A bacterial vesicle-based pneumococcal vaccine against influenza-mediated secondary Streptococcus pneumoniae pulmonary infection.

Streptococcus pneumoniae (Spn) is a common pathogen causing a secondary bacterial infection following influenza, which leads to severe morbidity and mortality during seasonal and pandemic influenza. Therefore, there is an urgent need to develop bacterial vaccines that prevent severe post-influenza bacterial pneumonia. Here, an improved Yersinia pseudotuberculosis strain (designated as YptbS46) possessing an Asd+ plasmid pSMV92 could synthesize high amounts of the Spn pneumococcal surface protein A (PspA) antigen and monophosphoryl lipid A as an adjuvant. The recombinant strain produced outer membrane vesicles (OMVs) enclosing a high amount of PspA protein (designated as OMV-PspA). A prime-boost intramuscular immunization with OMV-PspA induced both memory adaptive and innate immune responses in vaccinated mice, reduced the viral and bacterial burden, and provided complete protection against influenza-mediated secondary Spn infection. Also, the OMV-PspA immunization afforded significant cross-protection against the secondary Spn A66.1 infection and long-term protection against the secondary Spn D39 challenge. Our study implies that an OMV vaccine delivering Spn antigens can be a new promising pneumococcal vaccine candidate.

LUNG group 2 innate lymphoid cells as a new adjuvant target to enhance intranasal vaccine efficacy against influenza.

Group 2 innate lymphoid cells (ILC2) are a relatively new class of innate immune cells. Lung ILC2 are early responders that secrete type 2 cytokines in response to danger 'alarmin' signals such as interleukin (IL)-33 and thymic stromal lymphopoietin. Being an early source of type 2 cytokines, ILC2 are a critical regulator of type 2 immune cells of both innate and adaptive immune responses. The immune regulatory functions of ILC2 were mostly investigated in diseases where T helper 2 inflammation predominates. However, in recent years, it has been appreciated that the role of ILC2 extends to other pathological conditions such as cancer and viral infections. In this review, we will focus on the potential role of lung ILC2 in the induction of mucosal immunity against influenza virus infection and discuss the potential utility of ILC2 as a target for mucosal vaccination.

Virus-based vaccine vectors with distinct replication mechanisms differentially infect and activate dendritic cells.

The precise mechanism by which many virus-based vectors activate immune responses remains unknown. Dendritic cells (DCs) play key roles in priming T cell responses and controlling virus replication, but their functions in generating protective immunity following vaccination with viral vectors are not always well understood. We hypothesized that highly immunogenic viral vectors with identical cell entry pathways but unique replication mechanisms differentially infect and activate DCs to promote antigen presentation and activation of distinctive antigen-specific T cell responses. To evaluate differences in replication mechanisms, we utilized a rhabdovirus vector (vesicular stomatitis virus; VSV) and an alphavirus-rhabdovirus hybrid vector (virus-like vesicles; VLV), which replicates like an alphavirus but enters the cell via the VSV glycoprotein. We found that while virus replication promotes CD8+ T cell activation by VLV, replication is absolutely required for VSV-induced responses. DC subtypes were differentially infected in vitro with VSV and VLV, and displayed differences in activation following infection that were dependent on vector replication but were independent of interferon receptor signaling. Additionally, the ability of the alphavirus-based vector to generate functional CD8+ T cells in the absence of replication relied on cDC1 cells. These results highlight the differential activation of DCs following infection with unique viral vectors and indicate potentially discrete roles of DC subtypes in activating the immune response following immunization with vectors that have distinct replication mechanisms.

Fear of Influenza Resurgence amid COVID-19 Pandemic: Need for Effective Flu Vaccine Still Exists.

As influenza season was approaching in 2020, public health officials feared that influenza would worsen the COVID-19 situation [...].

The Interleukin-33-Group 2 Innate Lymphoid Cell Axis Represents a Potential Adjuvant Target To Increase the Cross-Protective Efficacy of Influenza Vaccine.

