Induction of mucosal B-cell memory by intranasal immunization of mice with respiratory syncytial virus.
ABSTRACT: The capacity of live or inactivated respiratory syncytial virus (RSV) to induce B-cell memory in respiratory-associated lymphoid tissues of mice was examined. Eight weeks after primary inoculation with either live or inactivated RSV, adult BALB/c mice were challenged with 4x10(5) PFU of RSV. Protection from viral shedding and mucosal production of RSV-specific antibodies were examined at various time points after challenge. We found that primary immunization with live, but not inactivated, RSV induced complete and durable protection upon challenge within the upper and lower respiratory tract. Also, primary immunization with live, but not inactivated, RSV enhanced the production of mucosal RSV-specific immunoglobulin A (IgA) upon challenge. Secondary mucosal IgA responses were characterized by (i) the early production of mucosal IgA by B cells that reside in organized nasal-associated lymphoid tissues, cervical lymph nodes, and bronchial lymph nodes, and (ii) the subsequent production of RSV-specific IgA by mucosal effector tissues, such as the tracheal lamina propria and lung. These findings suggest that primary infection of mice with live RSV might induce mucosal IgA-committed memory B cells. A greater understanding of the characteristics of RSA-specific mucosal memory B cells may facilitate the development of an RSV vaccine.
Project description:Respiratory syncytial virus (RSV) infection is the most frequent cause of hospitalization in infants and young children worldwide. Although mucosal RSV vaccines can reduce RSV disease burden, little is known about mucosal immune response capabilities in children. Neonatal or adult mice were infected with RSV; a subset of neonatal mice received interferon alpha (IFN-?) (intranasal) prior to RSV infection. B cells, B cell activating factor (BAFF) and IgA were measured by flow cytometry. RSV specific IgA was measured in nasal washes. Nasal associated lymphoid tissue (NALT) and lungs were stained for BAFF and IgA. Herein, we show in a mouse model of RSV infection that IFN-? plays a dual role as an antiviral and immune modulator and age-related differences in IgA production upon RSV infection can be overcome by IFN-? administration. IFN-? administration before RSV infection in neonatal mice increased RSV-specific IgA production in the nasal mucosa and induced expression of the B-cell activating factor BAFF in NALT. These findings are important, as mucosal antibodies at the infection site, and not serum antibodies, have been shown to protect human adults from experimental RSV infection.
Project description:Background: Infections with respiratory syncytial virus (RSV) cause significant morbidity and hospitalization in older adults. We studied the humoral, mucosal and B cell responses of an investigational adjuvanted RSV sF vaccine, MEDI7510, in older adults. Methods: In a substudy of a randomized (1:1), double-blind, placebo-controlled study of MEDI7510 in adults ?60 years of age, we collected blood and nasal secretions at days 0, 8, 29, 91 and 180 post-vaccination to measure F-specific IgG and IgA antibodies by ELISA, and plasmablasts and memory B cells by IgA/IgG dual-color fluorospot. Results: The 27 vaccine- and 18 placebo-recipients had a mean age of 73 years and included 24 women. Among vaccinees, 93% had significant increases in F-specific plasma IgG 85% had increased plasma IgA; 74% had increased nasal IgG and 26% nasal IgA; 93% had IgG and 89% IgA plasmablasts on Day 8 post-immunization; and 82% had IgG and 7.4% IgA memory B cell responses to the vaccine. Vaccinees <70 years of age and women had the highest responses to the vaccine. Conclusions: This adjuvanted vaccine generated robust humoral immune responses in older adults, including RSV F-specific systemic and mucosal antibodies and memory B cells. Nevertheless, age ?70 years was associated with decreased immunogenicity of the adjuvanted vaccine.
Project description:The RSV vaccine field suffered a major set-back when children were vaccinated with a formalin-inactivated RSV vaccine (FI-RSV). Unexpectedly, the vaccinated children fared worse than unvaccinated children when they were naturally infected with RSV. Mouse models were then developed that implicated the CD4+ T helper cell population as a contributor to adverse events. Today, the T cell is viewed with much caution in the RSV field, and its induction by vaccination is sometimes discouraged. Here we re-emphasize the beneficial role of the CD4+ T cell. Experiments were performed with RSV-infected nude mice that received CD4+ T cells by adoptive transfer. Data demonstrated that CD4+ T cells were necessary for the induction of mucosal and systemic RSV-specific antibodies, for the establishment of RSV-specific IgG and IgA antibody secreting cells in the upper and lower respiratory tract, and for RSV clearance.
