective efficacy of these therapeutics.

Formaldehyde and Glutaraldehyde Inactivation of Bacterial Tier 1 Select Agents in Tissues.

For safety, designated Select Agents in tissues must be inactivated and viability tested before the tissue undergoes further processing and analysis. In response to the shipping of samples of "inactivated" Bacillus anthracis that inadvertently contained live spores to nonregulated entities and partners worldwide, the Federal Register now mandates in-house validation of inactivation procedures and standardization of viability testing to detect live organisms in samples containing Select Agents that have undergone an inactivation process. We tested and validated formaldehyde and glutaraldehyde inactivation procedures for animal tissues infected with virulent B. anthracis, Burkholderia pseudomallei, Francisella tularensis, and Yersinia pestis. We confirmed that our fixation procedures for tissues containing these Tier 1 Select Agents resulted in complete inactivation and that our validated viability testing methods do not interfere with detection of live organisms. Institutions may use this work as a guide to develop and conduct their own testing to comply with the policy.

A Standard Method To Inactivate Bacillus anthracis Spores to Sterility via Gamma Irradiation.

In 2015, a laboratory of the United States Department of Defense (DoD) inadvertently shipped preparations of gamma-irradiated spores of Bacillus anthracis that contained live spores. In response, a systematic evidence-based method for preparing, concentrating, irradiating, and verifying the inactivation of spore materials was developed. We demonstrate the consistency of spore preparations across multiple biological replicates and show that two different DoD institutions independently obtained comparable dose-inactivation curves for a monodisperse suspension of B. anthracis spores containing 3 × 1010 CFU. Spore preparations from three different institutions and three strain backgrounds yielded similar decimal reduction (D10) values and irradiation doses required to ensure sterility (DSAL) to the point at which the probability of detecting a viable spore is 10-6 Furthermore, spores of a genetically tagged strain of B. anthracis strain Sterne were used to show that high densities of dead spores suppress the recovery of viable spores. Together, we present an integrated method for preparing, irradiating, and verifying the inactivation of spores of B. anthracis for use as standard reagents for testing and evaluating detection and diagnostic devices and techniques.IMPORTANCE The inadvertent shipment by a U.S. Department of Defense (DoD) laboratory of live Bacillus anthracis (anthrax) spores to U.S. and international destinations revealed the need to standardize inactivation methods for materials derived from biological select agents and toxins (BSAT) and for the development of evidence-based methods to prevent the recurrence of such an event. Following a retrospective analysis of the procedures previously employed to generate inactivated B. anthracis spores, a study was commissioned by the DoD to provide data required to support the production of inactivated spores for the biodefense community. The results of this work are presented in this publication, which details the method by which spores can be prepared, irradiated, and tested, such that the chance of finding residual living spores in any given preparation is 1/1,000,000. These irradiated spores are used to test equipment and methods for the detection of agents of biological warfare and bioterrorism.

Effectiveness and toxicity of high-dose cyclophosphamide in obese versus non-obese patients receiving allogeneic hematopoietic stem cell transplant.

PURPOSE: To determine if there is a difference in toxicity and effectiveness between obese and non-obese patients who receive high-dose cyclophosphamide (Cy) prior to allogeneic hematopoietic stem cell transplant (allo-HCT). METHODS: Patients were included in this study if they were at least 18 years of age and received high-dose Cy in combination with total body irradiation (CyTBI) or busulfan (BuCy) prior to allo-HCT between 1 January 2008 and 29 February 2012. The primary endpoint was the difference in overall toxicity between obese and non-obese patients. Secondary objectives examined differences in effectiveness between groups assessed by relapse at day +100, relapse at 1 year, death at 1 year, chimerisms at days +30, +60, and +90, and incidence of acute graft versus host disease (aGVHD). RESULTS: Sixty-one patients met the inclusion criteria, 28 obese and 33 non-obese. Overall toxicity was greater in obese patients compared to non-obese patients (82% vs. 52%, OR 4.3 [95% CI 1.3-14.1]; p = 0.01), which was driven by a greater incidence of renal dysfunction (79% vs. 48%, OR 3.9 [95% CI 1.3-12.1]; p = 0.02). There were no differences in rates of grade 3 or 4 toxicity, hepatic dysfunction, or any measure of effectiveness between groups. CONCLUSION: Obese patients receiving high-dose Cy and allo-HCT are at increased risk for toxicity, although there appears to be no difference in the rate of relapse or survival between obese and non-obese patients.

