Respiratory Immunity Research Articles

Influenza vaccine with Surfacten, a modified pulmonary surfactant, induces systemic and mucosal immune responses without side effects in minipigs

Immune responses and side effects of intranasally administered flu vaccine with the commercial product Surfacten, a modified bovine pulmonary surfactant, were investigated in minipigs. The use of minipigs was based on the anatomical resemblance of nasal lymph nodes, the principal antigen uptake site of respiratory mucosal immunity, between pig and human. Intranasal instillation of HA vaccine adjuvanted with Surfacten elicited significantly higher serum hemagglutination inhibition titers than the antigen alone, with wide cross-neutralizing activities of secretory IgA in nasal washes. No significant induction of inflammatory cytokines or migration of inflammatory cells was observed at the site of immunization or serum after the first immunization. These data suggest the potential usefulness of Surfacten for mucosal vaccination.

Parenteral Feeding Depletes Pulmonary Lymphocyte Populations

The effect of parenteral nutrition (PN) on lymphocyte mass in the lung is unknown, but reduced mucosal lymphocytes are hypothesized to play a role in the reduced immunoglobulin A-mediated immunity in both gut and lung. The ability to transfer and track cells between mice may allow study of diet-induced mucosal immune function. The objectives of this study are to characterize lung T-cell populations following parenteral feeding and to study distribution patterns of transferred donor lung T cells in recipient mice. In experiment 1, cannulated male Balb/c mice are randomized to receive chow or PN for 5 days. Lung lymphocytes are obtained via collagenase digestion, and flow cytometric analysis is used to identify total T (CD3+) and B (CD45/B220+) cells. In experiment 2, isolated lung T cells from chow-fed male Balb/c mice are pooled and labeled in vitro with a fluorescent dye (carboxyfluorescein diacetate succinimidyl ester [CFSE]), and 1.1 x 10(8) CFSE+ cells (3.1 x 10(6) T cells) are transferred to chow-fed Balb/c recipients. Cells recovered from recipient lungs and intestinal lamina propria (LP) are analyzed by flow cytometry to determine CFSE/CD3+ T cells at 1, 2, and 7 days. In experiment 3, cells are transferred to PN-fed recipients. In experiment 1, PN significantly decreases lung T- and B-cell populations compared with chow feeding. In experiment 2, CFSE+ T-cell retention is highest on day 1 in lung and LP, and decreases on day 2. Cells are gone by day 7; 98.1% of retained donor lung T cells migrate to recipient lungs and 1.9% to the intestine on day 1. Similar results are seen in experiment 3 after transfer of cells to PN-fed recipients. PN reduces pulmonary lymphocyte populations consistent with impaired respiratory immunity. Transferred lung T cells preferentially localize to recipient lungs rather than intestine with maximal accumulation at 24 hours. Limited cross-talk of transferred lung T cells to the intestine indicates that mucosal lymphocyte traffic might be programmed to localize to specific effector sites.

Immunization With a Bivalent Adenovirus-vectored Tuberculosis Vaccine Provides Markedly Improved Protection Over Its Monovalent Counterpart Against Pulmonary Tuberculosis

Recombinant virus-vectored vaccines hold great promise for tuberculosis (TB) vaccination strategies. However, there is a lack of side-by-side comparative investigations to dissect the functional differences and support the advantage of multivalent virus-vectored vaccine over its monovalent counterpart. We previously successfully developed a monovalent adenovirus (Ad)-vectored vaccine expressing Ag85a (AdAg85a) and demonstrated its superior protective efficacy in models of pulmonary TB. In this study, we have developed a bivalent Ad TB vaccine expressing Ag85a and TB10.4 antigens as a fusion protein (AdAg85a:TB10.4) and compared its T-cell-activating and immune protective efficacy with that by monovalent AdAg85a. A single intranasal (i.n.) administration of AdAg85a:TB10.4 induced robust T-cell responses toward the respective antigens within the airway lumen and spleen, although the level of Ag85a-specific T-cell responses in the airway lumen triggered by bivalent AdAg85a:TB10.4 was lower than that by its monovalent counterpart at earlier time points. Thus, a single i.n. delivery of AdAg85a:TB10.4 conferred a markedly improved and sustained level of protection in the lung against Mycobacterium tuberculosis (M.tb) challenge over that by AdAg85a or by conventional BCG immunization with similarly induced levels of protection in the spleen. Our results indicate a unique advantage of multivalent viral-vectored TB vaccines for immunization against pulmonary TB.

