Development and Evaluation of an Immunoinformatics-Based Multi-Peptide Vaccine against Acinetobacter baumannii Infection.

Multi-drug-resistant (MDR) Acinetobacter baumannii is an opportunistic pathogen associated with hospital-acquired infections. Due to its environmental persistence, virulence, and limited treatment options, this organism causes both increased patient mortality and incurred healthcare costs. Thus, prophylactic vaccination could be ideal for intervention against MDR Acinetobacter infection in susceptible populations. In this study, we employed immunoinformatics to identify peptides containing both putative B- and T-cell epitopes from proteins associated with A. baumannii pathogenesis. A novel Acinetobacter Multi-Epitope Vaccine (AMEV2) was constructed using an A. baumannii thioredoxin A (TrxA) leading protein sequence followed by five identified peptide antigens. Antisera from A. baumannii infected mice demonstrated reactivity to rAMEV2, and subcutaneous immunization of mice with rAMEV2 produced high antibody titer against the construct as well as peptide components. Immunization results in increased frequency of IL-4-secreting splenocytes indicative of a Th2 response. AMEV2-immunized mice were protected against intranasal challenge with a hypervirulent strain of A. baumannii and demonstrated reduced bacterial burden at 48 h. In contrast, all mock vaccinated mice succumbed to infection within 3 days. Results presented here provide insight into the effectiveness of immunoinformatic-based vaccine design and its potential as an effective strategy to combat the rise of MDR pathogens.

Galleria mellonella Model of Coccidioidomycosis for Drug Susceptibility Tests and Virulence Factor Identification.

Coccidioidomycosis (CM) can manifest as respiratory and disseminated diseases that are caused by dimorphic fungal pathogens, such as Coccidioides species. The inhaled arthroconidia generated during the saprobic growth phase convert into multinucleated spherules in the lungs to complete the parasitic lifecycle. Research on coccidioidal virulence and pathogenesis primarily employs murine models typically associated with low lethal doses (LD100 < 100 spores). However, the Galleria model has recently garnered attention due to its immune system bearing both structural and functional similarities to the innate system of mammals. Our findings indicate that Coccidioides posadasii can convert and complete the parasitic cycle within the hemocoel of the Galleria larva. In Galleria, the LD100 is between 0.5 and 1.0 × 106 viable spores for the clinical isolate Coccidioides posadasii C735. Furthermore, we demonstrated the suitability of this model for in vivo antifungal susceptibility tests to validate the bioreactivity of newly discovered antifungals against Coccidioides. Additionally, we utilized this larva model to screen a Coccidioides posadasii mutant library showing attenuated virulence. Similarly, the identified attenuated coccidioidal mutants displayed a loss of virulence in a commonly used murine model of coccidioidomycosis. In this study, we demonstrated that Galleria larvae can be applied as a model for studying Coccidioides infection.

Development of an Imaging Flow Cytometry Method for Fungal Cytological Profiling and Its Potential Application in Antifungal Drug Development.

Automated imaging techniques have been in increasing demand for the more advanced analysis and efficient characterization of cellular phenotypes. The success of the image-based profiling method hinges on assays that can rapidly and simultaneously capture a wide range of phenotypic features. We have developed an automated image acquisition method for fungal cytological profiling (FCP) using an imaging flow cytometer that can objectively measure over 250 features of a single fungal cell. Fungal cells were labeled with calcofluor white and FM4-64FX, which bind to the cell wall and lipophilic membrane, respectively. Images of single cells were analyzed using IDEAS® software. We first acquired FCPs of fungal cells treated with fluconazole, amphotericin B, and caspofungin, each with a distinct mode of action, to establish FCP databases of profiles associated with specific antifungal treatment. Once fully established, we investigated the potential application of this technique as a screening methodology to identify compounds with novel antifungal activity against Candida albicans and Cryptococcus neoformans. Altogether, we have developed a rapid, powerful, and novel image-profiling method for the phenotypic characterization of fungal cells, also with potential applications in antifungal drug development.

Insights into Acinetobacter baumannii protective immunity.

