Ozone sauna therapy and pulsed electromagnetic field therapy could potentially improve outcome in women with diminished ovarian reserve undergoing assisted reproductive technology.

There are limited treatment options for women with severely diminished ovarian reserve (DOR) who experience repeatedly failed in vitro fertilization (IVF) cycles and with persistently thin endometrial lining thickness (EMT) during frozen embryo transfer cycles. Therefore, a large majority of patients resort to using donor oocytes and gestational carriers. Data from existing animal and human studies suggest that ozone sauna therapy (OST) and pulsed electromagnetic field therapy (PEMF) are emerging as potential therapeutic adjuncts for female reproduction. This study was conducted to assess the fertility outcome of OST + PEMF in vivo in patients undergoing IVF/frozen embryo transfe and the effects of OST in vitro on human granulosa cell (GC) function. Forty-four women with DOR underwent their 1st IVF cycle (Cycle 1), and then were administered transdermal and intravaginal OST + PEMF, twice a week for 3 weeks, followed by a 2nd IVF cycle (Cycle 2) using the same protocol as in Cycle 1. GCs collected from another six women who underwent egg retrieval were equally split and cultured with OST (test) or placed in room temperature (control) outside the OST chamber in the same room. The results demonstrated that Cycles 1 and 2 had no significant difference in the number of days of stimulation, baseline hormones measured, number of oocytes retrieved or peak estradiol levels. However, the number of embryos formed after OST + PEMF in Cycle 2 was significantly higher than the Cycle 1. Furthermore, EMT measured in Cycle 2 demonstrated a significant increase compared to Cycle 1 and all patients reached a satisfactory EMT of approximately 7 mm. In vitro studies demonstrated that OST led to a 5-fold significant increase in the aromatase enzyme while a significant 50% reduction was noted in the side-chain cleavage enzyme in GCs. Both OST + PEMF are known for their vasodilatory, anti-inflammatory, and antioxidant actions, which could enhance endometrial receptivity and increase the number of formed embryos without increasing the number of oocytes retrieved, suggesting an improvement in oocyte quality. Finally, ozone can alter genes involved in steroidogenesis suggesting that it could improve ovarian function.

Synthetic Antibody Mimics Based on Cancer-Targeting Immunostimulatory Peptides.

De novo cancer-targeting immunostimulatory peptides have been designed and developed as synthetic antibody mimics. A series of bifunctional peptides incorporating NKp30-binding and NK-cell-activating domains were synthesized as linear dimers and then extended into branching trimeric peptides by the incorporation of GRP78-targeting and tumor-cell-binding sequences. A selected trimeric peptide from this small set of peptides displayed binding capabilities on GRP78+ HepG2 and A549 target cells. Cell binding diminished in the presence of an anti-GRP78 peptide blocker, thus suggesting GRP78-binding dependence. Similarly, the selected trimeric peptide was also found to exhibit NK cell binding in an NKp30-dependent manner, which translated into NK cell activation as indicated by cytokine secretion. In co-culture, fluorescence microscopy revealed that the target GFP-expressing A549 cells were visibly associated with the effector NK cells when pre-activated with lead trimeric peptide. Accordingly, A549 cells were found to be compromised, as evidenced by the loss of GFP signal and notable detection of early-/late-stage apoptosis. Investigation of the immunological markers related to toxicity revealed detectable secretion of pro-inflammatory cytokines and chemokines, including IFN-γ, TNF-α, and IL-8. Furthermore, administration of peptide-activated NK cells into A549-tumor-bearing mice resulted in a consistent decrease in tumor growth when compared to the untreated control group. Taken together, the identification of a lead trimeric peptide capable of targeting and activating NK cells' immunotoxicity directly towards GRP78+ /B7H6- tumors provides a novel proof-of-concept for the development of cancer-targeting immunostimulatory peptide ligands that mimic antibody-targeting and -activating functions related to cancer immunotherapy applications.

Isotype-specific outcomes in Fc gamma receptor targeting of PspA using fusion proteins as a vaccination strategy against Streptococcus pneumoniae infection.

