Ann Harmsen

Oral Vaccination with Salmonella Simultaneously Expressing Yersinia pestis F1 and V Antigens Protects against Bubonic and Pneumonic Plague

The gut provides a large area for immunization enabling the development of mucosal and systemic Ab responses. To test whether the protective Ags to Yersinia pestis can be orally delivered, the Y. pestis caf1 operon, encoding the F1-Ag and virulence Ag (V-Ag) were cloned into attenuated Salmonella vaccine vectors. F1-Ag expression was controlled under a promoter from the caf1 operon; two different promoters (P), PtetA in pV3, PphoP in pV4, as well as a chimera of the two in pV55 were tested. F1-Ag was amply expressed; the chimera in the pV55 showed the best V-Ag expression. Oral immunization with Salmonella-F1 elicited elevated secretory (S)-IgA and serum IgG titers, and Salmonella-V-Ag(pV55) elicited much greater S-IgA and serum IgG Ab titers than Salmonella-V-Ag(pV3) or Salmonella-V-Ag(pV4). Hence, a new Salmonella vaccine, Salmonella-(F1+V)Ags, made with a single plasmid containing the caf1 operon and the chimeric promoter for V-Ag allowed the simultaneous expression of F1 capsule and V-Ag. Salmonella-(F1+V)Ags elicited elevated Ab titers similar to their monotypic derivatives. For bubonic plague, mice dosed with Salmonella-(F1+V)Ags and Salmonella-F1-Ag showed similar efficacy (>83% survival) against ∼1000 LD50 Y. pestis. For pneumonic plague, immunized mice required immunity to both F1- and V-Ags because the mice vaccinated with Salmonella-(F1+V)Ags protected against 100 LD50 Y. pestis. These results show that a single Salmonella vaccine can deliver both F1- and V-Ags to effect both systemic and mucosal immune protection against Y. pestis.

Inducible Bronchus-Associated Lymphoid Tissue Elicited by a Protein Cage Nanoparticle Enhances Protection in Mice against Diverse Respiratory Viruses

Background Destruction of the architectural and subsequently the functional integrity of the lung following pulmonary viral infections is attributable to both the extent of pathogen replication and to the host-generated inflammation associated with the recruitment of immune responses. The presence of antigenically disparate pulmonary viruses and the emergence of novel viruses assures the recurrence of lung damage with infection and resolution of each primary viral infection. Thus, there is a need to develop safe broad spectrum immunoprophylactic strategies capable of enhancing protective immune responses in the lung but which limits immune-mediated lung damage. The immunoprophylactic strategy described here utilizes a protein cage nanoparticle (PCN) to significantly accelerate clearance of diverse respiratory viruses after primary infection and also results in a host immune response that causes less lung damage. Methodology/Principal Findings Mice pre-treated with PCN, independent of any specific viral antigens, were protected against both sub-lethal and lethal doses of two different influenza viruses, a mouse-adapted SARS-coronavirus, or mouse pneumovirus. Treatment with PCN significantly increased survival and was marked by enhanced viral clearance, accelerated induction of viral-specific antibody production, and significant decreases in morbidity and lung damage. The enhanced protection appears to be dependent upon the prior development of inducible bronchus-associated lymphoid tissue (iBALT) in the lung in response to the PCN treatment and to be mediated through CD4+ T cell and B cell dependent mechanisms. Conclusions/Significance The immunoprophylactic strategy described utilizes an infection-independent induction of naturally occurring iBALT prior to infection by a pulmonary viral pathogen. This strategy non-specifically enhances primary immunity to respiratory viruses and is not restricted by the antigen specificities inherent in typical vaccination strategies. PCN treatment is asymptomatic in its application and importantly, ameliorates the damaging inflammation normally associated with the recruitment of immune responses into the lung.

Biomimetic Antigenic Nanoparticles Elicit Controlled Protective Immune Response to Influenza

Here we present a biomimetic strategy toward nanoparticle design for controlled immune response through encapsulation of conserved internal influenza proteins on the interior of virus-like particles (VLPs) to direct CD8+ cytotoxic T cell protection. Programmed encapsulation and sequestration of the conserved nucleoprotein (NP) from influenza on the interior of a VLP, derived from the bacteriophage P22, results in a vaccine that provides multistrain protection against 100 times lethal doses of influenza in an NP specific CD8+ T cell-dependent manner. VLP assembly and encapsulation of the immunogenic NP cargo protein is the result of a genetically programmed self-assembly making this strategy amendable to the quick production of vaccines to rapidly emerging pathogens. Addition of adjuvants or targeting molecules were not required for eliciting the protective response.