The potential role of locally induced matrix metalloproteinase 3 in respiratory mucosal immunity (39.17)

Abstract Recent studies suggest that matrix metalloproteinase 3 (MMP3) is involved in intestinal immunity against bacterial infections as well as in immune cell migrations in delayed hypersensitivity. The goal of this study is to assess the potential role of MMP3 in the development of mucosal immunity at respiratory tract using CpG oligodeoxynucleotide (ODN) as a model system. Groups of BALB/c mice were administrated intranasally with either CpG ODN or GpC ODN or PBS. MMP3 was up-regulated at both 1 and 3 days post immunization in bronchoalveolar lavages (BAL) and respiratory mucosal tissues of CpG ODN immunized mice as determined by ELISA, Western blot and immunohistochemistry (IHC) staining. Furthermore, an increase of both CD11c+ antigen presenting cells and CD4+ T cells was observed in lungs of CpG vaccinated mice compared with GpC ODN or PBS controls. To confirm that MMP3 expression is associated with increased immune cell migration in lungs, mice were treated with MMP3-specific inhibitor II 6hr after CpG immunization. Lung tissues were collected 3 days later and subjected to IHC analysis to determine the expression of MMP3 and the number of CD11c antigen presenting cells and CD4+ T cells. Results showed that MMP3 expression was significantly reduced after treatment with inhibitor II, suggesting that MMP3 inhibitor effectively reduced the expression of MMP3. Accordingly, a reduced number of CD11c+ antigen presenting cells and CD4+ T helper cells was observed in inhibitor-treated mice, indicating a possible role of MMP3 in immune cell migration. Ongoing experiments are focused on validating these observations and investigating the mechanistic basis of this MMP3-associated mucosal immunity. It is anticipated that the results may provide new insights on the molecular basis of mucosal immunity at respiratory tract. (Supported by NSF/EPSCoR EPS-0091948)

Alveolar Type II Epithelial Cells Present Antigen to CD4+T Cells and Induce Foxp3+Regulatory T Cells

Although the contribution of alveolar type II epithelial cells (AECIIs) in respiratory immunity has become increasingly appreciated, their precise function in the induction and regulation of T-cell reactivity to self-antigen remains poorly understood. To investigate the role of AECII in the initiation of T-cell reactivity to alveolar self-antigen, and to clarify their function in the peripheral induction of Foxp3(+) regulatory CD4(+) T cells. To dissect the complex cellular and molecular functions of AECIIs in lung inflammation and immune regulation, we use a transgenic mouse model for CD4(+) T-cell-mediated pulmonary inflammation. Here we report that AECIIs present endogenously expressed antigen on major histocompatibility complex class II molecules to CD4(+) T cells. Epithelial antigen display was sufficient to induce primary T-cell activation and pulmonary inflammation. Upon inflammation, AECIIs induce the differentiation of Foxp3(+) regulatory T cells by a mechanism involving antiproliferative soluble factors, including transforming growth factor (TGF)-beta. Whereas, in acute inflammation, TGF-beta appears to be the dominant factor to induce regulatory T cells, other AECII-derived factors can substitute for and/or synergize with TGF-beta in chronic pulmonary inflammations. AECIIs are capable of priming naive CD4(+) T cells, demonstrating an active participation of these cells in respiratory immunity. Moreover, AECIIs display as yet unrecognized functions in balancing inflammatory and regulatory T-cell responses in the lung by connecting innate and adaptive immune mechanisms to establish peripheral T-cell tolerance to respiratory self-antigen.

Phagocytic clearance of apoptotic cells: role in lung disease

Apoptosis and apoptotic clearance are matched processes that are centered in the maintenance of homeostasis. Similar to apoptosis, apoptotic cell clearance is a conserved mechanism that is highly efficient and redundant, highlighting its overall functional importance in homeostasis. Increasing evidence suggests that the mismatch between apoptosis and apoptotic cell clearance underlies pathologic conditions including inflammatory lung diseases, such as chronic obstructive pulmonary disease, cystic fibrosis, asthma, acute lung injury/acute respiratory distress syndrome and cancer immunity. Although direct causality has yet to be established, this paradigm opens novel approaches towards the understanding and treatment of lung diseases. Glucocorticoids, statins and macrolide antibiotics, which are already in use for treating lung conditions, have a positive effect on apoptotic clearance and are among novel agents that are potential candidates for treatment of these disorders.

