Crystal L. Loving

Murine Aerosol Challenge Model of Anthrax

ABSTRACT The availability of relevant and useful animal models is critical for progress in the development of effective vaccines and therapeutics. The infection of rabbits and non-human primates with fully virulent Bacillus anthracis spores provides two excellent models of anthrax disease. However, the high cost of procuring and housing these animals and the specialized facilities required to deliver fully virulent spores limit their practical use in early stages of product development. Conversely, the small size and low cost associated with using mice makes this animal model more practical for conducting experiments in which large numbers of animals are required. In addition, the availability of knockout strains and well-characterized immunological reagents makes it possible to perform studies in mice that cannot be performed easily in other species. Although we, along with others, have used the mouse aerosol challenge model to examine the outcome of B. anthracis infection, a detailed characterization of the disease is lacking. The current study utilizes a murine aerosol challenge model to investigate disease progression, innate cytokine responses, and histological changes during the course of anthrax after challenge with aerosolized spores. Our results show that anthrax disease progression in a complement-deficient mouse after challenge with aerosolized Sterne spores is similar to that described for other species, including rabbits and non-human primates, challenged with fully virulent B. anthracis. Thus, the murine aerosol challenge model is both useful and relevant and provides a means to further investigate the host response and mechanisms of B. anthracis pathogenesis.

Role of Anthrax Toxins in Dissemination, Disease Progression, and Induction of Protective Adaptive Immunity in the Mouse Aerosol Challenge Model

ABSTRACT Anthrax toxins significantly contribute to anthrax disease pathogenesis, and mechanisms by which the toxins affect host cellular responses have been identified with purified toxins. However, the contribution of anthrax toxin proteins to dissemination, disease progression, and subsequent immunity after aerosol infection with spores has not been clearly elucidated. To better understand the role of anthrax toxins in pathogenesis in vivo and to investigate the contribution of antibody to toxin proteins in protection, we completed a series of in vivo experiments using a murine aerosol challenge model and a collection of in-frame deletion mutants lacking toxin components. Our data show that after aerosol exposure to Bacillus anthracis spores, anthrax lethal toxin was required for outgrowth of bacilli in the draining lymph nodes and subsequent progression of infection beyond the lymph nodes to establish disseminated disease. After pulmonary exposure to anthrax spores, toxin expression was required for the development of protective immunity to a subsequent lethal challenge. However, immunoglobulin (immunoglobulin G) titers to toxin proteins, prior to secondary challenge, did not correlate with the protection observed upon secondary challenge with wild-type spores. A correlation was observed between survival after secondary challenge and rapid anamnestic responses directed against toxin proteins. Taken together, these studies indicate that anthrax toxins are required for dissemination of bacteria beyond the draining lymphoid tissue, leading to full virulence in the mouse aerosol challenge model, and that primary and anamnestic immune responses to toxin proteins provide protection against subsequent lethal challenge. These results provide support for the utility of the mouse aerosol challenge model for the study of inhalational anthrax.

Contribution of Bordetella bronchiseptica filamentous hemagglutinin and pertactin to respiratory disease in swine.

ABSTRACT Bordetella bronchiseptica is pervasive in swine populations and plays multiple roles in respiratory disease. Most studies addressing virulence factors of B. bronchiseptica are based on isolates derived from hosts other than pigs. Two well-studied virulence factors implicated in the adhesion process are filamentous hemagglutinin (FHA) and pertactin (PRN). We hypothesized that both FHA and PRN would serve critical roles in the adhesion process and be necessary for colonization of the swine respiratory tract. To investigate the role of FHA and PRN in Bordetella pathogenesis in swine, we constructed mutants containing an in-frame deletion of the FHA or the PRN structural gene in a virulent B. bronchiseptica swine isolate. Both mutants were compared to the wild-type swine isolate for their ability to colonize and cause disease in swine. Colonization of the FHA mutant was lower than that of the wild type at all respiratory tract sites and time points examined and caused limited to no disease. In contrast, the PRN mutant caused similar disease severity relative to the wild type; however, colonization of the PRN mutant was reduced relative to the wild type during early and late infection and induced higher anti-Bordetella antibody titers. Together, our results indicate that despite inducing different pathologies and antibody responses, both FHA and PRN are necessary for optimal colonization of the swine respiratory tract.

