RETRACTED: Skelton et al. Contribution of Host Immune Responses against Influenza D Virus Infection toward Secondary Bacterial Infection in a Mouse Model. Viruses 2019, 11, 994.

The Viruses Editorial Office retracts the article, "Contribution of Host Immune Responses Against Influenza D Virus Infection Toward Secondary Bacterial Infection in a Mouse Model" [...].

Diet-induced changes in metabolism influence immune response and viral shedding dynamics in Jamaican fruit bats.

Land-use change may drive viral spillover from bats into humans, partly through dietary shifts caused by decreased availability of native foods and increased availability of cultivated foods. We manipulated diets of Jamaican fruit bats to investigate whether diet influences shedding of a virus they naturally host. To reflect dietary changes experienced by wild bats during periods of nutritional stress, bats were fed either standard or putative suboptimal diets which were deprived of protein (suboptimal-sugar) and/or supplemented with fat (suboptimal-fat). Upon H18N11 influenza A-virus infection, bats fed the suboptimal-sugar diet shed the most viral RNA for the longest period, but bats fed the suboptimal-fat diet shed the least viral RNA for the shortest period. Unlike mice and humans, bats fed the suboptimal-fat diet displayed higher pre-infection levels of metabolic markers associated with gut health. Diet-driven heterogeneity in viral shedding may influence population-level viral dynamics in wild bats and alter risk of shedding and spillover to humans.

Pathophysiology of Influenza D Virus Infection in Specific-Pathogen-Free Lambs with or without Prior Mycoplasma ovipneumoniae Exposure.

Polymicrobial pneumonias occur frequently in cattle, swine, and sheep, resulting in major economic losses. Individual pathogens comprising these complex infections may be mild on their own but can instead exhibit synergism or increase host susceptibility. Two examples of such pathogens, Mycoplasma ovipneumoniae (M. ovipneumoniae) and influenza D viruses (IDVs), naturally infect domestic sheep. In sheep, the role of M. ovipneumoniae in chronic nonprogressive pneumonia is well-established, but the pathogenesis of IDV infection has not previously been studied. We utilized a specific-pathogen-free sheep flock to study the clinical response to IDV infection in naïve vs. M. ovipneumoniae-exposed lambs. Lambs were inoculated intranasally with M. ovipneumoniae or mock infection, followed after four weeks by infection with IDV. Pathogen shedding was tracked, and immunological responses were evaluated by measuring acute phase response and IDV-neutralizing antibody titers. While lamb health statuses remained subclinical, M. ovipneumoniae-exposed lambs had significantly elevated body temperatures during IDV infection compared to M. ovipneumoniae-naïve, IDV-infected lambs. Moreover, we found a positive correlation between prior M. ovipneumoniae burden, early-infection IDV shedding, and IDV-neutralizing antibody response. Our findings suggest that IDV infection may not induce clinical symptoms in domestic sheep, but previous M. ovipneumoniae exposure may promote mild IDV-associated inflammation.

An ADAM17-Neutralizing Antibody Reduces Inflammation and Mortality While Increasing Viral Burden in a COVID-19 Mouse Model.

Angiotensin Converting Enzyme 2 (ACE2) is the primary cell entry receptor for SARS-CoV and SARS-CoV-2 viruses. A disintegrin and metalloproteinase 17 (ADAM17) is a protease that cleaves ectodomains of transmembrane proteins, including that of ACE2 and the proinflammatory cytokine TNF-α, from cell surfaces upon cellular activation. We hypothesized that blockade of ADAM17 activity would alter COVID-19 pathogenesis. To assess this pathway, we blocked the function of ADAM17 using the monoclonal antibody MEDI3622 in the K18-hACE2 transgenic mouse model of COVID-19. Antibody-treated mice were healthier, less moribund, and had significantly lower lung pathology than saline-treated mice. However, the viral burden in the lungs of MEDI3622-treated mice was significantly increased. Thus, ADAM17 appears to have a critical anti-viral role, but also may promote inflammatory damage. Since the inflammatory cascade is ultimately the reason for adverse outcomes in COVID-19 patients, there may be a therapeutic application for the MEDI3622 antibody.