Mucosally Delivered Dendritic Cells Activate T Cells Independently of IL-12 and Endogenous APCs

In vitro manipulated dendritic cells (DC) have increasingly been used as a promising vaccine formulation against cancer and infectious disease. However, improved understanding of the immune mechanisms is needed for the development of safe and efficacious mucosal DC immunization. We have developed a murine model of respiratory mucosal immunization by using a genetically manipulated DC vaccine. Within 24 h of intranasal delivery, the majority of vaccine DCs migrated to the lung mucosa and draining lymph nodes and elicited a significant level of T cells capable of IFN-gamma secretion and CTL in the airway lumen as well as substantial T cell responses in the spleen. And such T cell responses were associated with enhanced protection against respiratory mucosal intracellular bacterial challenge. In comparison, parenteral i.m. DC immunization did not elicit marked airway luminal T cell responses and immune protection regardless of strong systemic T cell activation. Although repeated mucosal DC delivery boosted Ag-specific T cells in the airway lumen, added benefits to CD8 T cell activation and immune protection were not observed. By using MHC-deficient vaccine DCs, we further demonstrated that mucosal DC immunization-mediated CD8 and CD4 T cell activation does not require endogenous DCs. By using IL-12-deficient vaccine DCs, we also observed that IL-12(-/-) DCs failed to migrate to the lymph nodes but remained capable of T cell activation. Our observations indicate that mucosal delivery of vaccine DCs represents an effective approach to enhance mucosal T cell immunity, which may operate independent of vaccine IL-12 and endogenous DCs.

Stimulation of respiratory immunity by oral administration ofLactococcus lactis

This work demonstrates that non-recombinant Lactococcus lactis NZ, administered by the oral route at the proper dose, is able to improve resistance against pneumococcal infection. Lactococcus lactis NZ oral administration was able to improve pathogen lung clearance, increased survival of infected mice, and reduced lung injuries. This effect was related to an upregulation of the respiratory innate and specific immune responses. Administration of L. lactis NZ improved production of bronchoalveolar lavage (BAL) fluid TNF-alpha, enhanced recruitment of neutrophils into the alveolar spaces, and induced a higher activation of BAL phagocytes compared with the control group. Lactococcus lactis NZ administered orally stimulated the IgA cycle, increased IgA+ cells in intestine and bronchus, and improved production of BAL IL-4 and IL-10 during infection. Moreover, mice treated with L. lactis NZ showed higher levels of BAL anti-pneumococcal IgA and IgG. Taking into consideration that orally administered L. lactis NZ stimulates both the innate and the specific immune responses in the respiratory tract and that bacterial and viral antigens have been efficiently produced in this strain, L. lactis NZ is an excellent candidate for the development of an effective pneumococcal oral vaccine.

Mucosal immunization against ovine lentivirus using PEI–DNA complexes and modified vaccinia Ankara encoding the gag and/or env genes

Sheep were immunized against Visna/Maedi virus (VMV) gag and/or env genes via the nasopharynx-associated lymphoid tissue (NALT) and lung using polyethylenimine (PEI)–DNA complexes and modified vaccinia Ankara, and challenged with live virus via the lung. env immunization enhanced humoral responses prior to but not after VMV challenge. Systemic T cell proliferative and cytotoxic responses were generally low, with the responses following single gag gene immunization being significantly depressed after challenge. A transient reduction in provirus load in the blood early after challenge was observed following env immunization, whilst the gag gene either alone or in combination with env resulted in significantly elevated provirus loads in lung. However, despite this, a significant reduction in lesion score was observed in animals immunized with the single gag gene at post-mortem. Inclusion of IFN-γ in the immunization mixture in general had no significant effects. The results thus showed that protective effects against VMV-induced lesions can be induced following respiratory immunization with the single gag gene, though this was accompanied by an increased pulmonary provirus load.

