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Backgound: The introduction of the pneumococcal conjugate and polysaccharide vaccines have been valuable tools for combating invasive pneumococcal infection in children and healthy adults. Despite the available vaccination strategies, pneumococcal pneumonia and associated diseases continue to cause substantial morbidity and mortality, particularly in individuals with chronic disease and ageing populations. Next-generation pneumococcal vaccines will need to be highly immunogenic across patient populations providing both mucosal and systemic protective immunity. Mucosal immunization is an effective strategy for stimulating the immune response at the site of pathogen entry while increasing systemic immunity. In this study we utilized intranasal immunization with pneumococcal surface protein A (PspA), in combination with the mucosal adjuvant cholera toxin B (CTB), to characterize the immune components providing protection against S. pneumoniaechallenge.Methods: Mice were immunized intranasally with CTB and PspA individually, and in combination, followed by lethal bacterial challenge with S. pneumoniae, strain A66.1. Animals were monitored for survival and tested for lung bacterial burden, cytokine production as well as S. pneumoniae-specific antibody titer in mouse sera. The primary immunological contributor to the observed protection was confirmed by cytokine neutralization and serum passive transfer.Results: The combination of CTB and PspA provided complete protection against bacterial challenge, which coincided with a significant decrease in lung bacterial burden. Increases in the T-helper (Th) 1 cytokines, interferon (IFN)-γ and interleukin (IL)-2 were observed in the lung 24 h post-challenge while decreases in proinflammatory mediators IL-6 and tumor necrosis factor (TNF)-α were also recorded at the same time point. The adjuvanted PspA immunization induced significant titers of S. pneumoniae-specific antibody in the serum of mice prior to infection. Serum adoptive transfer passively protected animals against subsequent challenge while IFN-γ neutralization had no impact on the outcome of immunization, suggesting a primary role for antibody-mediated protection in the context of this immunization strategy.Conclusion: Mucosal immunization with CTB and PspA induced a local cellular immune response and systemic humoral immunity which resulted in effective reduction of pulmonary bacterial burden and complete protection against S. pneumoniaechallenge. While induction of the pleiotropic cytokine IFN-γ likely contributes to control of infection through activation of effector pathways, it was not required for protection. Instead, immunization with PspA and CTB-induced S. pneumoniae-specific antibodies in the serum prior to infection that were sufficient to protect against mucosal challenge.

ABSTRACTTargeting antigens (Ag) to Fcγ receptors (FcγR) intranasally (i.n.) enhances immunogenicity and protection against intracellular and extracellular pathogens. Specifically, we have demonstrated that targeting fixed (inactivated) Francisella tularensis(iFT) organisms to FcR in mice i.n., with MAb-iFT immune complexes, enhances F. tularensis-specific immune responses and protection against F. tularensischallenge. Furthermore, traditional adjuvant is not required. In addition, we have demonstrated that the increased immunogenicity following the targeting of iFT to FcR is due, in part, to enhanced dendritic cell (DC) maturation, enhanced internalization, and processing and presentation of iFT by DCs, as well as neonatal FcR (FcRn)-enhanced trafficking of iFT from the nasal passage to the nasal mucosa-associated lymphoid tissue (NALT). Using this immunization and challenge model, we expanded on these studies to identify specific in vivoimmune responses impacted and enhanced by FcR targeting of iFT i.n. Specifically, the results of this study demonstrate for the first time that targeting iFT to FcR increases the frequency of activated DCs within the lungs of MAb-iFT-immunized mice subsequent to F. tularensisLVS challenge. In addition, the frequency and number of gamma interferon (IFN-γ)-secreting effector memory (EM) CD4+T cells elicited by F. tularensisinfection (postimmunization) is increased in an interleukin 12 (IL-12)-dependent manner. In summary, these studies build significantly upon previously published work utilizing this vaccine platform. We have identified a number of additional mechanisms by which this novel, adjuvant-independent, FcR-targeted mucosal vaccine approach enhances immunity and protection against infection, while further validating its potential as a universal vaccine platform against mucosal pathogens.

Targeting an antigen to Fc receptors (FcR) can enhance the immune response to the antigen in the absence of adjuvant. Furthermore, we recently demonstrated that intranasal immunization with an Fc?R-targeted antigen enhances protection against a category A intracellular mucosal pathogen, Francisella tularensis. To determine if a similar strategy could be applied to the important pathogen Streptococcus pneumoniae, we used an improved mucosal FcR-targeting strategy that specifically targets human Fc?R type I (hFc?RI). A humanized single-chain antibody component in which the variable domain binds to hFc?RI [anti-hFc?RI (H22)] was linked in a fusion protein with the pneumococcal surface protein A (PspA). PspA is known to elicit protection against pneumococcal sepsis, carriage, and pneumonia in mouse models when administered with adjuvants. Anti-hFc?RI-PspA or recombinant PspA (rPspA) alone was used to intranasally immunize wild-type (WT) and hFc?RI transgenic (Tg) mice in the absence of adjuvant. The hFc?RI Tg mice receiving anti-hFc?RI-PspA exhibited elevated S. pneumoniae-specific IgA, IgG2c, and IgG1 antibodies in serum and bronchoalveolar lavage fluid. Neither immunogen was effective in protecting WT mice in the absence of adjuvant, but when PspA was targeted to hFc?RI as the anti-hFc?RI-PspA fusion, enhanced protection against lethal S. pneumoniaechallenge was observed in the hFc?RI Tg mice compared to mice given nontargeted rPspA alone. Immune sera from the anti-hFc?RI-PspA-immunized Tg mice showed enhanced complement C3 deposition on bacterial surfaces, and protection was dependent upon an active complement system. Immune serum also showed an enhanced bactericidal activity directed against S. pneumoniaethat appears to be lactoferrin mediated.

ABSTRACTAlthough cellular immunity is essential for host defense during intracellular bacterial infections, humoral immunity can also play a significant role in host defense during infection by some intracellular bacteria, including the ehrlichiae. Antibodies can protect susceptible SCID mice from fatal Ehrlichia chaffeensisinfection, an observation that has been hypothesized to involve the opsonization of bacteria released from host cells. To determine whether humoral immunity plays an essential role during ehrlichia infection in immunocompetent mice, we utilized a murine model of fatal monocytotropic ehrlichiosis caused by Ixodes ovatusehrlichia. Mice lacking either B cells or FcγRI were unable to resolve a low-dose (sublethal) I. ovatusehrlichia infection, which suggested that humoral immunity is essential for resistance. Polyclonal sera generated in I. ovatusehrlichia-infected mice recognized a conserved ehrlichia outer membrane protein and, when administered to infected mice, caused a significant decrease in bacterial infection. Mice experimentally depleted of complement, or deficient for complement receptors 1 and 2, were also susceptible to sublethal I. ovatusehrlichia infection, as were mice that lacked the phox91 subunit of NADPH oxidase. The data are consistent with a mechanism whereby bacteria released from infected cells are lysed directly by complement or undergo antibody-mediated FcγR-dependent phagocytosis and subsequent exposure to reactive oxygen intermediates. The findings suggest mechanisms whereby antibodies contribute to immunity against intracellular bacteria in immunocompetent mice.