Experimental infection of specific-pathogen-free domestic lambs with Mycoplasma ovipneumoniae causes asymptomatic colonization of the upper airways that is resistant to antibiotic treatment.

Mycoplasma ovipneumoniae (M. ovipneumoniae) is a respiratory pathogen associated with mild to moderate respiratory disease in domestic lambs and severe pneumonia outbreaks in wild ruminants such as bighorn sheep. However, whether M. ovipneumoniae by itself causes clinical respiratory disease in domestic sheep in the absence of secondary bacterial pathogens is still unclear. The goal of our study was to better understand the role of M. ovipneumoniae as a respiratory pathogen in domestic sheep and to explore potential antibiotic treatment approaches. Therefore, we inoculated four 4-month-old, specific-pathogen-free lambs with fresh nasal wash fluids from M. ovipneumoniae-infected sheep. The lambs were monitored for M. ovipneumoniae colonization, M. ovipneumoniae-specific antibodies, clinical signs, and cellular and molecular correlates of lung inflammation for eight weeks. All lambs then were treated with gamithromycin and observed for an additional four weeks. M. ovipneumoniae inoculation resulted in stable colonization of the upper respiratory tract in all M. ovipneumoniae-inoculated, but in none of the four mock-infected control lambs. All M. ovipneumoniae-infected lambs developed a robust antibody response to M. ovipneumoniae within 2 weeks. However, we did not observe significant signs of respiratory disease, evidence of lung damage or inflammation in any of the infected lambs. Interestingly, treatment with gamithromycin, which blocked growth of the M. ovipneumoniae in vitro, failed to reduce M. ovipneumoniae colonization. These observations indicate that, in the absence of co-infections, M. ovipneumoniae caused asymptomatic colonization of the upper respiratory tract that was resistant to clearance by the host immune response and by gamithromycin treatment.

Immune suppressive activity of myeloid-derived suppressor cells in cancer requires inactivation of the type I interferon pathway.

Myeloid-derived suppressor cells (MDSC) are pathologically activated neutrophils and monocytes with potent immune suppressive activity. These cells play an important role in accelerating tumor progression and undermining the efficacy of anti-cancer therapies. The natural mechanisms limiting MDSC activity are not well understood. Here, we present evidence that type I interferons (IFN1) receptor signaling serves as a universal mechanism that restricts acquisition of suppressive activity by these cells. Downregulation of the IFNAR1 chain of this receptor is found in MDSC from cancer patients and mouse tumor models. The decrease in IFNAR1 depends on the activation of the p38 protein kinase and is required for activation of the immune suppressive phenotype. Whereas deletion of IFNAR1 is not sufficient to convert neutrophils and monocytes to MDSC, genetic stabilization of IFNAR1 in tumor bearing mice undermines suppressive activity of MDSC and has potent antitumor effect. Stabilizing IFNAR1 using inhibitor of p38 combined with the interferon induction therapy elicits a robust anti-tumor effect. Thus, negative regulatory mechanisms of MDSC function can be exploited therapeutically.

A Self-Adjuvanted, Modular, Antigenic VLP for Rapid Response to Influenza Virus Variability.

The continuous evolution of influenza A virus (IAV) requires the influenza vaccine formulations to be updated annually to provide adequate protection. Recombinant protein-based vaccines provide safer, faster, and a more scalable alternative to the conventional embryonated egg approach for developing vaccines. However, these vaccines are typically poorer in immunogenicity than the vaccines containing inactivated or attenuated influenza viruses and require administration of a large antigen dosage together with potent adjuvants. The presentation of protein antigens on the surface of virus-like particles (VLP) provides an attractive strategy to rapidly induce stronger antigen-specific immune responses. Here we have examined the immunogenic potential and protective efficacy of P22 VLPs conjugated with multiple copies of the globular head domain of the hemagglutinin (HA) protein from the PR8 strain of IAV in a murine model of influenza pathogenesis. Using a covalent attachment strategy (SpyTag/SpyCatcher), we conjugated the HA globular head, which was recombinantly expressed in a genetically modified E. coli strain and found to refold as a monomer, to preassembled P22 VLPs. Immunization of mice with this P22-HAhead conjugate provided full protection from morbidity and mortality following infection with a homologous IAV strain. Moreover, the P22-HAhead conjugate also elicited an accelerated and enhanced HA head specific IgG response, which was significantly higher than the soluble HA head, or the admixture of P22 and HA head without the need for adjuvants. Thus, our results show that the HA head can be easily prepared by in vitro refolding in a modified E. coli strain, maintaining its intact structure and enabling the induction of a strong immune response when conjugated to P22 VLPs, even when presented as a monomer. These results also demonstrate that the P22 VLPs can be rapidly modified in a modular fashion, resulting in an effective vaccine construct that can generate protective immunity without the need for additional adjuvants.