Interleukin-33 (IL-33) is a multifunctional cytokine that mediates type 2-dominated immune responses. In contrast, the role of IL-33 during viral vaccination, which often aims to induce type 1 immunity, has not been fully investigated. Here, we examined the effects of IL-33 on influenza vaccine responses. We found that intranasal coadministration of IL-33 with an inactivated influenza virus vaccine increases vaccine efficacy against influenza virus infection, not only with the homologous strain but also with heterologous strains, including the 2009 H1N1 influenza virus pandemic strain. Cross-protection was dependent on group 2 innate lymphoid cells (ILC2s), as the beneficial effect of IL-33 on vaccine efficacy was abrogated in ILC2-deficient C57BL/6 Il7rCre/+ Rorafl/fl mice. Furthermore, mechanistic studies revealed that IL-33-activated ILC2s potentiate vaccine efficacy by enhancing mucosal humoral immunity, particularly IgA responses, potentially in a Th2 cytokine-dependent manner. Our results demonstrate that IL-33-mediated activation of ILC2s is a critical early event that is important for the induction of mucosal humoral immunity, which in turn is responsible for cross-strain protection against influenza. Thus, we reveal a previously unrecognized role for the IL-33-ILC2 axis in establishing broadly protective and long-lasting humoral mucosal immunity against influenza, knowledge that may help in the development of a universal influenza vaccine. IMPORTANCE Current influenza vaccines, although capable of protecting against predicted viruses/strains included in the vaccine, are inept at providing cross-protection against emerging/novel strains. Thus, we are in critical need of a universal vaccine that can protect against a wide range of influenza viruses. Our novel findings show that a mucosal vaccination strategy involving the activation of lung ILC2s is highly effective in eliciting cross-protective humoral immunity in the lungs. This suggests that the biology of lung ILC2s can be exploited to increase the cross-reactivity of commercially available influenza subunit vaccines.

Impact of Pre-Existing Immunity to Influenza on Live-Attenuated Influenza Vaccine (LAIV) Immunogenicity.

During the previous influenza seasons, between 2010 and 2016, the live attenuated influenza vaccine (LAIV) provided variable efficacy against influenza in the U.S., causing the recommendation against the use of the LAIV. In striking contrast, pre-clinical studies have repeatedly demonstrated superior efficacy of LAIV against mismatched influenza viruses, compared to inactivated influenza vaccines (IIV). This disparity in reported vaccine efficacies between pre-clinical and clinical studies may in part be explained by limitations of the animal models of influenza. In particular, the absence of pre-existing immunity in animal models has recently emerged as a potential explanation for the discrepancies between preclinical findings and human studies. This commentary focuses on the potential impact of pre-existing immunity on LAIV induced immunogenicity with an emphasis on cross-protective immunity.

SARS-CoV-2 Vaccines: Inactivation by Gamma Irradiation for T and B Cell Immunity.

Despite accumulating preclinical data demonstrating a crucial role of cytotoxic T cell immunity during viral infections, ongoing efforts on developing COVID-19 vaccines are mostly focused on antibodies. In this commentary article, we discuss potential benefits of cytotoxic T cells in providing long-term protection against COVID-19. Further, we propose that gamma-ray irradiation, which is a previously tested inactivation method, may be utilized to prepare an experimental COVID-19 vaccine that can provide balanced immunity involving both B and T cells.

Impact of Pre-Existing Immunity on Live Attenuated Influenza Vaccine-Induced Cross-Protective Immunity.

The efficacy of the intranasally (i.n.) delivered live attenuated influenza vaccine (LAIV) is variable and, in some seasons, suboptimal. In this study, we report that LAIV exhibits cross-protective efficacy in mice, potentially associated with cellular immunity as opposed to antigen-specific antibody responses. However, pre-exposure to the intramuscularly (i.m.) delivered inactivated influenza vaccine (IIV) severely impaired LAIV-induced cross-protection against heterologous challenge, potentially by inhibiting replication of LAIV. Our findings suggest that pre-existing immunity afforded by IIV suppresses cross-protective T cell immunogenicity of LAIV.

A Triple-challenge Mouse Model of Allergic Airway Disease, Primary Influenza Infection, and Secondary Bacterial Infection.

Asthma is a global problem that affects millions of individuals. An increased risk of respiratory viral and bacterial infections is one of the complications of asthma. We recently reported that mice with ovalbumin-induced allergic airway disease (AAD) are protected against influenza-Streptococcus pneumoniae co-infection. Here, we describe in detail a protocol on how to induce AAD and influenza-S. pneumoniae co-infection in mice and to evaluate the specific roles of asthma on immunity to viral and bacterial pathogens in the hope of translating findings to benefit asthmatic individuals.

Evaluation of Pneumococcal Surface Protein A as a Vaccine Antigen against Secondary Streptococcus pneumoniae Challenge during Influenza A Infection.