Acinetobacter baumannii is a nosocomic opportunistic Gram-negative bacteria known for its extensive drug-resistant phenotype. A. baumannii hospital-acquired infections are major contributors to increased costs and mortality observed during the COVID-19 pandemic. With few effective antimicrobials available for treatment of this pathogen, immune-based therapy becomes an attractive strategy to combat multi-drug resistant Acinetobacter infection. Immunotherapeutics is a field of growing interest with advances in vaccines and monoclonal antibodies providing insight into the protective immune response required to successfully combat this pathogen. This review focuses on current knowledge describing the adaptive immune response to A. baumannii, the importance of antibody-mediated protection, developments in cell-mediated protection, and their respective therapeutic application going forward. With A. baumannii's increasing resistance to most current antimicrobials, elucidating an effective host adaptive immune response is paramount in the guidance of future immunotherapeutic development.

Influence of Fetuin-A on Chlamydia muridarum Pulmonary Infection.

Fetuin-A is an acute phase glycoprotein shown to counter in a regulatory manner proinflammatory cytokine production to maintain homeostasis during inflammation. We report here that in wild-type mice 12 days after Chlamydia muridarum (Cm) intranasal challenge, fetuin-A content in the lungs decreased 46%, while INF-γ increased 44%, consistent with a negative regulatory role of fetuin-A in inflammation. Importantly, the observed increased IFN-γ production was abrogated in fetuin-A-deficient AHSG mice suggesting that IFN-γ induction following Cm infection is fetuin-A dependent. Assessment of expression of genes associated with inflammation revealed fetuin-A-dependent upregulation of TBX21 (a Th1 cell-specific transcription factor) in the lungs of Cm-infected WT mice that correlated with IFN-γ induction. Additionally, the effect of fetuin-A deficiency in mounting an adaptive immune response to Cm infection was demonstrated using a splenocyte recall assay. Although preliminary in nature, these findings are suggestive of fetuin-A involvement following Cm pulmonary infection and underscores the need to investigate further the role of fetuin-A in the immune response and the consequences of its gene deletion.

Isotyping and quantitation of the humoral immune response to SARS-CoV-2.

Understanding the immune response to SARS-CoV-2 is important for development of effective diagnostics and vaccines. We report here a broad antibody response to SARS-CoV-2 spike protein receptor binding domain (RBD) in 100 convalescent patient plasma samples. Antibody isotypes IgA, IgM, and IgG exhibited significantly higher anti-RBD titers when compared to SARS-CoV-2 negative controls. IgG subtyping indicated IgG1 and IgG3 to be most abundant. Greater than 90 % of SARS-CoV-2 positive plasma samples tested exhibited significant neutralization capacity using a surrogate virus neutralization assay. Of the IgG subclasses, IgG1 and IgG3 exhibited the highest viral neutralization capacity; whereas, IgG2 and IgG4 viral neutralization was not observed. Comparison of SARS-CoV-2 elicited total IgG binding to emerging variant (alpha, beta, and delta) RBDs indicated decreased binding. Furthermore, neutralization by SARS-CoV-2 convalescent plasma of delta and omicron variant RBDs was significantly decreased suggesting that neutralizing antibodies in convalescent plasma are less effective in inhibiting variants currently in circulation.

Thioredoxin-mediated alteration of protein content and cytotoxicity of Acinetobacter baumannii outer membrane vesicles.

Acinetobacter baumannii is a Gram-negative bacterium responsible for many hospital-acquired infections including ventilator-associated pneumonia and sepsis. We have previously identified A. baumannii thioredoxin A protein (TrxA) as a virulence factor with a multitude of functions including reduction of protein disulfides. TrxA plays an important role in resistance to oxidative stress facilitating host immune evasion in part by alteration of type IV pili and cell surface hydrophobicity. Other virulence factors such as outer membrane vesicles (OMV) shed by bacteria have been shown to mediate bacterial intercellular communication and modulate host immune response. To investigate whether OMVs can be modulated by TrxA, we isolated OMVs from wild type (WT) and TrxA-deficient (ΔtrxA) A. baumannii clinical isolate Ci79 and carried out a functional and proteomic comparison. Despite attenuation of ΔtrxA in a mouse challenge model, pulmonary inoculation of ΔtrxA OMVs resulted in increased lung permeability compared to WT OMVs. Furthermore, ΔtrxA OMVs induced more J774 macrophage-like cell death than WT OMVs. This ΔtrxA OMV-mediated cell death was abrogated when cells were incubated with protease-K-treated OMVs suggesting OMV proteins were responsible for cytotoxicity. We therefore compared WT and mutant OMV proteins using proteomic analysis. We observed that up-regulated and unique ΔtrxA OMV proteins consisted of many membrane bound proteins involved in small molecule transport as well as proteolytic activity. Bacterial OmpA, metalloprotease, and fimbrial protein have been shown to enhance mammalian cell apoptosis through various mechanisms. Differential packaging of these proteins in ΔtrxA OMVs may contribute to the increased cytotoxicity observed in this study.