Adverse Childhood Experiences and Immune System Inflammation in Adults Residing on the Blackfeet Reservation: The Moderating Role of Sense of Belonging to the Community.

BACKGROUND: Previous research documents an association between adverse childhood experiences (ACEs) and immune system inflammation. High chronic inflammation is believed to be one biological pathway through which childhood adversity may affect health into adulthood. The Blackfeet tribal community has high rates of childhood trauma and community members are disproportionately affected by inflammatory diseases. PURPOSE: To investigate whether belonging to the tribal community may moderate the relationship between childhood trauma and immune system inflammation in the Blackfeet tribal community. METHODS: In a sample of 90 adults residing on the Blackfeet reservation, we measured ACEs belonging to the tribal community and two markers of immune system inflammation, interleukin-6 (IL-6) and C-reactive protein (CRP). RESULTS: We found that independent of age, gender, annual income, body mass index, and depressive symptoms, belonging to the tribal community and ACEs interacted to predict levels of both IL-6 and CRP (B= -.37, t[81] = -3.82, p < .001, R2 change = .07 and B = -.29, t[81] = -2.75, p = .01, R2 change = .08, respectively). The association between ACEs and markers of immune system inflammation was statistically significant for community members who reported low levels of belonging to the community. CONCLUSIONS: The findings of this study have important implications for intervention research seeking to reduce risk for inflammatory diseases for at-risk populations. Fostering stronger connections to the larger tribal community may positively affect risk for inflammatory diseases. Future work should examine the behavioral and psychosocial pathways through which stronger connections to community may confer health benefits.

Contribution of Host Immune Responses Against Influenza D Virus Infection Toward Secondary Bacterial Infection in a Mouse Model.

Influenza D viruses (IDV) are known to co-circulate with viral and bacterial pathogens in cattle and other ruminants. Currently, there is limited knowledge regarding host responses to IDV infection and whether IDV infection affects host susceptibility to secondary bacterial infections. To begin to address this gap in knowledge, the current study utilized a combination of in vivo and in vitro approaches to evaluate host cellular responses against primary IDV infection and secondary bacterial infection with Staphylococcus aureus (S. aureus). Primary IDV infection in mice did not result in clinical signs of disease and it did not enhance the susceptibility to secondary S. aureus infection. Rather, IDV infection appeared to protect mice from the usual clinical features of secondary bacterial infection, as demonstrated by improved weight loss, survival, and recovery when compared to S. aureus infection alone. We found a notable increase in IFN-ß expression following IDV infection while utilizing human alveolar epithelial A549 cells to analyze early anti-viral responses to IDV infection. These results demonstrate for the first time that IDV infection does not increase the susceptibility to secondary bacterial infection with S. aureus, with evidence that anti-viral immune responses during IDV infection might protect the host against these potentially deadly outcomes.

Development of a Biomedical Program of Research in the Blackfeet Community: Challenges and Rewards.

American Indian (AI) communities have high levels of stress and trauma and are disproportionately affected by numerous preventable diseases. Here, we describe an academic-community partnership based on a collaboration between Blackfeet Community College students and faculty in Psychology and Immunology at Montana State University (MSU). The collaboration, which has spanned over 5 years, was sparked by community interest in the relationship between stress and disease on the Blackfeet reservation. Specifically, community members wanted to understand how the experience of psychological stress and trauma may affect disease risk in their community and identify factors that promote resilience. In doing so, they hoped to identify pathways through which health could be improved for individual community members. Here, we discuss all stages of the collaborative process, including development of measures and methods and themes of research projects, challenges for community members and non-indigenous collaborators, future directions for research, and the lessons learned. Finally, we note the ways in which this partnership and experience has advanced the science of community engagement in tribal communities, with the hope that our experiences will positively affect future collaborations between indigenous community members and non-indigenous scientists.