Nod1/Nod2-Mediated Recognition Plays a Critical Role in Induction of Adaptive Immunity to Anthrax after Aerosol Exposure

ABSTRACT Toll-like receptors and Nod-like receptors (NLR) play an important role in sensing invading microorganisms for pathogen clearance and eliciting adaptive immunity for protection against rechallenge. Nod1 and Nod2, members of the NLR family, are capable of detecting bacterial peptidoglycan motifs in the host cytosol for triggering proinflammatory cytokine production. In the current study, we sought to determine if Nod1/Nod2 are involved in sensing Bacillus anthracis infection and eliciting protective immune responses. Using mice deficient in both Nod1 and Nod2 proteins, we showed that Nod1/Nod2 are involved in detecting B. anthracis for production of tumor necrosis factor alpha, interleukin-1α (IL-1α), IL-1β, CCL5, IL-6, and KC. Proinflammatory responses were higher when cells were exposed to viable spores than when they were exposed to irradiated spores, indicating that recognition of vegetative bacilli through Nod1/Nod2 is significant. We also identify a critical role for Nod1/Nod2 in priming responses after B. anthracis aerosol exposure, as mice deficient in Nod1/Nod2 were impaired in their ability to mount an anamnestic antibody response and were more susceptible to secondary lethal challenge than wild-type mice.

Virulence, Transmission, and Heterologous Protection of Four Isolates of Haemophilus parasuis

ABSTRACT Haemophilus parasuis causes Glässers disease, a syndrome of polyserositis, meningitis, and arthritis in swine. Previous studies with H. parasuis have revealed virulence disparity among isolates and inconsistent heterologous protection. In this study, virulence, direct transmission, and heterologous protection of 4 isolates of H. parasuis (SW114, 12939, MN-H, and 29755) were evaluated using a highly susceptible pig model. In an initial experiment, isolates 12939, MN-H, and 29755 caused Glässers disease, while strain SW114 failed to cause any clinical signs of disease. One pig from each group challenged with MN-H or 29755 failed to develop clinical disease but was able to transmit H. parasuis to noninfected pigs, which subsequently developed Glässers disease. Pigs colonized with SW114, 29755, or MN-H that were free of clinical disease were protected from a subsequent challenge with isolate 12939. In a following experiment, pigs vaccinated with strain SW114 given as either a bacterin intramuscularly or a live intranasal vaccine were protected from subsequent challenge with isolate 12939; however, some pigs given live SW114 developed arthritis. Overall these studies demonstrated that pigs infected with virulent isolates of H. parasuis can remain healthy and serve as reservoirs for transmission to naive pigs and that heterologous protection among H. parasuis isolates is possible. In addition, further attenuation of strain SW114 is necessary if it is to be used as a live vaccine.

Characterization and Vaccine Potential of Outer Membrane Vesicles Produced by Haemophilus parasuis.

Haemophilus parasuis is a Gram-negative bacterium that colonizes the upper respiratory tract of swine and is capable of causing a systemic infection, resulting in high morbidity and mortality. H. parasuis isolates display a wide range of virulence and virulence factors are largely unknown. Commercial bacterins are often used to vaccinate swine against H. parasuis, though strain variability and lack of cross-reactivity can make this an ineffective means of protection. Outer membrane vesicles (OMV) are spherical structures naturally released from the membrane of bacteria and OMV are often enriched in toxins, signaling molecules and other bacterial components. Examination of OMV structures has led to identification of virulence factors in a number of bacteria and they have been successfully used as subunit vaccines. We have isolated OMV from both virulent and avirulent strains of H. parasuis, have examined their protein content and assessed their ability to induce an immune response in the host. Vaccination with purified OMV derived from the virulent H. parasuis Nagasaki strain provided protection against challenge with a lethal dose of the bacteria.

Anamnestic Protective Immunity to Bacillus anthracis Is Antibody Mediated but Independent of Complement and Fc Receptors

ABSTRACT The threat of bioterrorist use of Bacillus anthracis has focused urgent attention on the efficacy and mechanisms of protective immunity induced by available vaccines. However, the mechanisms of infection-induced immunity have been less well studied and defined. We used a combination of complement depletion along with immunodeficient mice and adoptive transfer approaches to determine the mechanisms of infection-induced protective immunity to B. anthracis. B- or T-cell-deficient mice lacked the complete anamnestic protection observed in immunocompetent mice. In addition, T-cell-deficient mice generated poor antibody titers but were protected by the adoptive transfer of serum from B. anthracis-challenged mice. Adoptively transferred sera were protective in mice lacking complement, Fc receptors, or both, suggesting that they operate independent of these effectors. Together, these results indicate that antibody-mediated neutralization provides significant protection in B. anthracis infection-induced immunity.

