James A. Wiley

Antigen-Specific CD8+ T Cells Persist in the Upper Respiratory Tract Following Influenza Virus Infection

Because little is known about lymphocyte responses in the nasal mucosa, lymphocyte accumulation in the nasal mucosa, nasal-associated lymphoid tissue (NALT), and cervical lymph nodes (CLN) were determined after primary and heterosubtypic intranasal influenza challenge of mice. T cell accumulation peaked in the nasal mucosa on day 7, but peaked slightly earlier in the CLN (day 5) and later (day 10) in the NALT. Tetrameric staining of nasal mucosal cells revealed a peak accumulation of CD8 T cells specific for either the H-2Db influenza nucleoprotein epitope 366–374 (DbNP366) or the H-2Db polymerase 2 protein epitope 224–233 (DbPA224) at 7 days. By day 13, DbPA224-specific CD8 T cells were undetectable in the mucosa, whereas DbNP366-specific CD8 T cells persisted for at least 35 days in the mucosa and spleen. After heterosubtypic virus challenge, the accumulation of CD8 T cells in the nasal mucosa was quicker, more intense, and predominantly DbNP366 specific relative to the primary inoculation. The kinetics and specificity of the CD8 T cell response were similar to those in the CLN, but the responses in the NALT and spleen were again slower and more protracted. These results indicate that similar to what was reported in the lung, DbNP366-specific CD8 T cells persist in the nasal mucosa after primary influenza infection and predominate in an intensified nasal mucosal response to heterosubtypic challenge. In addition, differences in the kinetics of the CD8 T cell responses in the CLN, NALT, and spleen suggest different roles of these lymphoid tissues in the mucosal response.

Production of Interferon-γ by Influenza Hemagglutinin-Specific CD8 Effector T Cells Influences the Development of Pulmonary Immunopathology

This study examined the inflammation, lung function impairment, and immune protection associated with either wild-type or interferon (IFN)-gamma-deficient Tc1- or Tc2-CD8 effector cells responding to influenza pneumonia. The adoptive transfer of influenza hemagglutinin-specific Tc1 effectors afforded protection and elicited only minimal impairment of lung function. IFN-gamma-deficient Tc1 effector cells were equally protective, but were associated with an eosinophil influx and slightly more lung function impairment early in the response. Relative to Tc1, Tc2 effector cells were less protective, elicited an eosinophil influx and a greater impairment of lung functions. IFN-gamma-deficient Tc2 effector cells were not protective and were associated with the severest impairment of lung function throughout the response, an accumulation of neutrophils, and extensive pulmonary vasculitis and alveolar hemorrhaging. Deletion of IFN-gamma was associated with a delay in effector cell recruitment and the elicitation of a more intense inflammatory response that resulted in more severe lung function impairment in the recipients of either Tc1 or Tc2 IFN-gamma-deficient effector cells. Thus, during influenza infections, IFN-gamma production by the responding CD8 T cells is associated with effector cell recruitment and mitigation of the associated inflammation and of the resulting impairment in lung functions but is not necessary for optimal protection.

Influenza-pseudotyped Gag virus-like particle vaccines provide broad protection against highly pathogenic avian influenza challenge

Influenza-pseudotyped Gag virus-like particles (VLPs) were produced via the expression of influenza hemagglutinin (HA), neuraminidase (NA) and the murine leukemia virus Gag product in the baculovirus-insect cell expression system. Hemagglutination specific activities of sucrose gradient-purified VLPs were similar to those of egg-grown influenza viruses but particle morphologies were gamma retrovirus-like in the form of consistent 100nm spheres. Immunization of mice and ferrets demonstrated robust immunogenicity and protection from challenge with no measurable morbidity. Ferret data were striking in that immunization with H5N1 VLPs representing either A/Vietnam/1203/04 or A/Indonesia/5/05 resulted in solid protection against highly pathogenic A/Vietnam/1203/04 challenge with no detectable virus in the upper respiratory tract post-challenge in either group. H1N1 VLP immunization of ferrets resulted in partial protection against H5N1 challenge with markedly accelerated virus clearance from the upper respiratory tract relative to controls. The immunogenicity of influenza-pseudotyped VLPs was not dependent on the adjuvant properties of replication competent contaminating baculovirus. These data demonstrate robust vaccine protection of Gag-based, influenza-pseudotyped VLPs carrying a variety of influenza antigens and suggest applicability toward a number of additional respiratory viruses.