A Novel Role for PDZ-Binding Motif of Influenza A Virus Nonstructural Protein 1 in Regulation of Host Susceptibility to Postinfluenza Bacterial Superinfections.

Influenza A viruses (IAVs) have multiple mechanisms for altering the host immune response to aid in virus survival and propagation. While both type I and II interferons (IFNs) have been associated with increased bacterial superinfection (BSI) susceptibility, we found that in some cases type I IFNs can be beneficial for BSI outcome. Specifically, we have shown that antagonism of the type I IFN response during infection by some IAVs can lead to the development of deadly BSI. The nonstructural protein 1 (NS1) from IAV is well known for manipulating host type I IFN responses, but the viral proteins mediating BSI severity remain unknown. In this study, we demonstrate that the PDZ-binding motif (PDZ-bm) of the NS1 C-terminal region from mouse-adapted A/Puerto Rico/8/34-H1N1 (PR8) IAV dictates BSI susceptibility through regulation of IFN-α/ß production. Deletion of the NS1 PDZ-bm from PR8 IAV (PR8-TRUNC) resulted in 100% survival and decreased bacterial burden in superinfected mice compared with 0% survival in mice superinfected after PR8 infection. This reduction in BSI susceptibility after infection with PR8-TRUNC was due to the presence of IFN-ß, as protection from BSI was lost in Ifn-ß-/- mice, resembling BSI during PR8 infection. PDZ-bm in PR8-infected mice inhibited the production of IFN-ß posttranscriptionally, and both delayed and reduced expression of the tunable interferon-stimulated genes. Finally, a similar lack of BSI susceptibility, due to the presence of IFN-ß on day 7 post-IAV infection, was also observed after infection of mice with A/TX98-H3N2 virus that naturally lacks a PDZ-bm in NS1, indicating that this mechanism of BSI regulation by NS1 PDZ-bm may not be restricted to PR8 IAV. These results demonstrate that the NS1 C-terminal PDZ-bm, like the one present in PR8 IAV, is involved in controlling susceptibility to BSI through the regulation of IFN-ß, providing new mechanisms for NS1-mediated manipulation of host immunity and BSI severity.

Handling Stress and Sample Storage Are Associated with Weaker Complement-Mediated Bactericidal Ability in Birds but Not Bats.

Variation in immune defense influences infectious disease dynamics within and among species. Understanding how variation in immunity drives pathogen transmission among species is especially important for animals that are reservoir hosts for zoonotic pathogens. Bats, in particular, have a propensity to host serious viral zoonoses without developing clinical disease themselves. The immunological adaptations that allow bats to host viruses without disease may be related to their adaptations for flight (e.g., in metabolism and mediation of oxidative stress). A number of analyses report greater richness of zoonotic pathogens in bats than in other taxa, such as birds (i.e., mostly volant vertebrates) and rodents (i.e., nonvolant small mammals), but immunological comparisons between bats and these other taxa are rare. To examine interspecific differences in bacterial killing ability (BKA), a functional measure of overall constitutive innate immunity, we use a phylogenetic meta-analysis to compare how BKA responds to the acute stress of capture and to storage time of frozen samples across the orders Aves and Chiroptera. After adjusting for host phylogeny, sample size, and total microbe colony-forming units, we find preliminary evidence that the constitutive innate immune defense of bats may be more resilient to handling stress and storage time than that of birds. This pattern was also similar when we analyzed the proportion of nonnegative and positive effect sizes per species, using phylogenetic comparative methods. We discuss potential physiological and evolutionary mechanisms by which complement proteins may differ between species orders and suggest future avenues for comparative field studies of immunity between sympatric bats, birds, and rodents in particular.

IFNAR2 Is Required for Anti-influenza Immunity and Alters Susceptibility to Post-influenza Bacterial Superinfections.