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Organ distribution of transgene expression following intranasal mucosal delivery of recombinant replication-defective adenovirus gene transfer vector

It is believed that respiratory mucosal immunization triggers more effective immune protection than parenteral immunization against respiratory infection caused by viruses and intracellular bacteria. Such understanding has led to the successful implementation of intranasal immunization in humans with a live cold-adapted flu virus vaccine. Furthermore there has been an interest in developing effective mucosal-deliverable genetic vaccines against other infectious diseases. However, there is a concern that intranasally delivered recombinant viral-based vaccines may disseminate to the CNS via the olfactory tissue. Initial experimental evidence suggests that intranasally delivered recombinant adenoviral gene transfer vector may transport to the olfactory bulb. However, there is a lack of quantitative studies to compare the relative amounts of transgene products in the respiratory tract, lung, olfactory bulb and brain after intranasal mucosal delivery of viral gene transfer vector. To address this issue, we have used fluorescence macroscopic imaging, luciferase quantification and PCR approaches to compare the relative distribution of transgene products or adenoviral gene sequences in the respiratory tract, lung, draining lymph nodes, olfactory bulb, brain and spleen. Intranasal mucosal delivery of replication-defective recombinant adenoviral vector results in gene transfer predominantly in the respiratory system including the lung while it does lead to a moderate level of gene transfer in the olfactory bulb. However, intranasal inoculation of adenoviral vector leads to little or no viral dissemination to the major region of the CNS, the brain. These experimental findings support the efficaciousness of intranasal adenoviral-mediated gene transfer for the purpose of mucosal immunization and suggest that it may not be of significant safety concern.

Enteral Feeding of a Chemically Defined Diet Preserves Pulmonary Immunity but Not Intestinal Immunity: The Role of Lymphotoxin β Receptor

Compared with chow or a complex enteral diet (CED), IV administration of a parenteral nutrition solution (IV-PN) impairs intestinal and respiratory mucosal immunity, resulting in cellular and immunoglobulin A (IgA) defects in the intestine and impaired respiratory antiviral and antibacterial defenses. PN given intragastrically (IG-PN) impairs intestinal immunity similar to IV-PN but preserves antiviral defences and partially preserves antibacterial defenses. Lymphotoxin beta receptor (LTbetaR) is a molecule essential for development and organization of lymphoid tissue. It controls many molecules important in mucosal immune integrity. This study examines effects of route (IV or enteral) and type (PN, CED, or chow) on murine intestine and lung LTbetaR expression. Forty-three mice randomly received IV-PN (n = 12), IG-PN (n = 11), IV saline + chow (chow; n = 11), or a CED (n = 9). After 5 days of feeding, intestinal and lung samples were obtained and processed for levels of LTbetaR by Western blot. IV-PN significantly reduced intestinal and lung LTbetaR compared with CED and chow. IG-PN reduced LTbetaR levels only in the intestine but preserved lung levels. Route and type of nutrition differentially influence molecular events in the intestinal and respiratory mucosal immune systems. Enteral feeding with any diet (complex or chemically defined) maintains lung LTbetaR expression, whereas intestinal LTbetaR levels are maintained only with CEDs (chow and CED). We hypothesize that LTbetaR is responsible for the observed preservation of respiratory tract immunity with administration of a noncomplex, chemically defined enteral diet, whereas intestinal immunity is compromised with this diet.

Advances in the development of vaccines against Marburg and Ebola viruses.

Peter L Collins & Alexander Bukreyev† †Author for correspondence Laboratory of Infectious Disease, National Institute of Allergy & Infectious Diseases, National Institutes of Health, 50 South Drive, Room 6505, Bethesda, MA 20892-8007, USA Tel.: +1 301 594 1854; Fax: +1 301 496 8312; ab176v@nih.gov ‘The development of vaccines against MARV and EBOV ... is a high priority because of their high virulence, ability to spread from person to person and the possibility of their use in bioterrorism.’