Sex differences in immune protection in mice conferred by heterologous vaccines for pneumonic plague.

Background: Yersinia 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. Methods: In 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. Results: The 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. Conclusions: This 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.

Characterization of two affinity matured Anti-Yersinia pestis F1 human antibodies with medical countermeasure potential.

Yersinia pestis, the causative agent of plague and a biological threat agent, presents an urgent need for novel medical countermeasures due to documented cases of naturally acquired antibiotic resistance and potential person-to-person spread during a pneumonic infection. Immunotherapy has been proposed as a way to circumvent current and future antibiotic resistance. Here, we describe the development and characterization of two affinity matured human antibodies (αF1Ig AM2 and αF1Ig AM8) that promote survival of mice after exposure to aerosolized Y. pestis. We share details of the error prone PCR and yeast display technology-based affinity maturation process that we used. The resultant matured antibodies have nanomolar affinity for Y. pestis F1 antigen, are produced in high yield, and are resilient to 37°C stress for up to 6 months. Importantly, in vitro assays using a murine macrophage cell line demonstrated that αF1Ig AM2 and αF1Ig AM8 are opsonic. Even more importantly, in vivo studies using pneumonic plague mouse models showed that 100% of the mice receiving 500 µg of IgGs αF1Ig AM2 and αF1Ig AM8 survived lethal challenge with aerosolized Y. pestis CO92. Combined, these results provide evidence of the quality and robustness of αF1Ig AM2 and αF1Ig AM8 and support their development as potential medical countermeasures against plague.

Angiotensin-converting enzyme inhibitors for intermittent claudication associated with peripheral arterial disease.

OBJECTIVE: To review published literature regarding the effectiveness of angiotensin-converting enzyme (ACE) inhibitors for managing intermittent claudication (IC) associated with peripheral arterial disease (PAD). DATA SOURCES: A search of MEDLINE/PubMed (1966-July 2013) using the MeSH terms intermittent claudication and angiotensin-converting enzyme inhibitors was conducted. Limits included articles written in English with human participants. Additional data were identified through bibliographic reviews. STUDY SELECTION AND DATA EXTRACTION: All English-language articles identified from the data sources were evaluated. Clinical trials and meta-analyses were included if they evaluated the efficacy of ACE inhibitors for improving functional capacity of patients with PAD with IC. In all, 9 clinical trials and 1 meta-analysis were identified and included for review. ACE inhibitors evaluated in the studies were captopril, lisinopril, perindopril, quinapril, and ramipril. DATA SYNTHESIS: Current medications approved for treating the symptoms and improving function in PAD with IC have limited efficacy. It has been suggested that ACE inhibitors may be effective in PAD with IC. Though data evaluating ACE inhibitors as a class in this patient population are conflicting, results of the largest and longest trial reported that ramipril increases maximum walking time and pain-free walking time and improves quality of life in patients with PAD with IC. CONCLUSIONS: ACE inhibitors may provide some relief of IC symptoms when used in patients with PAD. The greatest functional benefit has been seen with ramipril; it is unknown whether other agents in the class would show similar results. Well-controlled and designed studies with sufficient power and using diverse patient populations are needed.

Polyclonal Antibodies Derived from Transchromosomic Bovines Vaccinated with the Recombinant F1-V Vaccine Increase Bacterial Opsonization In Vitro and Protect Mice from Pneumonic Plague.