Influenza virus infections particularly when followed by bacterial superinfections (BSI) result in significant morbidities and mortalities especially during influenza pandemics. Type I interferons (IFNs) regulate both anti-influenza immunity and host susceptibility to subsequent BSIs. These type I IFNs consisting of, among others, 14 IFN-α's and a single IFN-ß, are recognized by and signal through the heterodimeric type I IFN receptor (IFNAR) comprised of IFNAR1 and IFNAR2. However, the individual receptor subunits can bind IFN-ß or IFN-α's independently of each other and induce distinct signaling. The role of type I IFN signaling in regulating host susceptibility to both viral infections and BSI has been only examined with respect to IFNAR1 deficiency. Here, we demonstrate that despite some redundancies, IFNAR1 and IFNAR2 have distinct roles in regulating both anti-influenza A virus (IAV) immunity and in shaping host susceptibility to subsequent BSI caused by S. aureus. We found IFNAR2 to be critical for anti-viral immunity. In contrast to Ifnar1-/- mice, IAV-infected Ifnar2-/- mice displayed both increased and accelerated morbidity and mortality compared to WT mice. Furthermore, unlike IFNAR1, IFNAR2 was sufficient to generate protection from lethal IAV infection when stimulated with IFN-ß. With regards to BSI, unlike what we found previously in Ifnar1-/- mice, Ifnar2-/- mice were not susceptible to BSI induced on day 3 post-IAV, even though absence of IFNAR2 resulted in increased viral burden and an increased inflammatory environment. The Ifnar2-/- mice similar to what we previously found in Ifnar1-/- mice were less susceptible than WT mice to BSI induced on day 7 post-IAV, indicating that signaling through a complete receptor increases BSI susceptibility late during clinical IAV infection. Thus, our results support a role for IFNAR2 in induction of anti-IAV immune responses that are involved in altering host susceptibility to BSI and are essential for decreasing the morbidity and mortality associated with IAV infection. These results begin to elucidate some of the mechanisms involved in how the individual IFNAR subunits shape the anti-viral immune response. Moreover, our results highlight the importance of examining the contributions of entire receptors, as individual subunits can induce distinct outcomes as shown here.

Contributions of Influenza Virus Hemagglutinin and Host Immune Responses Toward the Severity of Influenza Virus: Streptococcus pyogenes Superinfections.

Influenza virus infections can be complicated by bacterial superinfections, which are medically relevant because of a complex interaction between the host, the virus, and the bacteria. Studies to date have implicated several influenza virus genes, varied host immune responses, and bacterial virulence factors, however, the host-pathogen interactions that predict survival versus lethal outcomes remain undefined. Previous work by our group showed that certain influenza viruses could yield a survival phenotype (A/swine/Texas/4199-2/98-H3N2, TX98), whereas others were associated with a lethal phenotype (A/Puerto Rico/8/34-H1N1, PR8). Based on this observation, we developed the hypothesis that individual influenza virus genes could contribute to a superinfection, and that the host response after influenza virus infection could influence superinfection severity. The present study analyzes individual influenza virus gene contributions to superinfection severity using reassortant viruses created using TX98 and PR8 viral genes. Host and pathogen interactions, relevant to survival and lethal phenotypes, were studied with a focus on pathogen clearance, host cellular infiltrates, and cytokine levels after infection. Specifically, we found that the hemagglutinin gene expressed by an influenza virus can contribute to the severity of a secondary bacterial infection, likely through modulation of host proinflammatory responses. Altogether, these results advance our understanding of molecular mechanisms underlying influenza virus-bacteria superinfections and identify viral and corresponding host factors that may contribute to morbidity and mortality.

Induction of Antiviral Immune Response through Recognition of the Repeating Subunit Pattern of Viral Capsids Is Toll-Like Receptor 2 Dependent.