Secondary bacterial pneumonia is responsible for significant morbidity and mortality during seasonal and pandemic influenza. Due to the unpredictability of influenza A virus evolution and the time-consuming process of manufacturing strain-specific influenza vaccines, recent efforts have been focused on developing anti-Streptococcus pneumoniae immunity to prevent influenza-related illness and death. Bacterial vaccination to prevent viral-bacterial synergistic interaction during co-infection is a promising concept that needs further investigation. Here, we show that immunization with pneumococcal surface protein A (PspA) fully protects mice against low-dose, but not high-dose, secondary bacterial challenge using a murine model of influenza A virus-S. pneumoniae co-infection. We further show that immunization with PspA is more broadly protective than the pneumococcal conjugate vaccine (Prevnar). These results demonstrate that PspA is a promising vaccine target that can provide protection against a physiologically relevant dose of S. pneumoniae following influenza infection.

Compartmentalized effects of aging on group 2 innate lymphoid cell development and function.

The effects of aging on innate immunity and the resulting impacts on immunosenescence remain poorly understood. Here, we report that aging induces compartmentalized changes to the development and function of group 2 innate lymphoid cells (ILC2), an ILC subset implicated in pulmonary homeostasis and tissue repair. Aging enhances bone marrow early ILC2 development through Notch signaling, but the newly generated circulating ILC2 are unable to settle in the lungs to replenish the concomitantly declining mature lung ILC2 pool in aged mice. Aged lung ILC2 are transcriptomically heterogeneous and functionally compromised, failing to produce cytokines at homeostasis and during influenza infection. They have reduced expression of Cyp2e1, a cytochrome P450 oxidase required for optimal ILC2 function. Transfer of lung ILC2 from young mice enhances resistance to influenza infection in old mice. These data highlight compartmentalized effects of aging on ILC and indicate that targeting tissue-resident ILCs might unlock therapies to enhance resistance to infections and diseases in the elderly.

Allergic Airway Disease Prevents Lethal Synergy of Influenza A Virus-Streptococcus pneumoniae Coinfection.

Fatal outcomes following influenza infection are often associated with secondary bacterial infections. Allergic airway disease (AAD) is known to influence severe complications from respiratory infections, and yet the mechanistic effect of AAD on influenza virus-Streptococcus pneumoniae coinfection has not been investigated previously. We examined the impact of AAD on host susceptibility to viral-bacterial coinfections. We report that AAD improved survival during coinfection when viral-bacterial challenge occurred 1 week after AAD. Counterintuitively, mice with AAD had significantly deceased proinflammatory responses during infection. Specifically, both CD4+ and CD8+ T cell interferon gamma (IFN-γ) responses were suppressed following AAD. Resistance to coinfection was also associated with strong transforming growth factor ß1 (TGF-ß1) expression and increased bacterial clearance. Treatment of AAD mice with IFN-γ or genetic deletion of TGF-ß receptor II expression reversed the protective effects of AAD. Using a novel triple-challenge model system, we show for the first time that AAD can provide protection against influenza virus-S. pneumoniae coinfection through the production of TGF-ß that suppresses the influenza virus-induced IFN-γ response, thereby preserving antibacterial immunity.IMPORTANCE Asthma has become one of the most common chronic diseases and has been identified as a risk factor for developing influenza. However, the impact of asthma on postinfluenza secondary bacterial infection is currently not known. Here, we developed a novel triple-challenge model of allergic airway disease, primary influenza infection, and secondary Streptococcus pneumoniae infection to investigate the impact of asthma on susceptibility to viral-bacterial coinfections. We report for the first time that mice recovering from acute allergic airway disease are highly resistant to influenza-pneumococcal coinfection and that this resistance is due to inhibition of influenza virus-mediated impairment of bacterial clearance. Further characterization of allergic airway disease-associated resistance against postinfluenza secondary bacterial infection may aid in the development of prophylactic and/or therapeutic treatment against coinfection.

Effects of Influenza on Alveolar Macrophage Viability Are Dependent on Mouse Genetic Strain.