Fractionation and Characterization of Biologically-active Polysaccharides from Artemisia tripartita

The leaves of Artemisia species have been traditionally used for prevention and treatment of a number of diseases. In this study, five polysaccharide fractions (designated A-I-A-V) were isolated from the leaves of Artemisia tripartita Rydb. by the sequential use of hot-water extraction, ethanol precipitation, ultra-filtration, and chromatography. The homogeneity and average molecular weight of each fraction were determined by high performance size-exclusion chromatography. Sugar composition analysis revealed that Artemisia polysaccharides consisted primarily of xylose, glucose, arabinose, galactose, and galactosamine. Moreover, all fractions contained at least 3.4% sulfate, and fractions A-II-A-V contained an arabinogalactan type II structure. All fractions exhibited macrophage-activating activity, enhancing production of intracellular reactive oxygen species and release of nitric oxide, interleukin 6, interleukin 10, tumor necrosis factor alpha, and monocyte chemotactic protein 1. In addition, all fractions exhibited scavenging activity for reactive oxygen species generated enzymatically or produced extracellularly by human neutrophils. Finally, fractions A-I and A-V exhibited complement-fixing activity. Taken together, our results provide a molecular basis to explain at least part of the beneficial therapeutic effects of Artemisia extracts, and suggest the possibility of using Artemisia polysaccharides as an immunotherapeutic adjuvant.

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.

Prion Shedding from Olfactory Neurons into Nasal Secretions

This study investigated the role of prion infection of the olfactory mucosa in the shedding of prion infectivity into nasal secretions. Prion infection with the HY strain of the transmissible mink encephalopathy (TME) agent resulted in a prominent infection of the olfactory bulb and the olfactory sensory epithelium including the olfactory receptor neurons (ORNs) and vomeronasal receptor neurons (VRNs), whose axons comprise the two olfactory cranial nerves. A distinct glycoform of the disease-specific isoform of the prion protein, PrPSc, was found in the olfactory mucosa compared to the olfactory bulb, but the total amount of HY TME infectivity in the nasal turbinates was within 100-fold of the titer in the olfactory bulb. PrPSc co-localized with olfactory marker protein in the soma and dendrites of ORNs and VRNs and also with adenylyl cyclase III, which is present in the sensory cilia of ORNs that project into the lumen of the nasal airway. Nasal lavages from HY TME-infected hamsters contained prion titers as high as 103.9 median lethal doses per ml, which would be up to 500-fold more infectious in undiluted nasal fluids. These findings were confirmed using the rapid PrPSc amplification QuIC assay, indicating that nasal swabs have the potential to be used for prion diagnostics. These studies demonstrate that prion infection in the olfactory epithelium is likely due to retrograde spread from the olfactory bulb along the olfactory and vomeronasal axons to the soma, dendrites, and cilia of these peripheral neurons. Since prions can replicate to high levels in neurons, we propose that ORNs can release prion infectivity into nasal fluids. The continual turnover and replacement of mature ORNs throughout the adult lifespan may also contribute to prion shedding from the nasal passage and could play a role in transmission of natural prion diseases in domestic and free-ranging ruminants.

