Edmund J. Gosselin

Flow Cytometric Analysis of Leukocytes in the Human Female Reproductive Tract: Comparison of Fallopian Tube, Uterus, Cervix, and Vagina

PROBLEM: The tissues of the human female reproductive tract (Fallopian tube, uterus, cervix, and vagina) may play different roles in the provision of mucosal immunity. The purpose of this study was to develop a uniform method suitable for quantitative comparison of the leukocytes from all these tissues.

Host-Adaptation of Francisella tularensis Alters the Bacterium's Surface-Carbohydrates to Hinder Effectors of Innate and Adaptive Immunity

Background The gram-negative bacterium Francisella tularensis survives in arthropods, fresh water amoeba, and mammals with both intracellular and extracellular phases and could reasonably be expected to express distinct phenotypes in these environments. The presence of a capsule on this bacterium has been controversial with some groups finding such a structure while other groups report that no capsule could be identified. Previously we reported in vitro culture conditions for this bacterium which, in contrast to typical methods, yielded a bacterial phenotype that mimics that of the bacteriums mammalian, extracellular phase. Methods/Findings SDS-PAGE and carbohydrate analysis of differentially-cultivated F. tularensis LVS revealed that bacteria displaying the host-adapted phenotype produce both longer polymers of LPS O-antigen (OAg) and additional HMW carbohydrates/glycoproteins that are reduced/absent in non-host-adapted bacteria. Analysis of wildtype and OAg-mutant bacteria indicated that the induced changes in surface carbohydrates involved both OAg and non-OAg species. To assess the impact of these HMW carbohydrates on the access of outer membrane constituents to antibody we used differentially-cultivated bacteria in vitro to immunoprecipitate antibodies directed against outer membrane moieties. We observed that the surface-carbohydrates induced during host–adaptation shield many outer membrane antigens from binding by antibody. Similar assays with normal mouse serum indicate that the induced HMW carbohydrates also impede complement deposition. Using an in vitro macrophage infection assay, we find that the bacterial HMW carbohydrate impedes TLR2-dependent, pro-inflammatory cytokine production by macrophages. Lastly we show that upon host-adaptation, the human-virulent strain, F. tularensis SchuS4 also induces capsule production with the effect of reducing macrophage-activation and accelerating tularemia pathogenesis in mice. Conclusion F. tularensis undergoes host-adaptation which includes production of multiple capsular materials. These capsules impede recognition of bacterial outer membrane constituents by antibody, complement, and Toll-Like Receptor 2. These changes in the host-pathogen interface have profound implications for pathogenesis and vaccine development.

Mucosal Immunization with an Unadjuvanted Vaccine That Targets Streptococcus pneumoniae PspA to Human Fcγ Receptor Type I Protects against Pneumococcal Infection through Complement- and Lactoferrin-Mediated Bactericidal Activity

ABSTRACT 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 FcγR-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 FcγR type I (hFcγRI). A humanized single-chain antibody component in which the variable domain binds to hFcγRI [anti-hFcγRI (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-hFcγRI-PspA or recombinant PspA (rPspA) alone was used to intranasally immunize wild-type (WT) and hFcγRI transgenic (Tg) mice in the absence of adjuvant. The hFcγRI Tg mice receiving anti-hFcγRI-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 hFcγRI as the anti-hFcγRI-PspA fusion, enhanced protection against lethal S. pneumoniae challenge was observed in the hFcγRI Tg mice compared to mice given nontargeted rPspA alone. Immune sera from the anti-hFcγRI-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. pneumoniae that appears to be lactoferrin mediated.

Differential Requirements for Protection against Mucosal Challenge with Francisella tularensis in the Presence versus Absence of Cholera Toxin B and Inactivated F. tularensis

Francisella tularensis is a category A biothreat agent for which there is no approved vaccine and the correlates of protection are not well understood. In particular, the relationship between the humoral and cellular immune response to F. tularensis and the relative importance of each in protection is controversial. Yet, understanding this relationship will be crucial to the development of an effective vaccine against this organism. We demonstrate, for the first time, a differential requirement for humoral vs cellular immunity in vaccine-induced protection against F. tularensis infection, and that the requirement for Ab observed in some protection studies, may be overcome through the induction of enhanced cellular immunity. Specifically, following intranasal/mucosal immunization of mice with inactivated F. tularensis organisms plus the cholera toxin B subunit, we observe increased production of IgG2a/2c vs IgG1 Ab, as well as IFN-γ, indicating induction of a Th1 response. In addition, the requirement for F. tularensis-specific IgA Ab production, observed in studies following immunization with inactivated F. tularensis alone, is eliminated. Thus, these data indicate that enhanced Th1 responses can supersede the requirement for anti-F. tularensis-specific IgA. This observation also has important ramifications for vaccine development against this organism.