Secondary bacterial coinfections following influenza virus pose a serious threat to human health. Therefore, it is of significant clinical relevance to understand the immunological causes of this increased susceptibility. Influenza-induced alterations in alveolar macrophages (AMs) have been shown to be a major underlying cause of the increased susceptibility to bacterial superinfection. However, the mechanisms responsible for this remain under debate, specifically in terms of whether AMs are depleted in response to influenza infection or are maintained postinfection, but with disrupted phagocytic activity. The data presented in this article resolves this issue by showing that either mechanism can differentially occur in individual mouse strains. BALB/c mice exhibited a dramatic IFN-γ-dependent reduction in levels of AMs following infection with influenza A, whereas AM levels in C57BL/6 mice were maintained throughout the course of influenza infection, although the cells displayed an altered phenotype, namely an upregulation in CD11b expression. These strain differences were observed regardless of whether infection was performed with low or high doses of influenza virus. Furthermore, infection with either the H1N1 A/California/04/2009 (CA04) or H1N1 A/PR8/1934 (PR8) virus strain yielded similar results. Regardless of AM viability, both BALB/c and C57BL/6 mice showed a high level of susceptibility to postinfluenza bacterial infection. These findings resolve the apparent inconsistencies in the literature, identify mouse strain-dependent differences in the AM response to influenza infection, and ultimately may facilitate translation of the mouse model to clinical application.

Detrimental Influence of Alveolar Macrophages on Protective Humoral Immunity during Francisella tularensis SchuS4 Pulmonary Infection.

Opsonizing antibody is a critical component of the host protective immune response against many respiratory pathogens. However, the role of antibodies in protection against pulmonary infection with highly virulent Francisella tularensis strain SchuS4 is unclear, and the mechanism that allows F. tularensis to evade antibody-mediated bacterial clearance is not fully understood. We have now found that depletion of alveolar macrophages reveals an otherwise cryptic protective effect of opsonizing antibody. While antibody opsonization alone failed to confer any survival benefit against SchuS4 lung infection, significant protection was observed when mice were depleted of alveolar macrophages prior to infection. Blood immune signature analyses and bacterial burden measurements indicated that the treatment regimen blocked establishment of productive, systemic infection. In addition, protection was found to be dependent upon neutrophils. The results show for the first time a protective effect of opsonizing antibodies against highly virulent F. tularensis SchuS4 pulmonary infection through depletion of alveolar macrophages, the primary bacterial reservoir, and prevention of systemic dissemination. These findings have important implications for the potential use of therapeutic antibodies against intracellular pathogens that may escape clearance by residing within mucosal macrophages.

Defective anti-polysaccharide IgG vaccine responses in IgA deficient mice.

We report that IgA-/- mice exhibit specific defects in IgG antibody responses to various polysaccharide vaccines (Francisella tularensis LPS and Pneumovax), but not protein vaccines such as Fluzone. This defect further included responses to polysaccharide-protein conjugate vaccines (Prevnar and Haemophilus influenzae type b-tetanus toxoid vaccine). In agreement with these findings, IgA-/- mice were protected from pathogen challenge with protein- but not polysaccharide-based vaccines. Interestingly, after immunization with live bacteria, IgA+/+ and IgA-/- mice were both resistant to lethal challenge and their IgG anti-polysaccharide antibody responses were comparable. Immunization with live bacteria, but not purified polysaccharide, induced production of serum B cell-activating factor (BAFF), a cytokine important for IgG class switching; supplementing IgA-/- cell cultures with BAFF enhanced in vitro polyclonal IgG production. Taken together, these findings show that IgA deficiency impairs IgG class switching following vaccination with polysaccharide antigens and that live bacterial immunization can overcome this defect. Since IgA deficient patients also often show defects in antibody responses following immunization with polysaccharide vaccines, our findings could have relevance to the clinical management of this population.

Protective Role for Macrophages in Respiratory Francisella tularensis Infection.

Francisella tularensis causes lethal pneumonia following infection of the lungs by targeting macrophages for intracellular replication; however, macrophages stimulated with interferon gamma (IFN-γ) can resist infection in vitro We therefore hypothesized that the protective effect of IFN-γ against F. tularensisin vivo requires macrophages receptive to stimulation. We found that the lethality of pulmonary F. tularensis LVS infection was exacerbated under conditions of alveolar macrophage depletion and in mice with a macrophage-specific defect in IFN-γ signaling (termed mice with macrophages insensitive to IFN-γ [MIIG mice]). We previously found that treatment with exogenous interleukin 12 (IL-12) protects against F. tularensis infection; this protection was lost in MIIG mice. MIIG mice also exhibited reduced neutrophil recruitment to the lungs following infection. Systemic neutrophil depletion was found to render wild-type mice highly sensitive to respiratory F. tularensis infection, and depletion beginning at 3 days postinfection led to more pronounced sensitivity than depletion beginning prior to infection. Furthermore, IL-12-mediated protection required NADPH oxidase activity. These results indicate that lung macrophages serve a critical protective role in respiratory F. tularensis LVS infection. Macrophages require IFN-γ signaling to mediate protection, which ultimately results in recruitment of neutrophils to further aid in survival from infection.