Role of Type I IFNs in Pulmonary Complications of Pneumocystis murina Infection

Despite the advent of highly active antiretroviral therapy, pulmonary complications in AIDS are a common clinical problem. Pneumocystis jiroveci infection causes a life-threatening pneumonia, especially in individuals with CD4 T cell deficiencies as occurs in AIDS. Although Pneumocystis sp. is an extracellular fungal pathogen, CD8 T cells are the predominant lymphocyte recruited to the lung in CD4-deficient humans and mice during Pneumocystis pneumonia, and we have found that these CD8 T cells are responsible for subsequent lung damage in CD4 T cell-depleted mice. Comparing CD4 T cell-depleted IFN-α receptor knockout (KO) mice to wild-type mice, we found that this CD8 T cell recruitment and lung damage is type I IFN (IFN-αβ) dependent. However, in both CD4 competent, wild-type and IFN-α receptor (IFNAR) KO mice, Pneumocystis infection leads to an eosinophilic granulocyte influx with bronchial epithelial changes as seen in asthma. This response is delayed in IFNAR KO mice, as is pathogen clearance. Although the inflammation is transient in wild-type animals and resolves upon Pneumocystis clearance, it is more severe and persists through day 35 postinfection in IFNAR KO mice, leading to fibrosis. In addition, IFNAR KO, but not wild-type, mice mount a Pneumocystis-specific IgE response, an indicator of allergic sensitization. Thus, in the absence of IFNAR signaling and CD4 T cells, Pneumocystis-mediated lung damage does not occur, whereas in CD4-competent animals, the absence of IFNAR signaling results in an exacerbated Th2 response, asthma-like symptoms, and fibrosis. Therefore, both CD4 T cell- and type I IFN-mediated mechanisms can determine pulmonary complications from Pneumocystis infection.

A virus-like particle vaccine platform elicits heightened and hastened local lung mucosal antibody production after a single dose

Abstract We show that a model antigen, ovalbumin (OVA), can be chemically conjugated to the exterior of a small heat shock protein (sHsp) cage that has structural similarities to virus-like particles (VLPs). OVA–sHsp conjugation efficiency was dependent upon the stoichiometry and the length of the small molecule linker utilized, and the attachment position on the sHsp cage. When conjugated OVA–sHsp was delivered intranasally to naïve mice, the resulting immune response to OVA was accelerated and intensified, and OVA-specific IgG1 responses were apparent within 5 days after a single immunizing dose, illustrating its utility for vaccine development. If animals were pretreated with a disparate VLP, P22 (a non-replicative bacteriophage capsid), before OVA–sHsp conjugate immunization, OVA-specific IgG1 responses were apparent already by 4 days after a single immunizing dose of conjugate in OVA-naïve mice. Additionally, the mice pretreated with P22 produced high titer mucosal IgA, and isotype-switched OVA-specific serum IgG. Similarly, sHsp pretreatment enhanced the accumulation of lung germinal center B cells, T follicular helper cells, and increased polymeric Ig receptor expression, priming the lungs for subsequent IgG and IgA responses to influenza virus challenge. Thus, sHsp nanoparticles elicited quick and intense antibody responses and these accelerated responses could similarly be induced to antigen chemically conjugated to the sHsp. Pretreatment of mice with P22 further accelerated the onset of the antibody response to OVA–sHsp, demonstrating the utility of conjugating antigens to VLPs for pre-, or possibly post-exposure prophylaxis of lung, all without the need for adjuvant.

Virus-Like Particle-Induced Protection Against MRSA Pneumonia Is Dependent on IL-13 and Enhancement of Phagocyte Function

The importance of the priming of the lung environment by past infections is being increasingly recognized. Exposure to any given antigen can either improve or worsen the outcome of subsequent lung infections, depending on the immunological history of the host. Thus, an ability to impart transient alterations in the lung environment in anticipation of future insult could provide an important novel therapy for emerging infectious diseases. In this study, we show that nasal administration of virus-like particles (VLPs) before, or immediately after, lethal challenge with methicillin-resistant Staphylococcus aureus (MRSA) of mice i) ensures complete recovery from lung infection and near absolute clearance of bacteria within 12 hours of challenge, ii) reduces host response-induced lung tissue damage, iii) promotes recruitment and efficient bacterial clearance by neutrophils and CD11c+ cells, and iv) protects macrophages from MRSA-induced necrosis. VLP-mediated protection against MRSA relied on innate immunity. Complete recovery occurred in VLP-dosed mice with severe combined immunodeficiency, but not in wild-type mice depleted of either Ly6G+ or CD11c+ cells. Early IL-13 production associated with VLP-induced CD11c+ cells was essential for VLP-induced protection. These results indicate that VLP-induced alteration of the lung environment protects the host from lethal MRSA pneumonia by enhancing phagocyte recruitment and killing and by reducing inflammation-induced tissue damage via IL-13–dependent mechanisms.