Biochemical characterization of a recombinant acid phosphatase from Acinetobacter baumannii.

Genomic sequence analysis of Acinetobacter baumannii revealed the presence of a putative Acid Phosphatase (AcpA; EC 3.1.3.2). A plasmid construct was made, and recombinant protein (rAcpA) was expressed in E. coli. PAGE analysis (carried out under denaturing/reducing conditions) of nickel-affinity purified protein revealed the presence of a near-homogeneous band of approximately 37 kDa. The identity of the 37 kDa species was verified as rAcpA by proteomic analysis with a molecular mass of 34.6 kDa from the deduced sequence. The dependence of substrate hydrolysis on pH was broad with an optimum observed at 6.0. Kinetic analysis revealed relatively high affinity for PNPP (Km = 90 µM) with Vmax, kcat, and Kcat/Km values of 19.2 pmoles s-1, 4.80 s-1(calculated on the basis of 37 kDa), and 5.30 x 104 M-1s-1, respectively. Sensitivity to a variety of reagents, i.e., detergents, reducing, and chelating agents as well as classic acid phosphatase inhibitors was examined in addition to assessment of hydrolysis of a number of phosphorylated compounds. Removal of phosphate from different phosphorylated compounds is supportive of broad, i.e., 'nonspecific' substrate specificity; although, the enzyme appears to prefer phosphotyrosine and/or peptides containing phosphotyrosine in comparison to serine and threonine. Examination of the primary sequence indicated the absence of signature sequences characteristic of Type A, B, and C nonspecific bacterial acid phosphatases.

Modulation of Immune Response to Chlamydia muridarum by Host miR-135a.

Previously, our laboratory established the role of small, noncoding RNA species, i.e., microRNA (miRNA) including miR-135a in anti-chlamydial immunity in infected hosts. We report here chlamydial infection results in decreased miR-135a expression in mouse genital tissue and a fibroblast cell line. Several chemokine and chemokine receptor genes (including CXCL10, CCR5) associated with chlamydial pathogenesis were identified in silico to contain putative miR-135a binding sequence(s) in the 3' untranslated region. The role of miR-135a in the host immune response was investigated using exogenous miR-135a mimic to restore the immune phenotype associated with decreased miR-135a following Chlamydia muridarum (Cm) infection. We observed miR-135a regulation of Cm-primed bone marrow derived dendritic cells (BMDC) via activation of Cm-immune CD4+ T cells for clonal expansion and CCR5 expression. Using a transwell cell migration assay, we explore the role of miR-135a in regulation of genital tract CXCL10 expression and recruitment of CXCR3+ CD4+ T cells via the CXCL10/CXCR3 axis. Collectively, data reported here support miR-135a affecting multiple cellular processes in response to chlamydial infection.

CARD9-Associated Dectin-1 and Dectin-2 Are Required for Protective Immunity of a Multivalent Vaccine against Coccidioides posadasii Infection.