Streptococcus pneumoniae (Spn) remains a considerable threat to public health despite the availability of antibiotics and polysaccharide conjugate vaccines. The lack of mucosal immunity in addition to capsular polysaccharide diversity, has proved to be problematic in developing a universal vaccine against Spn. Targeting antigen to Fc receptors is an attractive way to augment both innate and adaptive immunity against mucosal pathogens, by promoting interactions with activating Fcγ receptors (FcγR) that mediate diverse immunomodulatory functions. The effect of targeting FcγR is highly influenced by the IgG subclass, which bares differential affinities for activating and inhibitory FcγR. In the current study we demonstrate targeting activating FcγR with fusion proteins consisting of PspA and IgG2a Fc enhance PspA-specific immune responses, and effectively protect against mucosal Spn challenge. Specifically, targeting PspA to FcγR polarized alveolar macrophage to the AM1 phenotype and increased conventional dendritic cell subsets in the lung in addition to augmenting Th1 cytokines and PspA-specific IgG and IgA. In contrast, fusion proteins consisting of PspA fused to the IgG1 Fc provided minimal benefit over administration of PspA alone, as a result of interaction with the inhibitory FcγRIIB. Protective efficacy of the IgG1 fusion protein was significantly enhanced in animals deficient for FcγRIIB accompanied by increased B cell maturation and proliferation levels in these animals. These studies demonstrate FcγR targeting is an effective strategy for inducing potent cellular and humoral responses via mucosal immunization with Fc fusion proteins, however, careful consideration of the Fc region utilized is required since Fc isotype subclass heavily influenced immunization induced effector functions and survival against lethal Spn challenge. Fc-engineering with specific attention to FcγRIIB engagement presents a valuable vaccine strategy for protecting against Spn infection.

Macrophage subtype and cytokine expression characterization during the acute inflammatory phase of mouse bone fracture repair.

Fracture repair is a complex process requiring heterotypic interactions between osteogenic cells and immune cells. Recent evidence indicates that macrophages are critically involved in fracture repair. Polarized macrophage populations differentially promote and regulate inflammation in other tissues, but little is known about the various macrophage subtypes and their signaling activities following a bone fracture. The authors hypothesized that classically activated (M1 subtype) and alternatively activated (M2 subtype) macrophages are active during the early repair process to initiate and regulate the inflammatory response. To test our hypothesis, bone marrow was collected from intact femurs (naïve group), contralateral and fractured femurs of mice on days 0, 1, 2, 4, and 7 postfracture. Macrophages were isolated from the bone marrow and macrophage subtypes were identified using flow cytometry with antibodies to F4/80, MHC II, CD86, CD11c, and CD40. Bone marrow cytokine levels were measured using xMAP. Flow cytometry revealed dynamic changes in M1 subtype (F4/80+/MHC II+/CD86+), M2 subtype (F4/80+/MHC II-/CD86-), and dendritic cell (DCs; MHCII+/CD11c+/CD40+) populations following fracture as compared to naïve controls. M1 subtype levels were correlated with IL-1α, IL-1ß, IL-2, IL-17, Eotaxin, and MCP-1, while DCs were correlated with IL-6, G-CSF, LIF, KC, and VEGF-A. The results indicate that M1 and M2 subtypes and DCs are recruited to the fracture site early during the repair process and consequently may work in tandem to regulate the inflammatory response required to recruit osteogenic cells needed for later stages of repair.

Utilization of cholera toxin B as a mucosal adjuvant elicits antibody-mediated protection against S. pneumoniae infection in mice.

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. pneumoniae challenge. 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. pneumoniae challenge. 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.

Synthesis and immunological evaluation of a low molecular weight saccharide with TLR-4 agonist activity.

The paucity of FDA approved adjuvants renders the synthesis, characterization, and use of new compounds as vaccine adjuvants, a necessity. For this purpose, a novel saccharide analog has been synthesized from glucosamine, pyruvylated galactose and 1,4-cyclohexanediol and its biological efficacy was determined in innate immune cells. More specifically, we assessed the production of pro-inflammatory cytokines from the murine monocyte cell line, Raw 264.7 and from C57 BL/6 mouse peritoneal macrophages following exposure to the saccharide analog. Our data conclude that the novel saccharide has immunostimulatory activity on mouse macrophages as indicated by the elevated levels of IL-6 and TNF-α in culture supernatants. This effect was TLR-4-dependent but TLR-2-independent. Our data, suggest TLR-4 agonism; a key feature of vaccine adjuvants.

B7H6-derived peptides trigger TNF-α-dependent immunostimulatory activity of lymphocytic NK92-MI cells.