Immunité et vaccinations antivirales : exemple de la muqueuse respiratoire

Objective As the mucosal surfaces of the respiratory tract represent a major portal of entry for most human viruses and many bacteria, they seem to be a critical component of the mammalian immunologic repertoire. Thus, vaccines stimulating this local immunity could represent an interesting approach to prevent these infections. After detailing the different mechanisms implied in this mucosal immunity, the aim of this study is to analyze the basis of such a vaccination and the different vaccines available to mucosal respiratory tract use. Mucosal immunity The major antibody isotype in external secretions is secretory immunoglobin A (S-IgA); the role of IgM (S-IgM) and IgG (S-IgG) are actually questionned. It is, however, interesting that the major effector cells in the mucosal surfaces are not IgA B cells, but T lymphocytes that may represent up to 80% of the entire mucosal lymphoid cell population. Immunoprophylaxis by the mucosal route Passive antibodies were shown to protect against mucosal viral infections, such as those caused by RSV, but very high quantities of passive antibodies are needed to restrict virus replication on mucosal surface. In general, factors which favor development of mucosal antibody and cell mediated immune responses include the oral or respiratory immunization and the replicating nature of the vaccine agents. However, to date only a few vaccines have become available to mucosal respiratory tract use, and cold-adapted influenza virus vaccines is the only one available using nasal route. Other parenteral licensed vaccines have not been recommended for mucosal administration. Some of them have been experimentally used with nasal administration of replicating agents (varicella and measles vaccines) or non replicating agents (influenza inactivated vaccine), but have been found to induce a very low mucosal response. Conclusion Based on the experience with existing vaccines, the development of mucosal immunity or administration of vaccines via the mucosal route is clearly not a prerequisite today for control or prevention of most viral infectious respiratory diseases or diseases with respiratory tract as a route of contamination. But the example of live attenuated intranasal influenza vaccine inducing both systemic and local immune response without immunopathology, is promising for the future of the mucosal immunization against respiratory viral infections.

Successful Topical Respiratory Tract Immunization of Primates against Ebola Virus

Ebola virus causes outbreaks of severe viral hemorrhagic fever with high mortality in humans. The virus is highly contagious and can be transmitted by contact and by the aerosol route. These features make Ebola virus a potential weapon for bioterrorism and biological warfare. Therefore, a vaccine that induces both systemic and local immune responses in the respiratory tract would be highly beneficial. We evaluated a common pediatric respiratory pathogen, human parainfluenza virus type 3 (HPIV3), as a vaccine vector against Ebola virus. HPIV3 recombinants expressing the Ebola virus (Zaire species) surface glycoprotein (GP) alone or in combination with the nucleocapsid protein NP or with the cytokine adjuvant granulocyte-macrophage colony-stimulating factor were administered by the respiratory route to rhesus monkeys--in which HPIV3 infection is mild and asymptomatic--and were evaluated for immunogenicity and protective efficacy against a highly lethal intraperitoneal challenge with Ebola virus. A single immunization with any construct expressing GP was moderately immunogenic against Ebola virus and protected 88% of the animals against severe hemorrhagic fever and death caused by Ebola virus. Two doses were highly immunogenic, and all of the animals survived challenge and were free of signs of disease and of detectable Ebola virus challenge virus. These data illustrate the feasibility of immunization via the respiratory tract against the hemorrhagic fever caused by Ebola virus. To our knowledge, this is the first study in which topical immunization through respiratory tract achieved prevention of a viral hemorrhagic fever infection in a primate model.

αE Integrin Compensates for β7 Deficiency in the Nasal Passages (NP) Following Nasal Immunization with Cholera Toxin (CT) (41.10)

Abstract Nasal immunization stimulates robust Ab responses in the head and neck lymph nodes (HNLN) and nasal-associated lymphoid tissue (NALT). Studies showed that B lymphocyte trafficking in the HNLN and NALT is both L-selectin- and β7-dependent. β7 integrin generally associates with αE or α4 integrins to mediate lymphocyte retention in the epithelium or the mucosa, respectively. To study the contribution of β7 integrin to upper respiratory immunity, β7−/− mice were nasally dosed with CT on days 0, 7, and 14. Mucosal IgA or serum IgG Ab titers were not different from B6 β7+/+ mice at any of the time points examined til day 42. Vaginal IgG responses were slightly enhanced in β7−/− mice, and absent in L-selectin−/− mice. However, the absence of β7 did alter the types of supporting B cells found in the NP. Increased expression of αE was observed on day 42 despite the absence of its co-receptor, β7. This increased αE expression was not observed in B6 β7+/+ mice. αE+ B cells continued to be found in the β7−/− small intestinal lamina propria. Minimal αE+ B cells were found in the HNLN. From these studies, although mucosal Ab responses remain intact in β7−/− mice, it does appear that different homing receptor-dependent mechanisms are employed. This work is supported by NIH AI-55563.