Upper Respiratory Tract Resistance to Influenza Infection Is Not Prevented by the Absence of Either Nasal-Associated Lymphoid Tissue or Cervical Lymph Nodes

The murine nasal-associated lymphoid tissue (NALT) and cervical lymph nodes (CLN) are involved in the generation of local immune responses within the upper respiratory tract (URT). However, their involvement in these responses does not imply the necessity for resistance to URT infections. We surgically removed NALT or CLN to address the necessity of these lymphatic tissues for the development of a local protective immune response after a URT influenza infection. No histological evidence of the re-establishment of either tissue was detected after surgery and the subsequent infection. Removal of NALT did not elicit changes in serum or nasal mucosa-associated influenza-specific Ig levels. However, increases in PR8-specific serum IgG and nasal mucosa-associated IgA were detected after removal of CLN. Recruitment of influenza-specific CD4 T cells into the nasal mucosa was not altered by removal of NALT. The removal of NALT or CLN did not alter the recruitment of influenza-specific CD8 T cells into the URT. However, increased levels of influenza-specific CD8 T cells were observed in the tracheal-bronchial lymph nodes after CLN surgery. The rate of viral clearance from nasal mucosa and lungs was not altered by removal of NALT or CLN. These studies demonstrate that despite the participation of NALT and CLN in the generation of local immunity to influenza infections, neither tissue is essential for the development of protective immunity and viral clearance in URT.

Neutrophils Select Hypervirulent CovRS Mutants of M1T1 Group A Streptococcus during Subcutaneous Infection of Mice

ABSTRACT Pathogen mutants arise during infections. Mechanisms of selection for pathogen variants are poorly understood. We tested whether neutrophils select mutations in the two-component regulatory system CovRS of group A Streptococcus (GAS) during infection using the lack of production of the protease SpeB (SpeB activity negative [SpeBA−]) as a marker. Depletion of neutrophils by antibodies RB6-8C5 and 1A8 reduced the percentage of SpeBA− variants (SpeBA−%) recovered from mice infected with GAS strain MGAS2221 by >76%. Neutrophil recruitment and SpeBA−% among recovered GAS were reduced by 95% and 92%, respectively, in subcutaneous MGAS2221 infection of CXCR2−/− mice compared with control mice. In air sac infection with MGAS2221, levels of neutrophils and macrophages in lavage fluid were reduced by 49% and increased by 287%, respectively, in CXCR2−/− mice compared with control mice, implying that macrophages play an insignificant role in the reduction of selection for SpeBA− variants in CXCR2−/− mice. One randomly chosen SpeBA− mutant outcompeted MGAS2221 in normal mice but was outcompeted by MGAS2221 in neutropenic mice and had enhancements in expression of virulence factors, innate immune evasion, skin invasion, and virulence. This and nine other SpeBA− variants from a mouse all had nonsynonymous covRS mutations that resulted in the SpeBA− phenotype and enhanced expression of the CovRS-controlled secreted streptococcal esterase (SsE). Our findings are consistent with a model that neutrophils select spontaneous covRS mutations that maximize the potential of GAS to evade neutrophil responses, resulting in variants with enhanced survival and virulence. To our knowledge, this is the first report of the critical contribution of neutrophils to the selection of pathogen variants.

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.

Accelerated Shedding of Prions following Damage to the Olfactory Epithelium

ABSTRACT In this study, we investigated the role of damage to the nasal mucosa in the shedding of prions into nasal samples as a pathway for prion transmission. Here, we demonstrate that prions can replicate to high levels in the olfactory sensory epithelium (OSE) in hamsters and that induction of apoptosis in olfactory receptor neurons (ORNs) in the OSE resulted in sloughing off of the OSE from nasal turbinates into the lumen of the nasal airway. In the absence of nasotoxic treatment, olfactory marker protein (OMP), which is specific for ORNs, was not detected in nasal lavage samples. However, after nasotoxic treatment that leads to apoptosis of ORNs, both OMP and prion proteins were present in nasal lavage samples. The cellular debris that was released from the OSE into the lumen of the nasal airway was positive for both OMP and the disease-specific isoform of the prion protein, PrPSc. By using the real-time quaking-induced conversion assay to quantify prions, a 100- to 1,000-fold increase in prion seeding activity was observed in nasal lavage samples following nasotoxic treatment. Since neurons replicate prions to higher levels than other cell types and ORNs are the most environmentally exposed neurons, we propose that an increase in ORN apoptosis or damage to the nasal mucosa in a host with a preexisting prion infection of the OSE could lead to a substantial increase in the release of prion infectivity into nasal samples. This mechanism of prion shedding from the olfactory mucosa could contribute to prion transmission.