Coccidioides species are fungal pathogens that can cause a widely varied clinical manifestation from mild pulmonary symptom to disseminated, life-threatening disease. We have previously created a subunit vaccine by encapsulating a recombinant coccidioidal Ag (rCpa1) in glucan-chitin particles (GCPs) as an adjuvant-delivery system. The GCP-rCpa1 vaccine has shown to elicit a mixed Th1 and Th17 response and confers protection against pulmonary coccidioidomycosis in mice. In this study, we further delineated the vaccine-induced protective mechanisms. Depletion of IL-17A in vaccinated C57BL/6 mice prior to challenge abrogated the protective efficacy of GCP-rCpa1 vaccine. Global transcriptome and Ingenuity Pathway Analysis of murine bone marrow-derived macrophages after exposure to this vaccine revealed the upregulation of proinflammatory cytokines (TNF-α, IL-6, and IL-1ß) that are associated with activation of C-type lectin receptors (CLR) Dectin-1- and Dectin-2-mediated CARD9 signaling pathway. The GCP formulation of rCpa1 bound soluble Dectin-1 and Dectin-2 and triggered ITAM signaling of corresponding CLR reporter cells. Furthermore, macrophages that were isolated from Dectin-1 -/-, Dectin-2 -/-, and CARD9 -/- mice significantly reduced production of inflammatory cytokines in response to the GCP-rCpa1 vaccine compared with those of wild-type mice. The GCP-rCpa1 vaccine had significantly reduced protective efficacy in Dectin-1 -/-, Dectin-2 -/-, and CARD9 -/- mice that showed decreased acquisition of Th cells in Coccidioides-infected lungs compared with vaccinated wild-type mice, especially Th17 cells. Collectively, we conclude that the GCP-rCpa1 vaccine stimulates a robust Th17 immunity against Coccidioides infection through activation of the CARD9-associated Dectin-1 and Dectin-2 signal pathways.

Characterization of an Uncinocarpus reesii-expressed recombinant tube precipitin antigen of Coccidioides posadasii for serodiagnosis.

Early and accurate diagnosis of coccidioidomycosis, also known as Valley fever, is critical for appropriate disease treatment and management. Current serodiagnosis is based on the detection of patient serum antibodies that react with tube precipitin (TP) and complement fixation (CF) antigens of Coccidioides. IgM is the first class of antibodies produced by hosts in response to coccidioidal insults. The highly glycosylated ß-glucosidase 2 (BGL2) is a major active component of the TP antigen that stimulates IgM antibody responses during early Coccidioides infection. The predominant IgM epitope on BGL2 is a unique 3-O-methyl-mannose moiety that is not produced by commonly used protein expression systems. We genetically engineered and expressed a recombinant BGL2 (rBGL2ur), derived from Coccidioides, in non-pathogenic Uncinocarpus reesii, a fungus phylogenetically related to the Coccidioides pathogen. The rBGL2ur protein was purified from the culture medium of transformed U. reesii by nickel affinity chromatography, and the presence of 3-O-methyl mannose was demonstrated by gas chromatography. Seroreactivity of the purified rBGL2ur protein was tested by enzyme-linked immunosorbent assays using sera from 90 patients with coccidioidomycosis and 134 control individuals. The sensitivity and specificity of the assay with rBGL2ur were 78.8% and 87.3%, respectively. These results were comparable to those obtained using a proprietary MiraVista Diagnostic (MVD) IgM (63.3% sensitivity; 96.3% specificity), but significantly better than the ID-TP assay using non-concentrated patient sera (33.3% sensitivity; 100% specificity). Expression of rBGL2ur in U. reesii retains its antigenicity for coccidioidomycosis serodiagnosis and greatly reduces biosafety concerns for antigen production, as Coccidioides spp. are biological safety level 3 agents.

Thioredoxin-A is a virulence factor and mediator of the type IV pilus system in Acinetobacter baumannii.

The Gram-negative pathogen, Acinetobacter baumannii has emerged as a global nosocomial health threat affecting the majority of hospitals in the U.S. and abroad. The redox protein thioredoxin has been shown to play several roles in modulation of cellular functions affecting various virulence factors in Gram-negative pathogens. This study aims to explore the role of thioredoxin-A protein (TrxA) in A. baumannii virulence. We determined that deletion of the TrxA gene did not significantly affect resistance to environmental stressors such as temperature, salt, and pH. However, TrxA was critical for survival in the presence of elevated levels of hydrogen peroxide. Lack of TrxA was associated with decreased expression of type IV pili related genes and an inability to undergo normal twitching motility. Interestingly, the TrxA-null mutant was able to form biofilms better than the wildtype (WT) and was observed to be significantly less virulent than the WT in a pulmonary infection model. These results are supportive of thioredoxin playing a key role in A. baumannii virulence.

Thioredoxin Modulates Cell Surface Hydrophobicity in Acinetobacter baumannii.