Development of tolerogenic dendritic cells and regulatory T cells favors exponential bacterial growth and survival during early respiratory tularemia

Tularemia is a vector‐borne zoonosis caused by Ft, a Gram‐negative, facultative intracellular bacterium. Ft exists in two clinically relevant forms, the European biovar B (holarctica), which produces acute, although mild, self‐limiting infections, and the more virulent United States biovar A (tularensis), which is often associated with pneumonic tularemia and more severe disease. In a mouse model of tularemia, respiratory infection with the virulence‐attenuated Type B (LVS) or highly virulent Type A (SchuS4) strain engenders peribronchiolar and perivascular inflammation. Paradoxically, despite an intense neutrophilic infiltrate and high bacterial burden, Th1‐type proinflammatory cytokines (e.g., TNF, IL‐1β, IL‐6, and IL‐12) are absent within the first ∼72 h of pulmonary infection. It has been suggested that the bacterium has the capacity to actively suppress or block NF‐κB signaling, thus causing an initial delay in up‐regulation of inflammatory mediators. However, our previously published findings and those presented herein contradict this paradigm and instead, strongly support an alternative hypothesis. Rather than blocking NF‐κB, Ft actually triggers TLR2‐dependent NF‐κB signaling, resulting in the development and activation of tDCs and the release of anti‐inflammatory cytokines (e.g., IL‐10 and TGF‐β). In turn, these cytokines stimulate development and proliferation of Tregs that may restrain Th1‐type proinflammatory cytokine release early during tularemic infection. The highly regulated and overall anti‐inflammatory milieu established in the lung is permissive for unfettered growth and survival of Ft. The capacity of Ft to evoke such a response represents an important immune‐evasive strategy.

Fc receptor-targeted mucosal vaccination as a novel strategy for the generation of enhanced immunity against mucosal and non-mucosal pathogens

Numerous studies have demonstrated that targeting immunogens to Fcγ receptors (FcγR) on antigen (Ag)-presenting cells (APC) can enhance humoral and cellular immunity in vitro and in vivo. FcγR are classified based on their molecular weight, IgG-Fc binding affinities, IgG subclass binding specificity, and cellular distribution and they consist of activating and inhibitory receptors. However, despite the potential advantages of targeting Ag to FcR at mucosal sites, very little is known regarding the role of FcR in mucosal immunity or the efficacy of FcR-targeted mucosal vaccines. In addition, recent work has suggested that FcRn is present in the lungs of adult mice and humans and can transport FcRn-targeted Ag to FcγR-bearing APC within mucosal lymphoid tissue. In this review we will discuss the need for new vaccine strategies, the potential for FcR-targeted vaccines to fill this need, the impact of activating versus inhibitory FcγR on FcR-targeted vaccination, the significance of focusing on mucosal immunity, as well as caveats that could impact the use of FcR targeting as a mucosal vaccine strategy.

A two-component modular approach for enhancing T-cell activation utilizing a unique anti-FcγRI-streptavidin construct and microspheres coated with biotinylated-antigen

The professional antigen presenting cell (APC) plays an essential role in the initiation and propagation of the acquired immune response. Thus, much work has been done in designing strategies that target vaccine antigen (Ag) to APC. Utilizing recombinant DNA technology, we have created a unique two-component system that delivers biotinylated Ag to the Fc gamma receptor type I (FcgammaRI) on APC. Our studies demonstrate that we can successfully engineer FcgammaRI-specific targeting element proteins that simultaneously bind both biotin and recognize FcgammaRI. Additionally, we are able to engineer biotinylated Ag, which form functional elements when adsorbed onto latex microspheres. Furthermore, the targeting and functional element components bind to each other and successfully form two-component immunogens. T-cell activation in response to targeted Ag-laden microspheres is 10- to 100-fold greater than the response to the non-targeted Ag-laden microspheres. This enhancement is 100- to 1000-fold greater than the responses generated to soluble Ag. Thus, our results suggest that specific targeting of Ag-laden microspheres to FcgammaRI may significantly enhance the adjuvant properties of microparticulate delivery systems. Further development of this system may help to elucidate the mechanisms involved in generating enhanced responses to APC-targeted vaccines and significantly advance vaccine technology.