Vaccine-Associated Enhanced Respiratory Disease Does Not Interfere with the Adaptive Immune Response Following Challenge with Pandemic A/H1N1 2009

The implications of sequential prime and challenge with mismatched influenza A viruses is a concern in mammals, including humans. We evaluated the ability of pigs affected with vaccine-associated enhanced respiratory disease (VAERD) to generate a humoral immune response against the heterologous challenge virus inciting the VAERD. Vaccinated and challenged (V/C) pigs were administered an inactivated swine δ-cluster H1N2 (MN08) vaccine with an HA similar to pre-2009 seasonal human viruses and challenged with heterologous A(H1N1) pandemic 2009 (H1N1pdm09). Vaccination induced MN08-specific hemagglutination inhibition (HI) antibody but not cross-reacting H1N1pdm09 HI antibody. However, vaccinated pigs demonstrated significantly higher post-challenge anti-H1N1pdm09 serum neutralizing (SN) antibodies at 14 and 21 days post inoculation (dpi) compared to nonvaccinated, challenged pigs (NV/C), indicating a priming effect of the vaccine. Serum and lung whole virus anti-H1N1pdm09u2009IgG ELISA antibodies in the vaccinated group were significantly higher than the challenge only pigs at all-time points evaluated. Lung IgA ELISA antibodies to both antigens were detected at 2, 5, and 21u2009dpi in vaccine-primed pigs, contrasted against mucosal ELISA antibody responses detected only at 21u2009dpi in the naïve-challenged group. Collectively, vaccine-primed pigs demonstrated a robust humoral immune response and elevated local adaptive cytokine levels, indicating VAERD does not adversely affect the induction of an immune response to challenge with heterologous virus despite the severe clinical disease and underlying lung pathology. Thus, original antigenic sin does not appear to be a component of VAERD.

Comparison of Human-Like H1 (δ-Cluster) Influenza A Viruses in the Swine Host.

Influenza A viruses cause acute respiratory disease in swine. Viruses with H1 hemagglutinin genes from the human seasonal lineage (δ-cluster) have been isolated from North American swine since 2003. The objective of this work was to study the pathogenesis and transmission of δ-cluster H1 influenza viruses in swine, comparing three isolates from different phylogenetic subclusters, geographic locations, and years of isolation. Two isolates from the δ2 subcluster, A/sw/MN/07002083/07 H1N1 (MN07) and A/sw/IL/00685/05 H1N1 (IL05), and A/sw/TX/01976/08 H1N2 (TX08) from the δ1 sub-cluster were evaluated. All isolates caused disease and were transmitted to contact pigs. Respiratory disease was apparent in pigs infected with MN07 and IL05 viruses; however, clinical signs and lung lesions were reduced in severity as compared to TX08. On day 5 following infection MN07-infected pigs had lower virus titers than the TX08 pigs, suggesting that although this H1N1 was successfully transmitted, it may not replicate as efficiently in the upper or lower respiratory tract. MN07 and IL05 H1N1 induced higher serum antibody titers than TX08. Greater serological cross-reactivity was observed for viruses from the same HA phylogenetic sub-cluster; however, antigenic differences between the sub-clusters may have implications for disease control strategies for pigs.

Bordetella bronchiseptica Colonization Limits Efficacy, but Not Immunogenicity, of Live-Attenuated Influenza Virus Vaccine and Enhances Pathogenesis After Influenza Challenge

Intranasally administered live-attenuated influenza virus (LAIV) vaccines provide significant protection against heterologous influenza A virus (IAV) challenge. However, LAIV administration can modify the bacterial microbiota in the upper respiratory tract, including alterations in species that cause pneumonia. We sought to evaluate the effect of Bordetella bronchiseptica colonization on LAIV immunogenicity and efficacy in swine, and the impact of LAIV and IAV challenge on B. bronchiseptica colonization and disease. LAIV immunogenicity was not significantly impacted by B. bronchiseptica colonization, but protective efficacy against heterologous IAV challenge in the upper respiratory tract was impaired. Titers of IAV in the nose and trachea of pigs that received LAIV were significantly reduced when compared to non-vaccinated, challenged controls, regardless of B. bronchiseptica infection. Pneumonia scores were higher in pigs colonized with B. bronchiseptica and challenged with IAV, but this was regardless of LAIV vaccination status. While LAIV vaccination provided significant protection against heterologous IAV challenge, the protection was not sterilizing and IAV replicated in the respiratory tract of all LAIV vaccinated pig. The interaction between IAV, B. bronchiseptica, and host led to development of acute-type B. bronchiseptica lesions in the lung. Thus, the data presented do not negate the efficacy of LAIV vaccination, but instead indicate that controlling B. bronchiseptica colonization in swine could limit the negative interaction between IAV and Bordetella on swine health.