Acinetobacter baumannii, a Gram-negative coccobacillus, has become a prevalent nosocomial health threat affecting the majority of hospitals both in the U.S. and around the globe. Microbial cell surface hydrophobicity (CSH) has previously been correlated with virulence, uptake by immune cells, and attachment to epithelial cells. A mutant strain of A. baumannii (ΔtrxA) lacking the redox protein thioredoxin A was found to be more hydrophobic than its wild type (WT) and complemented counterparts, as measured by both Microbial Adhesion to Hydrocarbon (MATH) and salt aggregation. The hydrophobicity of the mutant could be abrogated through treatment with sodium cyanoborohydride (SCBH). This modulation correlated with reduction of disulfide bonds, as SCBH was able to reduce 5,5'-dithio-bis-[2-nitrobenzoic acid] and treatment with the known disulfide reducer, ß-mercaptoethanol, also decreased ΔtrxA CSH. Additionally, the ΔtrxA mutant was more readily taken up than WT by J774 macrophages and this differential uptake could be abrogated though SCBH treatment. When partitioned into aqueous and hydrophobic phases, ΔtrxA recovered from the hydrophobic partition was phagocytosed more readily than from the aqueous phase further supporting the contribution of CSH to A. baumannii uptake by phagocytes. A second Gram-negative bacterium, Francisella novicida, also showed the association of TrxA deficiency (FnΔtrxA) with increased hydrophobicity and uptake by J774 cells. We previously have demonstrated that modification of the type IV pilus system (T4P) was associated with the A. baumannii ΔtrxA phenotype, and the Francisella FnΔtrxA mutant also was found to have a marked T4P deficiency. Interestingly, a F. novicida mutant lacking pilT also showed increased hydrophobicity over FnWT. Collective evidence presented in this study suggests that Gram-negative bacterial thioredoxin mediates CSH through multiple mechanisms including disulfide-bond reduction and T4P modulation.

Glucan-Chitin Particles Enhance Th17 Response and Improve Protective Efficacy of a Multivalent Antigen (rCpa1) against Pulmonary Coccidioides posadasii Infection.

Developing an effective and safe recombinant vaccine requires microbe-specific antigens combined with an adjuvant/delivery system to strengthen protective immunity. In this study, we designed and expressed a multivalent recombinant Coccidioides polypeptide antigen (rCpa1) that consists of three previously identified antigens (i.e., Ag2/Pra, Cs-Ag, and Pmp1) and five pathogen-derived peptides with high affinity for human major histocompatibility complex class II (MHC-II) molecules. The purified rCpa1 was encapsulated into four types of yeast cell wall particles containing ß-glucan, mannan, and chitin in various proportions or was mixed with an oligonucleotide (ODN) containing two methylated dinucleotide CpG motifs. This multivalent antigen encapsulated into glucan-chitin particles (GCP-rCpa1) showed significantly greater reduction of fungal burden for human HLA-DR4 transgenic mice than the other adjuvant-rCpa1 formulations tested. Among the adjuvants tested, both GCPs and ß-glucan particles (GPs) were capable of stimulating a mixed Th1 and Th17 response. Mice vaccinated with GCP-rCpa1 showed higher levels of interleukin 17 (IL-17) production in T-cell recall assays and earlier lung infiltration by activated Th1 and Th17 cells than GP-rCpa1-vaccinated mice. Both C57BL/6 and HLA-DR4 transgenic mice that were vaccinated with the GCP-rCpa1 vaccine showed higher survival rates than mice that received GCPs alone. Concurrently, the GCP-rCpa1 vaccine stimulated greater infiltration of the injection sites by macrophages, which engulf and process the vaccine for antigen presentation, than the GP-rCpa1 vaccine. This is the first attempt to systematically characterize the presentation of a multivalent coccidioidomycosis vaccine encapsulated with selected adjuvants that enhance the protective cellular immune response to infection.

Acinetobacter baumannii Gastrointestinal Colonization Is Facilitated by Secretory IgA Which Is Reductively Dissociated by Bacterial Thioredoxin A.