Respiratory syncytial virus and innate immunity: a complex interplay of exploitation and subversion

Respiratory syncytial virus causes significant disease in infants, the elderly and select groups of immunocompromised patients. Healthy individuals are also naturally infected with respiratory syncytial virus repeatedly throughout life. Therefore, safe and effective vaccines and therapies are needed. However, a number of factors have prevented development of such antiviral interventions to date. These include a failed vaccine trial, the very young age of the primary target population (neonates), the inability of natural infection to induce long-term protective immunity, and an incomplete understanding of virus–host interactions. The identification of pattern recognition receptors has led to significant increases in our understanding of induction and regulation of innate immune responses. This review will address the impact of these findings on respiratory syncytial virus research.

CD11b is required for the resolution of inflammation induced by Bordetella bronchiseptica respiratory infection

CD11b is a cell surface receptor that contributes to many cellular processes which are involved in the generation of a protective immune response against pathogenic organisms. In this work, the natural host-pathogen model of murine Bordetella bronchiseptica infection was used to explore the role of CD11b in respiratory immunity. Following intranasal inoculation, CD11b-/- mice rapidly succumb to B. bronchiseptica respiratory infection, highlighting the prominent role of CD11b in the generation of a protective immune response in this model. CD11b appears to be required for both the control of bacterial numbers and the regulation of cellular responses in the lungs. An increased accumulation of neutrophils in the lungs of CD11b-/- mice as compared with wild-type mice suggests that CD11b contributes to the regulation of cellular responses to respiratory infection. This accumulation may be explained by a decrease in apoptosis that is observed in the absence of CD11b following cellular interactions with B. bronchiseptica. Interestingly, this role for CD11b in the regulation of cellular accumulation appears to be critically important for the resolution of damage associated with the type III secretion system (TTSS) of B. bronchiseptica. These data provide new insight into the key role CD11b plays in the resolution of damage in the lower respiratory tract, as well as the B. bronchiseptica virulence determinant that induces it.

Use of recombinant virus-vectored tuberculosis vaccines for respiratory mucosal immunization

Recombinant virus-vectored TB vaccines represent the most promising vaccine platform for boosting the protective immunity mediated by parenteral BCG prime immunization. A major advantage associated with virus-vectored vaccines is that they are potent respiratory mucosa-deliverable vaccines. A recombinant replication-deficient adenoviral (Ad) vector was engineered to express Mycobacterium tuberculosis (M.tb) Ag85A. Single administration of this Ad vaccine via the intranasal, but not intramuscular, route provided potent immune protection from pulmonary M.tb challenge. Respiratory mucosal boosting immunization with Ad vaccine was effective in enhancing T-cell activation and immune protection following parenteral DNA or BCG prime immunization. We have also recently developed a recombinant vesicular stomatitis virus-vectored (VSV) TB vaccine. Ad and VSV vector systems will be complementary to each other for BCG prime-virus vaccine boost immunization protocols.

Respiratory immunity is an important component of protection elicited by subunit vaccination against pneumonic plague

Mice were vaccinated with a recombinant fusion protein, rF1-V, by an intramuscular prime followed by an intranasal boost, to evaluate protection against pneumonic plague. Forty-two days after the intranasal boost, the mice were challenged by aerosol exposure to Yersinia pestis. Survival after exposure depended upon the dose of rF1-V given i.n. with ≥80% survival in the highest dose groups. Pulmonary and serum antibody titers to V were the best predictors of outcome. For vaccinated mice that succumbed to the infection, death was delayed by 1–2 days compared to sham-inoculated controls. Weight loss early after exposure correlated with outcome. Pathology studies indicated a severe, necrotizing bronchopneumonia in vaccinated mice that succumbed to the infection, compatible with a prolonged disease course, while the lungs of sham-inoculated mice had only mild pneumonia, which is compatible with a more rapid disease course. Immunity in the respiratory tract appears to be critical for protection against primary pneumonia caused by Y. pestis.