Plague is an ancient disease that continues to be of concern to both the public health and biodefense research communities. Pneumonic plague is caused by hematogenous spread of Yersinia pestis bacteria from a ruptured bubo to the lungs or by directly inhaling aerosolized bacteria. The fatality rate associated with pneumonic plague is significant unless effective antibiotic therapy is initiated soon after an early and accurate diagnosis is made. As with all bacterial pathogens, drug resistance is a primary concern when developing strategies to combat these Yersinia pestis infections in the future. While there has been significant progress in vaccine development, no FDA-approved vaccine strategy exists; thus, other medical countermeasures are needed. Antibody treatment has been shown to be effective in animal models of plague. We produced fully human polyclonal antibodies in transchromosomic bovines vaccinated with the recombinant F1-V plague vaccine. The resulting human antibodies opsonized Y. pestis bacteria in the presence of RAW264.7 cells and afforded significant protection to BALB/c mice after exposure to aerosolized Y. pestis. These data demonstrate the utility of this technology to produce large quantities of non-immunogenic anti-plague human antibodies to prevent or possibly treat pneumonic plague in human.

Functional assays to screen and select monoclonal antibodies that target Yersinia pestis.

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.

The rLVS ΔcapB/iglABC vaccine provides potent protection in Fischer rats against inhalational tularemia caused by various virulent Francisella tularensis strains.

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.

Efficacy of Treatment with the Antibiotic Novobiocin against Infection with Bacillus anthracis or Burkholderia pseudomallei.

The microbial pathogens Burkholderia pseudomallei and Bacillus anthracis are unrelated bacteria, yet both are the etiologic agents of naturally occurring diseases in animals and humans and are classified as Tier 1 potential biothreat agents. B. pseudomallei is the gram-negative bacterial agent of melioidosis, a major cause of sepsis and mortality globally in endemic tropical and subtropical regions. B. anthracis is the gram-positive spore-forming bacterium that causes anthrax. Infections acquired by inhalation of these pathogens are challenging to detect early while the prognosis is best; and they possess innate multiple antibiotic resistance or are amenable to engineered resistance. Previous studies showed that the early generation, rarely used aminocoumarin novobiocin was very effective in vitro against a range of highly disparate biothreat agents. The objective of the current research was to begin to characterize the therapeutic efficacy of novobiocin in mouse models of anthrax and melioidosis. The antibiotic was highly efficacious against infections by both pathogens, especially B. pseudomallei. Our results supported the concept that specific older generation antimicrobials can be effective countermeasures against infection by bacterial biothreat agents. Finally, novobiocin was shown to be a potential candidate for inclusion in a combined pre-exposure vaccination and post-exposure treatment strategy designed to target bacterial pathogens refractory to a single medical countermeasure.

Layered and integrated medical countermeasures against Burkholderia pseudomallei infections in C57BL/6 mice.

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 we demonstrate that when used in combination, current vaccine strategies (recombinant protein subunits AhpC and/or Hcp1 plus capsular polysaccharide conjugated to CRM197 or the live attenuated vaccine strain B. pseudomallei 668 ΔilvI) and co-trimoxazole regimens can result in near uniform protection in a mouse model of melioidosis due to apparent synergy associated with distinct medical countermeasures. Our results 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. Layered and integrated medical countermeasures will provide novel treatment options for melioidosis 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.

Evaluation of two different vaccine platforms for immunization against melioidosis and glanders.

Burkholderia pseudomallei and the closely related species, Burkholderia mallei, produce similar multifaceted diseases which range from rapidly fatal to protracted and chronic, and are a major cause of mortality in endemic regions. Besides causing natural infections, both microbes are Tier 1 potential biothreat agents. Antibiotic treatment is prolonged with variable results, hence effective vaccines are urgently needed. The purpose of our studies was to compare candidate vaccines that target both melioidosis and glanders to identify the most efficacious one(s) and define residual requirements for their transition to the non-human primate aerosol model. Studies were conducted in the C57BL/6 mouse model to evaluate the humoral and cell-mediated immune response and protective efficacy of three Burkholderia vaccine candidates against lethal aerosol challenges with B. pseudomallei K96243, B. pseudomallei MSHR5855, and B. mallei FMH. The recombinant vaccines generated significant immune responses to the vaccine antigens, and the live attenuated vaccine generated a greater immune response to OPS and the whole bacterial cells. Regardless of the candidate vaccine evaluated, the protection of mice was associated with a dampened cytokine response within the lungs after exposure to aerosolized bacteria. Despite being delivered by two different platforms and generating distinct immune responses, two experimental vaccines, a capsule conjugate + Hcp1 subunit vaccine and the live B. pseudomallei 668 ΔilvI strain, provided significant protection and were down-selected for further investigation and advanced development.