Multidrug-resistant Acinetobacter baumannii is among the most common causes of infectious complications associated with combat-related trauma in military personnel serving overseas. However, little is currently known about its pathogenesis. While the gastrointestinal (GI) tract has been found to be a major reservoir for A. baumannii, as well as to potentially contribute to development of multidrug resistance, no studies have addressed the mechanisms involved in gut colonization. In this study, we address this critical gap in knowledge by first assessing the interaction between secretory IgA (SIgA), the principal humoral immune defense on mucosal surfaces, and the A. baumannii clinical isolate Ci79. Surprisingly, SIgA appeared to enhance A. baumannii GI tract colonization, in a process mediated by bacterial thioredoxin A (TrxA), as evidenced by reduction of bacterial attachment in the presence of TrxA inhibitors. Additionally, a trxA targeted deletion mutant (ΔtrxA) showed reduced bacterial burdens within the GI tract 24 h after oral challenge by in vivo live imaging, along with loss of thiol-reductase activity. Surprisingly, not only was GI tract colonization greatly reduced but the associated 50% lethal dose (LD50) of the ΔtrxA mutant was increased nearly 100-fold in an intraperitoneal sepsis model. These data suggest that TrxA not only mediates A. baumannii GI tract colonization but also may contribute to pathogenesis in A. baumannii sepsis following escape from the GI tract under conditions when the intestinal barrier is compromised, as occurs with cases of severe shock and trauma.IMPORTANCEAcinetobacter baumannii is an emerging bacterial pathogen recently classified as a serious threat to U.S. and global health by both the Centers for Disease Control and Prevention and the World Health Organization. It also is one of the leading causes of combat-related infections associated with injured military personnel serving overseas. Little is known regarding mechanisms of gastrointestinal tract colonization despite this site being shown to serve as a reservoir for multidrug-resistant (MDR) A. baumannii isolates. Here, we establish that secretory IgA, the major immunoglobulin of mucosal surfaces, promotes A. baumannii GI tract colonization via bacterial thioredoxin A as evidenced through significant reduction in colonization in IgA-deficient animals. Additionally, bacterial colonization and mortality were significantly reduced in animals challenged with a thioredoxin A-deficient A. baumannii mutant. Combined, these data suggest that thioredoxin A is a novel virulence factor, for which antithioredoxin therapies could be developed, for this important multidrug-resistant pathogen.

Repurposing Auranofin, Ebselen, and PX-12 as Antimicrobial Agents Targeting the Thioredoxin System.

As microbial resistance to drugs continues to rise at an alarming rate, finding new ways to combat pathogens is an issue of utmost importance. Development of novel and specific antimicrobial drugs is a time-consuming and expensive process. However, the re-purposing of previously tested and/or approved drugs could be a feasible way to circumvent this long and costly process. In this review, we evaluate the U.S. Food and Drug Administration tested drugs auranofin, ebselen, and PX-12 as antimicrobial agents targeting the thioredoxin system. These drugs have been shown to act on bacterial, fungal, protozoan, and helminth pathogens without significant toxicity to the host. We propose that the thioredoxin system could serve as a useful therapeutic target with broad spectrum antimicrobial activity.

Microbial co-infection alters macrophage polarization, phagosomal escape, and microbial killing.

Macrophages are important innate immune cells that respond to microbial insults. In response to multi-bacterial infection, the macrophage activation state may change upon exposure to nascent mediators, which results in different bacterial killing mechanism(s). In this study, we utilized two respiratory bacterial pathogens, Mycobacterium bovis (Bacillus Calmette Guerin, BCG) and Francisella tularensis live vaccine strain (LVS) with different phagocyte evasion mechanisms, as model microbes to assess the influence of initial bacterial infection on the macrophage response to secondary infection. Non-activated (M0) macrophages or activated M2-polarized cells (J774 cells transfected with the mouse IL-4 gene) were first infected with BCG for 24-48 h, subsequently challenged with LVS, and the results of inhibition of LVS replication in the macrophages was assessed. BCG infection in M0 macrophages activated TLR2-MyD88 and Mincle-CARD9 signaling pathways, stimulating nitric oxide (NO) production and enhanced killing of LVS. BCG infection had little effect on LVS escape from phagosomes into the cytosol in M0 macrophages. In contrast, M2-polarized macrophages exhibited enhanced endosomal acidification, as well as inhibiting LVS replication. Pre-infection with BCG did not induce NO production and thus did not further reduce LVS replication. This study provides a model for studies of the complexity of macrophage activation in response to multi-bacterial infection.

Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis.