Although viruses and viral capsids induce rapid immune responses, little is known about viral pathogen-associated molecular patterns (PAMPs) that are exhibited on their surface. Here, we demonstrate that the repeating protein subunit pattern common to most virus capsids is a molecular pattern that induces a Toll-like-receptor-2 (TLR2)-dependent antiviral immune response. This early antiviral immune response regulates the clearance of subsequent bacterial superinfections, which are a primary cause of morbidities associated with influenza virus infections. Utilizing this altered susceptibility to subsequent bacterial challenge as an outcome, we determined that multiple unrelated, empty, and replication-deficient capsids initiated early TLR2-dependent immune responses, similar to intact influenza virus or murine pneumovirus. These TLR2-mediated responses driven by the capsid were not dependent upon the capsid's shape, size, origin, or amino acid sequence. However, they were dependent upon the multisubunit arrangement of the capsid proteins, because unlike intact capsids, individual capsid subunits did not enhance bacterial clearance. Further, we demonstrated that even a linear microfilament protein built from repeating protein subunits (F-actin), but not its monomer (G-actin), induced similar kinetics of subsequent bacterial clearance as did virus capsid. However, although capsids and F-actin induced similar bacterial clearance, in macrophages they required distinct TLR2 heterodimers for this response (TLR2/6 or TLR2/1, respectively) and different phagocyte populations were involved in the execution of these responses in vivo Our results demonstrate that TLR2 responds to invading viral particles that are composed of repeating protein subunits, indicating that this common architecture of virus capsids is a previously unrecognized molecular pattern.IMPORTANCE Rapid and precise pathogen identification is critical for the initiation of pathogen-specific immune responses and pathogen clearance. Bacteria and fungi express common molecular patterns on their exteriors that are recognized by cell surface-expressed host pattern recognition receptors (PRRs) prior to infection. In contrast, viral molecular patterns are primarily nucleic acids, which are recognized after virus internalization. We found that an initial antiviral immune response is induced by the repeating subunit pattern of virus exteriors (capsids), and thus, induction of this response is independent of viral infection. This early response to viral capsids required the cell surface-expressed PRR TLR2 and allowed for improved clearance of subsequent bacterial infection that commonly complicates respiratory viral infections. Since the repeating protein subunit pattern is conserved across viral capsids, this suggests that it is not easy for a virus to change without altering fitness. Targeting this vulnerability could lead to development of a universal antiviral vaccine.

Differential Type I Interferon Signaling Is a Master Regulator of Susceptibility to Postinfluenza Bacterial Superinfection.

UNLABELLED: Bacterial superinfections are a primary cause of death during influenza pandemics and epidemics. Type I interferon (IFN) signaling contributes to increased susceptibility of mice to bacterial superinfection around day 7 post-influenza A virus (IAV) infection. Here we demonstrate that the reduced susceptibility to methicillin-resistant Staphylococcus aureus (MRSA) at day 3 post-IAV infection, which we previously reported was due to interleukin-13 (IL-13)/IFN-γ responses, is also dependent on type I IFN signaling and its subsequent requirement for protective IL-13 production. We found, through utilization of blocking antibodies, that reduced susceptibility to MRSA at day 3 post-IAV infection was IFN-ß dependent, whereas the increased susceptibility at day 7 was IFN-α dependent. IFN-ß signaling early in IAV infection was required for MRSA clearance, whereas IFN-α signaling late in infection was not, though it did mediate increased susceptibility to MRSA at that time. Type I IFN receptor (IFNAR) signaling in CD11c(+) and Ly6G(+) cells was required for the observed reduced susceptibility at day 3 post-IAV infection. Depletion of Ly6G(+) cells in mice in which IFNAR signaling was either blocked or deleted indicated that Ly6G(+) cells were responsible for the IFNAR signaling-dependent susceptibility to MRSA superinfection at day 7 post-IAV infection. Thus, during IAV infection, the temporal differences in type I IFN signaling increased bactericidal activity of both CD11c(+) and Ly6G(+) cells at day 3 and reduced effector function of Ly6G(+) cells at day 7. The temporal differential outcomes induced by IFN-ß (day 3) and IFN-α (day 7) signaling through the same IFNAR resulted in differential susceptibility to MRSA at 3 and 7 days post-IAV infection. IMPORTANCE: Approximately 114,000 hospitalizations and 40,000 annual deaths in the United States are associated with influenza A virus (IAV) infections. Frequently, these deaths are due to community-acquired Gram-positive bacterial species, many of which show increasing resistance to antibiotic therapy. Severe complications, including parapneumonic empyema and necrotizing pneumonia, can arise, depending on virulence factors expressed by either the virus or bacteria. Unfortunately, we are unable to control the expression of these virulence factors, making host responses a logical target for therapeutic interventions. Moreover, interactions between virus, host, and bacteria that exacerbate IAV-related morbidities and mortalities are largely unknown. Here, we show that type I interferon (IFN) expression can modulate susceptibility to methicillin-resistant Staphylococcus aureus (MRSA) infection, with IFN-ß reducing host susceptibility to MRSA infection while IFN-α increases susceptibility. Our data indicate that treatments designed to augment IFN-ß and/or inhibit IFN-α production around day 7 post-IAV infection could reduce susceptibility to deadly superinfections.