Project description:Respiratory syncytial virus (RSV) is an important viral pathogen that causes severe lower respiratory tract infection in infants, the elderly, and immunocompromised individuals. There are no licensed RSV vaccines to date. To prevent RSV infection, immune responses in both the upper and lower respiratory tracts are required. Previously, immunization with Venezuelan equine encephalitis virus replicon particles (VRPs) demonstrated effectiveness in inducing mucosal protection against various pathogens. In this study, we developed VRPs encoding RSV fusion (F) or attachment (G) glycoproteins and evaluated the immunogenicity and efficacy of these vaccine candidates in mice and cotton rats. VRPs, when administered intranasally, induced surface glycoprotein-specific virus neutralizing antibodies in serum and immunoglobulin A (IgA) antibodies in secretions at the respiratory mucosa. In addition, fusion protein-encoding VRPs induced gamma interferon (IFN-gamma)-secreting T cells in the lungs and spleen, as measured by reaction with an H-2K(d)-restricted CD8(+) T-cell epitope. In animals vaccinated with F protein VRPs, challenge virus replication was reduced below the level of detection in both the upper and lower respiratory tracts following intranasal RSV challenge, while in those vaccinated with G protein VRPs, challenge virus was detected in the upper but not the lower respiratory tract. Close examination of histopathology of the lungs of vaccinated animals following RSV challenge revealed no enhanced inflammation. Immunization with VRPs induced balanced Th1/Th2 immune responses, as measured by the cytokine profile in the lungs and antibody isotype of the humoral immune response. These results represent an important first step toward the use of VRPs encoding RSV proteins as a prophylactic vaccine for RSV.
Project description:Respiratory syncytial virus (RSV) is a high priority target for vaccine development. One concern in RSV vaccine development is that a non-live virus vaccine would predispose for enhanced disease similar to that seen with the formalin inactivated RSV (FI-RSV) vaccine. Since a mAb specific to RSV G protein can reduce pulmonary inflammation and eosinophilia seen after RSV infection of FI-RSV vaccinated mice, we hypothesized that RSV G peptides that induce antibodies with similar reactivity may limit enhanced disease after subunit or other non-live RSV vaccines. In support of this hypothesis, we show that FI-RSV vaccinated mice administered RSV G peptide vaccines had a significant reduction in enhanced disease after RSV challenge. These data support the importance of RSV G during infection to RSV disease pathogenesis and suggest that use of appropriately designed G peptide vaccines to reduce the risk of enhanced disease with non-live RSV vaccines merits further study.
Project description:T follicular helper (Tfh)-like cells with potent B-cell helping ability are mobilized into human circulation after parenteral vaccination and are generally held to reflect ongoing germinal center reactions. However, whether mucosal vaccination induces systemic Tfh responses and how such responses may relate to IgA production are unknown. We investigated the frequencies, phenotype and function of circulating Tfh-like CD4+CXCR5+ T cells (cTfh) in adults receiving an oral inactivated enterotoxigenic Escherichia coli vaccine. Subjects were classified as vaccine responders or weak/non-responders based on their intestine-derived antibody-secreting cell (ASC) IgA responses to major vaccine antigens. Oral immunization induced significantly increased proportions of cTfh cells expressing the cTfh activation marker inducible costimulator (ICOS) in ASC responders, but not in weak/non-responders. Vaccination also enhanced the expression of IL-21, Th17 markers and integrin ?7 by activated cTfh cells, supporting functionality and gut homing potential. cTfh cells promoted total and vaccine specific IgA production from cocultured B cells. Magnitudes of cTfh responses assessed within a week after primary vaccinations correlated with memory intestine-derived vaccine specific IgA responses 1-2 years later. We conclude that activated ICOS+ Tfh-like cells are mobilized into blood after oral vaccination and may be used as biomarkers of vaccine specific mucosal memory in humans.