MicroRNA regulation of a cancer network: consequences of the feedback loops involving miR-17-92, E2F, and Myc.

The transcription factors E2F and Myc participate in the control of cell proliferation and apoptosis, and can act as oncogenes or tumor suppressors depending on their levels of expression. Positive feedback loops in the regulation of these factors are predicted-and recently shown experimentally-to lead to bistability, which is a phenomenon characterized by the existence of low and high protein levels ("off" and "on" levels, respectively), with sharp transitions between levels being inducible by, for example, changes in growth factor concentrations. E2F and Myc are inhibited at the posttranscriptional step by members of a cluster of microRNAs (miRs) called miR-17-92. In return, E2F and Myc induce the transcription of miR-17-92, thus forming a negative feedback loop in the interaction network. The consequences of the coupling between the E2F/Myc positive feedback loops and the E2F/Myc/miR-17-92 negative feedback loop are analyzed using a mathematical model. The model predicts that miR-17-92 plays a critical role in regulating the position of the off-on switch in E2F/Myc protein levels, and in determining the on levels of these proteins. The model also predicts large-amplitude protein oscillations that coexist with the off steady state levels. Using the concept and model prediction of a "cancer zone," the oncogenic and tumor suppressor properties of miR-17-92 is demonstrated to parallel the same properties of E2F and Myc.

Biological Validation of a Chemical Effluent Decontamination System.

Introduction: Failure of an existing effluent decontamination system (EDS) prompted the consideration of commercial off-the-shelf solutions for decontamination of containment laboratory waste. A bleach-based chemical EDS was purchased to serve as an interim solution. Methods: Studies were conducted in the laboratory to validate inactivation of Bacillus spores with bleach in complex matrices containing organic simulants including fetal bovine serum, humic acid, and animal room sanitation effluent. Results: These studies demonstrated effective decontamination of >106 spores at a free chlorine concentration of ≥5700 parts per million with a 2-hour contact time. Translation of these results to biological validation of the bleach-based chemical EDS required some modifications to the system and its operation. Discussion: The chemical EDS was validated for the treatment of biosafety levels 3 and 4 waste effluent using laboratory-prepared spore packets along with commercial biological indicators; however, several issues and lessons learned identified during the process of onboarding are also discussed, including bleach product source, method of validation, dechlorination, and treated waste disposal.

Predictive and Experimental Immunogenicity of Burkholderia Collagen-like Protein 8-Derived Antigens.

Burkholderia pseudomallei is an infectious bacterium of clinical and biodefense concern, and is the causative agent of melioidosis. The mortality rate can reach up to 50% and affects 165,000 people per year; however, there is currently no vaccine available. In this study, we examine the antigen-specific immune response to a vaccine formulated with antigens derived from an outer membrane protein in B. pseudomallei, Bucl8. Here, we employed a number of bioinformatic tools to predict Bucl8-derived epitopes that are non-allergenic and non-toxic, but would elicit an immune response. From these data, we formulated a vaccine based on two extracellular components of Bucl8, the ß-barrel loops and extended collagen and non-collagen domains. Outbred CD-1 mice were immunized with vaccine formulations-composed of recombinant proteins or conjugated synthetic peptides with adjuvant-to assess the antigen-specific immune responses in mouse sera and lymphoid organs. We found that mice vaccinated with either Bucl8-derived components generated a robust TH2-skewed antibody response when antigen was combined with the adjuvant AddaVax, while the TH1 response was limited. Mice immunized with synthetic loop peptides had a stronger, more consistent antibody response than recombinant protein antigens, based on higher IgG titers and recognition of bacteria. We then compared peptide-based vaccines in an established C57BL/6 inbred mouse model and observed a similar TH2-skewed response. The resulting formulations will be applied in future studies examining the protection of Bucl8-derived vaccines.