Enhanced Antigen-Specific Antibody and Cytokine Responses When Targeting Antigen to Human FcGAMMA Receptor Type I Using an Anti-Human FcGAMMA Receptor Type I-Streptavidin Fusion Protein in an Adjuvant-Free System

There is a continuing need for alternatives to current human adjuvants. Recombinant protein vaccines, which target antigen to human Fc gamma receptor type I (hFcγRI) on hFcγRI-expressing antigen presenting cells, provide one potential alternative. Using a recombinant anti-hFcγRI-antigen fusion protein and adjuvant independent mouse model, we demonstrate enhanced antigen-specific antibody responses to low doses of antigen, when targeting antigen to hFcγRI in vivo. Enhanced antibody production to hFcγRI-targeted antigen is evident in both primary and secondary immune responses, as compared to that of non-targeted antibody. Furthermore, antigen isotype and cytokine responses following immunization with hFcγRI-targeted antigen, suggest enhancement of both Th1 and Th2 responses.

Lipoteichoic Acid Inhibits Interleukin-2 (IL-2) Function by Direct Binding to IL-2

ABSTRACT Lipoteichoic acid (LTA) is associated with the cell envelope of most gram-positive bacteria. Although previously thought to act mainly as a virulence factor by virtue of its adhesive nature, evidence is now provided that LTA can also suppress the function of interleukin-2 (IL-2), an autocrine growth factor for T cells. LTA from four separate bacterial strains lowered the levels of detectable IL-2 during a peripheral blood mononuclear cell response to the antigen tetanus toxoid (TT). T-cell proliferation in response to TT was similarly inhibited by LTA. In contrast, levels of detectable gamma interferon increased. In addition, LTA inhibited IL-2 detection by enzyme-linked immunosorbent assay (ELISA) and blocked the proliferative response of an IL-2-dependent T-cell line to soluble IL-2. Further studies using ELISA demonstrated that LTA blocks IL-2 detection and function by binding directly to IL-2. Flow cytometric analysis revealed that IL-2 binding to T cells is inhibited in the presence of purified LTA but not LTA plus anti-LTA monoclonal antibody. In summary, these studies demonstrate a novel effect of LTA on the immune response through direct binding to IL-2 and inhibition of IL-2 function. Importantly, gram-positive organisms from which LTA is obtained not only play an important role in the pathology of diseases such as bacterial endocarditis, septic shock, acute respiratory distress syndrome, and multiple organ failure but also comprise a significant portion of commensal populations within the human host. Inhibition of IL-2 function by LTA may represent yet another mechanism by which gram-positive bacteria dampen the host immune response and facilitate survival. Thus, LTA provides a potential target for therapeutic intervention when gram-positive organisms are involved.

Regulation of CD23 isoforms on B-chronic lymphocytic leukemia

CD23 is constitutively and atypically expressed on malignant B-cells in patients with chronic lymphocytic leukemia. It exists in two isoforms that differ only in a short amino acid sequence at the N-terminus. The CD23a isoform exhibits an endocytosis signal, that renders it more efficient in antigen uptake than CD23b. Therefore, we analyzed the regulation of CD23 isoforms and tested the ability to stimulate T-cell clones by targeting antigen to CD23 on CLL B-cells. Investigation of several stimulators to promote CD23a expression on CLL versus normal B-cells confirmed a different CD23 regulation in B-CLL. We did not find any evidence for a differential regulation of the two CD23 isoforms in B-CLL. However, CD23a is always predominantly expressed with a constant ratio of CD23a:CD23b. We show that antigen targeted to CD23 on CLL B-cells is very efficiently presented. Therefore, CD23 is likely to provide a suitable target for receptor-mediated antigen presentation in B-CLL which can be used to activate a T-cell response.