The rise of biologics that can stimulate immune responses towards the eradication of tumors has led to the evolution of cancer-based immunotherapy. Representatively, B7H6 has been recently identified as a protein ligand on tumor cells that binds specifically to the NKp30 receptor and triggers NK cell-derived cytokine production, which ultimately leads to tumor cell lysis and death. In an effort to develop effective immunotherapy approaches, the rational design of a novel class of immunostimulatory peptides (IPs) derived from the binding interface of B7H6:NKp30 is described in this study. The IPs comprised the B7H6 active site sequence for NKp30 binding and immunostimulatory activity. An aminohexanoic acid linker was also introduced at the N-terminus of the peptides for FITC-labeling by Fmoc-solid phase peptide synthesis. The peptides were characterized by LCMS to confirm identities and purities >95%. The secondary structures of the peptides were examined by CD spectroscopy in H2 O, PBS and a H2 O:TFE mixture which demonstrated versatile peptide structures which transitioned from random coil (H2 O) to α-helical (PBS) and turn-type (H2 O:TFE) conformations. Their biological properties were then evaluated by flow cytometry, enzyme-linked immunosorbent assays (ELISAs), and cell death assays. The occupancy of the synthetic peptides to a human NK cell line demonstrated comparable binding relative to the natural NKp30 ligand, B7H6, and the human anti-NKp30 monoclonal antibody (mAb), in a concentration dependent manner. A competitive binding assay between the human anti-NKp30 mAb or B7H6, and the synthetic peptides, demonstrated partial displacement of the ligands upon anti-NKp30 mAb treatment, suggesting NKp30 receptor specificities by the synthetic peptides. Moreover, the immunostimulatory activity of B7H6 was demonstrated by the secretion of the pro-inflammatory cytokines tumor necrosis factor-alfa (TNF-α) and interferon gamma (IFN-γ) by the human NK cell line. The immunostimulatory effects of IPs on the NK cells was assessed by the production of TNF-α alone as IFN-γ was undetectable. In a cell death assay, the IPs were found to be nontoxic, without any observable evidence of early or late stage apoptosis within the NK92-MI cells. Taking these findings together, this novel class of synthetic peptides may prove to be a promising lead in the development of a peptide-based immunotherapy approach, especially against B7H6 expressing tumors. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 658-672, 2016.

Cholera toxin B induced activation of murine macrophages exposed to a fixed bacterial immunogen.

OBJECTIVES: Previous studies have demonstrated that intranasal administration of inactivated (fixed) Francisella tularensis (iFt) live vaccine strain (LVS) in conjunction with the mucosal adjuvant, cholera toxin B (CTB), provides full protection against subsequent lethal challenge with Ft LVS and partial protection against the more virulent Ft SchuS4 strain. Understanding the mechanisms of CTB-induced immune stimulation that confer protection against Ft will be valuable to the development of an effective vaccine against this highly virulent fatal pathogen. In this study, an in vitro system was utilized to further elucidate the immunologic adjuvant effect of CTB when administered with the fixed bacterial immunogen iFt. METHODS: The murine macrophage cell line (RAW264.7) was treated with combinations of iFt and CTB. The treated RAW264.7 cells and their supernatants were collected and assessed for cell surface marker expression and cytokine secretion. In addition, the ability of RAW264.7 cells to present bacterial antigens (iFt or LVS) to an Ft-specific T-cell hybridoma cell line, following exposure to CTB, was analyzed. RESULTS: We found that RAW264.7 cells responded to treatment with iFt + CTB by an increased secretion of the proinflammatory cytokines interleukin 6 and tumor necrosis factor α and upregulation of the surface expression of toll-like receptor 4 and the costimulatory molecules CD80 and CD86. Furthermore, the experimental vaccine treatment iFt + CTB enhanced the ability of macrophages to present iFt antigens to an FT-specific T-cell hybridoma cell line, although they failed to do so with LVS. CONCLUSION: The adjuvant CTB administered in conjunction with iFt showed evidence of enhancing an antigen-specific proinflammatory response in vitro. These observations allow us to define, in part, the mechanisms of immune activation conferred by mucosal administration of iFt + CTB against lethal F. tularensis challenge.

Targeting of a Fixed Bacterial Immunogen to Fc Receptors Reverses the Anti-Inflammatory Properties of the Gram-Negative Bacterium, Francisella tularensis, during the Early Stages of Infection.