Prevention of Influenza Virus-Induced Immunopathology by TGF-ß Produced during Allergic Asthma.

Asthma is believed to be a risk factor for influenza infection, however little experimental evidence exists to directly demonstrate the impact of asthma on susceptibility to influenza infection. Using a mouse model, we now report that asthmatic mice are actually significantly more resistant to a lethal influenza virus challenge. Notably, the observed increased resistance was not attributable to enhanced viral clearance, but instead, was due to reduced lung inflammation. Asthmatic mice exhibited a significantly reduced cytokine storm, as well as reduced total protein levels and cytotoxicity in the airways, indicators of decreased tissue injury. Further, asthmatic mice had significantly increased levels of TGF-ß1 and the heightened resistance of asthmatic mice was abrogated in the absence of TGF-ß receptor II. We conclude that a transient increase in TGF-ß expression following acute asthma can induce protection against influenza-induced immunopathology.

Role of Interleukin-12 in Protection against Pulmonary Infection with Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a common pathogen associated with nosocomial pneumonia and is an increasing threat for severe community-acquired pneumonia. We have now investigated the role of interleukin-12 (IL-12) in protective immunity against lung infection with MRSA. The importance of IL-12 in protection from pulmonary MRSA infection was demonstrated by the finding that IL-12p35-deficient mice had a lower survival rate, higher bacterial burdens in lung and spleen, and decreased expression of interferon gamma (IFN-γ) in the lung compared to wild-type mice. These effects were completely reversed by replacement intranasal therapy with recombinant IL-12. Furthermore, exogenous IL-12 treatment of wild-type mice 24 h before pulmonary challenge with a lethal dose of MRSA significantly improved bacterial clearance and resulted in protection from death. The IL-12-treated mice had increased numbers of lung natural killer (NK) cells and neutrophils and higher levels of IFN-γ in the lung and serum compared to untreated mice. The major source of IL-12-driven IFN-γ expression in the lung was the NK cell, and the direct target of pulmonary IFN-γ was the lung macrophage, as shown using mice with a macrophage-specific defect in interferon gamma (IFN-γ) signaling (MIIG mice). Importantly, combination therapy with linezolid and IL-12 following intranasal MRSA infection significantly increased survival compared to that of mice receiving linezolid or IL-12 alone. These results indicate that IL-12-based immunotherapy may hold promise for treatment of MRSA pneumonia.

Allergic Lung Inflammation Reduces Tissue Invasion and Enhances Survival from Pulmonary Pneumococcal Infection in Mice, Which Correlates with Increased Expression of Transforming Growth Factor ß1 and SiglecF(low) Alveolar Macrophages.

Asthma is generally thought to confer an increased risk for invasive pneumococcal disease (IPD) in humans. However, recent reports suggest that mortality rates from IPD are unaffected in patients with asthma and that chronic obstructive pulmonary disease (COPD), a condition similar to asthma, protects against the development of complicated pneumonia. To clarify the effects of asthma on the subsequent susceptibility to pneumococcal infection, ovalbumin (OVA)-induced allergic lung inflammation (ALI) was induced in mice followed by intranasal infection with A66.1 serotype 3 Streptococcus pneumoniae. Surprisingly, mice with ALI were significantly more resistant to lethal infection than non-ALI mice. The heightened resistance observed following ALI correlated with enhanced early clearance of pneumococci from the lung, decreased bacterial invasion from the airway into the lung tissue, a blunted inflammatory cytokine and neutrophil response to infection, and enhanced expression of transforming growth factor ß1 (TGF-ß1). Neutrophil depletion prior to infection had no effect on enhanced early bacterial clearance or resistance to IPD in mice with ALI. Although eosinophils recruited into the lung during ALI appeared to be capable of phagocytizing bacteria, neutralization of interleukin-5 (IL-5) to inhibit eosinophil recruitment likewise had no effect on early clearance or survival following infection. However, enhanced resistance was associated with an increase in levels of clodronate-sensitive, phagocytic SiglecF(low) alveolar macrophages within the airways following ALI. These findings suggest that, while the risk of developing IPD may actually be decreased in patients with acute asthma, additional clinical data are needed to better understand the risk of IPD in patients with different asthma phenotypes.