Inducible Bronchus-Associated Lymphoid Tissue (iBALT) Synergizes with Local Lymph Nodes during Antiviral CD4+ T Cell Responses

BACKGROUND Exposure of the lungs to an antigen or pathogen elicits the formation of lymphoid satellite islands termed inducible bronchus-associated lymphoid tissue (iBALT). However, little is known about how the presence of iBALT, induced by a stimulus unrelated to the subsequent challenge agent, influences systemic immunity in distal locations, whether it be independently, antagonistically, or synergistically. Here, we determined the kinetics of the influenza-specific responses in the iBALT, tracheobronchial lymph node (TBLN), and spleen of mice with and without pre-formed iBALT. METHODS AND RESULTS Mice with VLP-induced iBALT or no pre-formed iBALT were challenged with influenza. We found that, as we have previously described, those mice whose lungs contained pre-formed iBALT were protected from morbidity, and furthermore, that these mice had increased dendritic cell, and alveolar macrophage accumulation in both the iBALT and TBLNs. This translated to similarly accelerated kinetics and intensified influenza-specific CD4(+), but not CD8(+) T cell responses in the iBALT, TBLN, and spleen. This expansion was then followed by a more rapid T cell contraction in all lymphoid tissues in the mice with pre-formed iBALT. CONCLUSIONS Thus, iBALT itself may not be responsible for the accelerated primary immune response we observe in mice with pre-formed iBALT, but may contribute to an overall accelerated local and systemic primary CD4(+), but not CD8(+) T cell response. Furthermore, less damaging immune responses observed in mice with pre-formed iBALT may be due to a quicker contraction of CD4(+) T cell responses in both local and systemic secondary lymphoid tissue.

Type I Interferon Signaling and B Cells Maintain Hemopoiesis during Pneumocystis Infection of the Lung

Loss of CD4 T cells is the hallmark of HIV infection. However, type I IFN-producing plasmacytoid dendritic cells may also be lost. This results in susceptibility to an opportunistic infection such as Pneumocystis pneumonia. In addition, regenerative bone marrow failure resulting in pancytopenia is another common problem in advanced stage AIDS. This may be linked to both the failing immune system and recurrent opportunistic infections. We generated lymphocyte-deficient type I IFN receptor-deficient mice (IFrag−/−) to study the effects on Pneumocystis infection of the lung. When IFrag−/− animals were infected with Pneumocystis they died between days 16 and 21 postinfection with minimal pneumonia but severe anemia due to complete bone marrow failure. This included the loss of uncommitted hemopoietic precursor cells. Bone marrow failure was prevented by the reconstitution of IFrag−/− mice with wild-type lymphocytes, especially B cells. T and B cells lacking type I IFN receptor signaling could only partially prevent bone marrow failure in response to Pneumocystis infection. However, the presence of T and B cells lacking type I IFN signaling resulted in compensatory extramedullary hemopoiesis in the liver and spleen. Lymphocyte support of the regenerative capacity of the bone marrow was provided by both type I IFN-dependent and -independent mechanisms that acted synergistically. Our findings point to the requirement of both type I IFNs and lymphocytes in the regenerative capabilities of the hemopoietic system under the pressure of Pneumocystis infection, but not during steady-state hemopoiesis. This may have implications in the management of pancytopenia in AIDS.

Regulation of IFN-γ by IL-13 dictates susceptibility to secondary postinfluenza MRSA pneumonia

Superinfection in mice at day 7 postinfluenza infection exacerbates bacterial pneumonia at least in part via downstream effects of increased IFN‐γ signaling. Here we show that up to 3 days postinfluenza infection, mice have reduced susceptibility to superinfection with methicillin‐resistant Staphylococcus aureus (MRSA), but that superinfection during that time exacerbated influenza disease. This was due to IL‐13 signaling that was advantageous for resolving MRSA infection via inhibition of IFN‐γ, but was detrimental to the clearance of influenza virus. However, if superinfection did not occur until the near resolution of influenza infection (day 7), IL‐13 signaling was inhibited, at least in part by upregulation of IL‐13 decoy receptor (IL‐13Rα2), which in turn caused increases in IFN‐γ signaling and exacerbation of bacterial infection. Understanding these cytokine sequelae is critical to development of immunotherapies for influenza‐MRSA coinfection since perturbations of these sequelae at the wrong time could increase susceptibility to MRSA and/or influenza.

CD11c+ cells primed with unrelated antigens facilitate an accelerated immune response to influenza virus in mice

Recent evidence suggests that an individuals unique history and sequence of exposures to pathogens and antigens may dictate downstream immune responses to disparate antigens. We show that the i.n. delivery of nonreplicative virus‐like particles (VLPs), which bear structural but no antigenic similarities to respiratory pathogens, acts to prime the lungs of both C56BL/6 and BALB/c mice, facilitating heightened and accelerated primary immune responses to high‐dose influenza challenge, thus providing a nonpathogenic model of innate imprinting. These responses correspond closely to those observed following natural infection with the opportunistic fungus, Pneumocystis murina, and are characterized by accelerated antigen processing by DCs and alveolar macrophages, an enhanced influx of cells to the local tracheobronchial lymph node, and early upregulation of T‐cell co‐stimulatory/adhesion molecules. CD11c+ cells, which have been directly exposed to VLPs or Pneumocystis are necessary in facilitating enhanced clearance of influenza virus, and the repopulation of the lung by Ly‐6C+ precursors relies on CCR2 expression. Thus, immune imprinting 72 h after VLP‐priming, or 2 weeks after Pneumocystis‐priming is CCR2‐mediated and results from the enhanced antigen processing, maturation, and trafficking abilities of DCs and alveolar macrophages, which cause accelerated influenza‐specific primary immune responses and result in superior viral clearance.