Production of pro-inflammatory cytokines by innate immune cells at the early stages of bacterial infection is important for host protection against the pathogen. Many intracellular bacteria, including Francisella tularensis, the agent of tularemia, utilize the anti-inflammatory cytokine IL-10, to evade the host immune response. It is well established that IL-10 has the ability to inhibit robust antigen presentation by dendritic cells and macrophages, thus suppressing the generation of protective immunity. The pathogenesis of F. tularensis is not fully understood, and research has failed to develop an effective vaccine to this date. In the current study, we hypothesized that F. tularensis polarizes antigen presenting cells during the early stages of infection towards an anti-inflammatory status characterized by increased synthesis of IL-10 and decreased production of IL-12p70 and TNF-α in an IFN-É£-dependent fashion. In addition, F. tularensis drives an alternative activation of alveolar macrophages within the first 48 hours post-infection, thus allowing the bacterium to avoid protective immunity. Furthermore, we demonstrate that targeting inactivated F. tularensis (iFt) to Fcγ receptors (FcÉ£Rs) via intranasal immunization with mAb-iFt complexes, a proven vaccine strategy in our laboratories, reverses the anti-inflammatory effects of the bacterium on macrophages by down-regulating production of IL-10. More specifically, we observed that targeting of iFt to FcγRs enhances the classical activation of macrophages not only within the respiratory mucosa, but also systemically, at the early stages of infection. These results provide important insight for further understanding the protective immune mechanisms generated when targeting immunogens to Fc receptors.

Downmodulation of vaccine-induced immunity and protection against the intracellular bacterium Francisella tularensis by the inhibitory receptor FcγRIIB.

Fc gamma receptor IIB (FcγRIIB) is the only Fc gamma receptor (FcγR) which negatively regulates the immune response, when engaged by antigen- (Ag-) antibody (Ab) complexes. Thus, the generation of Ag-specific IgG in response to infection or immunization has the potential to downmodulate immune protection against infection. Therefore, we sought to determine the impact of FcγRIIB on immune protection against Francisella tularensis (Ft), a Category A biothreat agent. We utilized inactivated Ft (iFt) as an immunogen. Naïve and iFt-immunized FcγRIIB knockout (KO) or wildtype (WT) mice were challenged with Ft-live vaccine strain (LVS). While no significant difference in survival between naïve FcγRIIB KO versus WT mice was observed, iFt-immunized FcγRIIB KO mice were significantly better protected than iFt-immunized WT mice. Ft-specific IgA in serum and bronchial alveolar lavage, as well as IFN-γ, IL-10, and TNF-α production by splenocytes harvested from iFt-immunized FcγRIIB KO, were also significantly elevated. In addition, iFt-immunized FcγRIIB KO mice exhibited a reduction in proinflammatory cytokine levels in vivo at 5 days after challenge, which correlates with increased survival following Ft-LVS challenge in published studies. Thus, these studies demonstrate for the first time the ability of FcγRIIB to regulate vaccine-induced IgA production and downmodulate immunity and protection. The immune mechanisms behind the above observations and their potential impact on vaccine development are discussed.

In vivo mechanisms involved in enhanced protection utilizing an Fc receptor-targeted mucosal vaccine platform in a bacterial vaccine and challenge model.

Targeting 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. tularensis challenge. 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 vivo immune 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. tularensis LVS challenge. In addition, the frequency and number of gamma interferon (IFN-γ)-secreting effector memory (EM) CD4(+) T cells elicited by F. tularensis infection (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.

Synthesis, characterization, and biological activity of poly(arginine)-derived cancer-targeting peptides in HepG2 liver cancer cells.

The solid-phase synthesis, structural characterization, and biological evaluation of a small library of cancer-targeting peptides have been determined in HepG2 hepatoblastoma cells. These peptides are based on the highly specific Pep42 motif, which has been shown to target the glucose-regulated protein 78 receptors overexpressed and exclusively localized on the cell surface of tumors. In this study, Pep42 was designed to contain varying lengths (3-12) of poly(arginine) sequences to assess their influence on peptide structure and biology. Peptides were effectively synthesized by 9-fluorenylmethoxycarbonyl-based solid-phase peptide synthesis, in which the use of a poly(ethylene glycol) resin provided good yields (14-46%) and crude purities >95% as analyzed by liquid chromatography-mass spectrometry. Peptide structure and biophysical properties were investigated using circular dichroism spectroscopy. Interestingly, peptides displayed secondary structures that were contingent on solvent and length of the poly(arginine) sequences. Peptides exhibited helical and turn conformations, while retaining significant thermal stability. Structure-activity relationship studies conducted by flow cytometry and confocal microscopy revealed that the poly(arginine) derived Pep42 sequences maintained glucose-regulated protein 78 binding on HepG2 cells while exhibiting cell translocation activity that was contingent on the length of the poly(arginine) strand. In single dose (0.15 mM) and dose-response (0-1.5 mM) cell viability assays, peptides were found to be nontoxic in human HepG2 liver cancer cells, illustrating their potential as safe cancer-targeting delivery agents.