Project description:Respiratory syncytial virus (RSV) is the single most important cause of serious lower respiratory tract disease in infants and young children worldwide and a high priority for vaccine development. Despite over 50 years of research, however, no vaccine is yet available. One block to vaccine development is an incomplete understanding of the aberrant memory response to the formalin-inactivated RSV vaccine (FI-RSV) given to children in the 1960s. This vaccine caused enhanced respiratory disease (ERD) with later natural RSV infection. Concern that any non-live virus vaccine may also cause ERD has blocked development of subunit vaccines for young children. A number of animal FI-RSV studies suggest various immune mechanisms behind ERD. However, other than limited data from the original FI-RSV trial, there is no information on the human ERD-associated responses. An in vitro model with human blood specimens may shed light on the immune memory responses likely responsible for ERD. Memory T cell responses to an antigen are guided by the innate responses, particularly dendritic cells that present an antigen in conjunction with co-stimulatory molecules and cytokine signaling. Our in vitro model involves human monocyte derived dendritic cells (moDC) and allogenic T cell cultures to assess innate responses that direct T cell responses. Using this model, we evaluated human responses to live RSV, FI-RSV, and subunit RSV G vaccines (G-containing virus-like particles, G-VLP). Similar to findings in animal studies, FI-RSV induced prominent Th2/Th17-biased responses with deficient type-1 responses compared to live virus. Responses to G-VLPs were similar to live virus, i.e. biased towards a Th1 and not a Th2/Th17. Also mutating CX3C motif in G gave a more pronounced moDC responses associated with type-1 T cell responses. This in vitro model identifies human immune responses likely associated with ERD and provides another pre-clinical tool to assess the safety of RSV vaccines.
Project description:Respiratory syncytial virus (RSV) is the leading cause of serious respiratory tract disease but there is no licensed RSV vaccine. Immunopathological mechanisms have long been suspected as operating in the development of severe RSV disease and have hampered the development of safe and effective vaccines. Here, we show that unlike intranasal immunization, sublingual immunization with RSV glycoprotein fragment containing the central conserved region (Gcf) primes the host for severe disease upon RSV challenge. This increased pathology does not require replication by the challenge virus and is associated with massive infiltration of inflammatory cells, extensive cell death, and excessive mucus production in the airway and lungs. This exacerbated RSV disease primed by sublingual Gcf immunization is distinct from the immunopathology by G-expressing vaccinia virus or formalin-inactivated RSV, and preceded by prominent IL-17 production. IL-17 deficiency abolished the enhanced disease. Our results suggest a novel mechanism of RSV vaccine-induced immunopathology by IL-17, and highlights the importance of vaccination site.
Project description:A role for interleukin-21 (IL-21) has recently been found in several diseases, but contribution to mucosal defences has not been described. In BALB/c mice infected with respiratory syncytial virus (RSV), IL-21 depletion had little effect in primary infection. However, depletion of mice during priming with recombinant vaccinia expressing RSV G protein (which primes RSV-specific T helper type 2 cells and causes lung eosinophilia during RSV infection) further exacerbated pathology during RSV challenge, with reduced viral clearance and impaired virus-specific serum antibody responses. This enhancement was accompanied by lymphocyte, neutrophil, and antigen-presenting cell recruitment to the lungs, with increased bronchoalveolar lavage interferon-? and IL-17 levels. Adoptive transfer of splenic CD4 T cells from depleted mice into naive recipients replicated these effects, indicating that IL-21 mediates its effects via CD4 T cells. Endogenous IL-21, therefore, has potent and specific effects on mucosal antiviral responses, assisting viral clearance, regulating pulmonary T- and B-cell responses, and inhibiting IL-17 production.
Project description:Respiratory syncytial virus (RSV) is an important human pathogen. Expression of virus structural proteins produces self-assembled virus-like nanoparticles (VLP). We investigated immune phenotypes after RSV challenge of immunized mice with VLP containing RSV F and G glycoproteins mixed with F-DNA (FdFG VLP). In contrast to formalin-inactivated RSV (FI-RSV) causing vaccination-associated eosinophilia, FdFG VLP immunization induced low bronchoalveolar cellularity, higher ratios of CD11c(+) versus CD11b(+) phenotypic cells and CD8(+) T versus CD4(+) T cells secreting interferon (IFN)-?, T helper type-1 immune responses, and no sign of eosinophilia upon RSV challenge. Furthermore, RSV neutralizing activity, lung viral clearance, and histology results suggest that FdFG VLP can be comparable to live RSV in conferring protection against RSV and in preventing RSV disease. This study provides evidence that a combination of recombinant RSV VLP and plasmid DNA may have a potential anti-RSV prophylactic vaccine inducing balanced innate and adaptive immune responses.