Pulmonary infection with the bacterium Francisella tularensis can lead to the serious and potentially fatal disease, tularemia, in humans. Due to the current lack of an approved tularemia vaccine for humans, research is focused on vaccine development utilizing appropriate animal models. The Fischer 344 rat has emerged as a model that reflects human susceptibility to F. tularensis infection, and thus is an attractive model for tularemia vaccine development. Intratracheal inoculation of the Fischer 344 rat with F. tularensis mimics pulmonary exposure in humans. The successful delivery into the rat trachea is critical for pulmonary delivery. A laryngoscope with illumination is used to properly intubate the tracheae of anesthetized rats; the correct placement within the trachea is determined by a simple device to detect breathing. Following intubation, the F. tularensis culture is delivered in a measured dose via syringe. This technique standardizes pulmonary delivery of F. tularensis within the rat trachea to evaluate vaccine efficacy.

Vaccination with a live attenuated Acinetobacter baumannii deficient in thioredoxin provides protection against systemic Acinetobacter infection.

Multi-drug resistant Acinetobacter baumannii (MDR-Ab), an opportunistic pathogen associated with nosocomial and combat related infections, has a high mortality due to its virulence and limited treatment options. Deletion of the thioredoxin gene (TrxA) from a clinical isolate of MDR-Ab resulted in a 100-fold increase in 50% lethal dose (LD50) in a systemic challenge murine model. Thus, we investigated the potential use of this attenuated strain as a live vaccine against MDR-Ab. Mice were vaccinated by subcutaneous (s.c.) injection of 2×105 CFU of the ΔtrxA mutant, boosted 14days later with an equivalent inoculum, and then challenged 30days post-vaccination by i.p. injection with 10 LD50 of the wild type (WT) Ci79 strain. Efficacy of vaccination was evaluated by monitoring MDR-Ab specific antibody titers and cytokine production, observing pathology and organ burdens after WT challenge, and measuring levels of serum pentraxin-3, a molecular correlate of A. baumannii infection severity, before and after challenge. Mice vaccinated with ΔtrxA were fully protected against the lethal challenge of WT. However, minimal immunoglobulin class switching was observed with IgM predominating. Spleens harvested from vaccinated mice exhibited negligible levels of IL-4, IFN-γ and IL-17 production when stimulated with UV-inactivated WT Ci79. Importantly, tissues obtained from vaccinated mice displayed reduced pathology and organ burden compared to challenged non-vaccinated mice. Additionally, serum pentraxin-3 concentrations were not increased 24h after challenge in vaccinated mice, correlating with reduction of WT MDR-Ab infection in ΔtrxA immunized mice. Furthermore, passive immunization with ΔtrxA-immune sera provided protection against lethal systemic Ci79 challenge. Collectively, the defined live attenuated ΔtrxA strain is a vaccine candidate against emerging MDR Acinetobacter infection.

Chlamydial protease-like activity factor mediated protection against C. trachomatis in guinea pigs.

We have comprehensively demonstrated using the mouse model that intranasal immunization with recombinant chlamydial protease-like activity factor (rCPAF) leads to a significant reduction in bacterial burden, genital tract pathology and preserves fertility following intravaginal genital chlamydial challenge. In the present report, we evaluated the protective efficacy of rCPAF immunization in guinea pigs, a second animal model for genital chlamydial infection. Using a vaccination strategy similar to the mouse model, we intranasally immunized female guinea pigs with rCPAF plus CpG deoxynucleotides (CpG; as an adjuvant), and challenged intravaginally with C. trachomatis serovar D (CT-D). Immunization with rCPAF/CpG significantly reduced vaginal CT-D shedding and induced resolution of infection by day 24, compared with day 33 in CpG alone treated and challenged animals. Immunization induced robust anti-rCPAF serum IgG 2 weeks following the last immunization, and was sustained at a high-level 4 weeks post challenge. Upregulation of antigen-specific IFN-γ gene expression was observed in rCPAF/CpG-vaccinated splenocytes. Importantly, a significant reduction in inflammation in the genital tissue in rCPAF/CpG-immunized guinea pigs compared with CpG-immunized animals was observed. Taken together, this study provides evidence of the protective efficacy of rCPAF as a vaccine candidate in a second animal model of genital chlamydial infection.