Regulatory T Cell Dysfunction Acquiesces to BTLA+ Regulatory B Cells Subsequent to Oral Intervention in Experimental Autoimmune Encephalomyelitis.

Regulatory T cells (Tregs) induced during autoimmunity often become quiescent and unable to resolve disease, suggesting inadequate activation. Resolution of established experimental autoimmune encephalomyelitis (EAE) can be achieved with myelin oligodendrocyte glycoprotein (MOG) fused to reovirus protein σ1 (MOG-pσ1), which activates Tregs, restoring protection, but requiring other regulatory cells to revitalize them. B cells have a dichotomous role in both the pathogenesis and recovery from EAE. Although inflammatory B cells contribute to EAE's pathogenesis, treatment of EAE mice with MOG-pσ1, but not OVA-pσ1, resulted in an influx of IL-10-producing B220(+)CD5(+) B regulatory cells (Bregs) enabling Tregs to recover their inhibitory activity, and in turn, leading to the rapid amelioration of EAE. These findings implicate direct interactions between Bregs and Tregs to facilitate this recovery. Adoptive transfer of B220(+)CD5(-) B cells from MOG-pσ1-treated EAE or Bregs from PBS-treated EAE mice did not resolve disease, whereas the adoptive transfer of MOG-pσ1-induced B220(+)CD5(+) Bregs greatly ameliorated EAE. MOG-pσ1-, but not OVA-pσ1-induced IL-10-producing Bregs, expressed elevated levels of B and T lymphocyte attenuator (BTLA) relative to CD5(-) B cells, as opposed to Tregs or effector T (Teff) cells, whose BTLA expression was not affected. These induced Bregs restored EAE Treg function in a BTLA-dependent manner. BTLA(-/-) mice showed more pronounced EAE with fewer Tregs, but upon adoptive transfer of MOG-pσ1-induced BTLA(+) Bregs, BTLA(-/-) mice were protected against EAE. Hence, this evidence shows the importance of BTLA in activating Tregs to facilitate recovery from EAE.

Symmetry Controlled, Genetic Presentation of Bioactive Proteins on the P22 Virus-like Particle Using an External Decoration Protein.

Viruses use spatial control of constituent proteins as a means of manipulating and evading host immune systems. Similarly, precise spatial control of proteins encapsulated or presented on designed nanoparticles has the potential to biomimetically amplify or shield biological interactions. Previously, we have shown the ability to encapsulate a wide range of guest proteins within the virus-like particle (VLP) from Salmonella typhimurium bacteriophage P22, including antigenic proteins from human pathogens such as influenza. Expanding on this robust encapsulation strategy, we have used the trimeric decoration protein (Dec) from bacteriophage L as a means of controlled exterior presentation on the mature P22 VLP, to which it binds with high affinity. Through genetic fusion to the C-terminus of the Dec protein, either the 17 kDa soluble region of murine CD40L or a minimal peptide designed from the binding region of the "self-marker" CD47 was independently presented on the P22 VLP capsid exterior. Both candidates retained function when presented as a Dec-fusion. Binding of the Dec domain to the P22 capsid was minimally changed across designed constructs, as measured by surface plasmon resonance, demonstrating the broad utility of this presentation strategy. Dec-mediated presentation offers a robust, modular means of decorating the exposed exterior of the P22 capsid in order to further orchestrate responses to internally functionalized VLPs within biological systems.