Reactivation of human polyomaviruses in immunocompromised states.

Infection with various human polyomaviruses (HPyVs) is prevalent, with rates as high as 80 % within the general population. Primary infection occurs during childhood through respiratory or urino-oral transmission. While the majority of individuals exhibit asymptomatic latent infection, those immunocompromised persons are at risk for viral reactivation and disease progression resulting in conditions such as progressive multifocal leukoencephalopathy (PML), trichodysplasia spinulosa, Merkel cell carcinoma, and polyomavirus associated nephropathy. Individuals with altered immune systems due to HIV, organ transplantation, lymphoproliferative diseases, and monoclonal antibody therapy are particularly susceptible to reactivation of various HPyVs. While the specific factors that induce lytic infection have yet to be defined, it is evident that dysfunctional host cellular immune responses allow active infection to occur. Immunosuppressant conditions, such as in chronic alcohol abuse, may serve as added risk factors for reactivation of HPyVs. Since the human HPyV family is rapidly expanding, continuing studies are needed to characterize the role that known and newly discovered HPyVs play in human disease.

Multiple mechanisms mediate enhanced immunity generated by mAb-inactivated F. tularensis immunogen.

We have previously demonstrated that immunization with the inactivated Francisella tularensis, a Category A intracellular mucosal pathogen, combined with IgG2a anti-F. tularensis monoclonal antibody (Ab), enhances protection against subsequent F. tularensis challenge. To understand the mechanism(s) involved, we examined the binding, internalization, presentation, and in vivo trafficking of inactivated F. tularensis in the presence and absence of opsonizing monoclonal Ab. We found that when inactivated F. tularensis is combined with anti-F. tularensis monoclonal Ab, presentation to F. tularensis-specific T cells is enhanced. This enhancement is Fc receptor (FcR)-dependent, and requires a physical linkage between the monoclonal Ab and the inactivated F. tularensis immunogen. This enhanced presentation is due, in part, to enhanced binding and internalization of inactivated F. tularensis by antigen(Ag)-presenting cells, and involves interactions with multiple FcR types. Furthermore, targeting inactivated F. tularensis to FcRs enhances dendritic cell maturation and extends the time period over which Ag-presenting cells stimulate T cells. In vivo trafficking studies reveal enhanced transport of inactivated F. tularensis immunogen to the nasal-associated lymphoid tissue in the presence of monoclonal Ab, which is FcRn-dependent. In summary, these are the first comprehensive studies using a single-vaccine protection model/immunogen to establish the array of mechanisms involved in enhanced immunity/protection mediated by an FcR-targeted mucosal immunogen. These results demonstrate that multiple cellular/immune mechanisms contribute to FcR-enhanced immunity.

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.

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.

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 bacterium's 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.

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 Fcgamma receptors (FcgammaR) on antigen (Ag)-presenting cells (APC) can enhance humoral and cellular immunity in vitro and in vivo. FcgammaR 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 FcgammaR-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 FcgammaR 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.

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-gamma, 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.

Utilization of Fc receptors as a mucosal vaccine strategy against an intracellular bacterium, Francisella tularensis.

Numerous studies have demonstrated that targeting Ag to Fc receptors (FcR) on APCs can enhance humoral and cellular immunity. However, studies are lacking that examine both the use of FcR-targeting in generating immune protection against infectious agents and the use of FcRs in the induction of mucosal immunity. Francisella tularensis is a category A intracellular mucosal pathogen. Thus, intense efforts are underway to develop a vaccine against this organism. We hypothesized that protection against mucosal infection with F. tularensis would be significantly enhanced by targeting inactivated F. tularensis live vaccine strain (iFt) to FcRs at mucosal sites, via intranasal immunization with mAb-iFt complexes. These studies demonstrate for the first time that: 1) FcR-targeted immunogen enhances immunogen-specific IgA production and protection against subsequent infection in an IgA-dependent manner, 2) FcgammaR and neonatal FcR are crucial to this protection, and 3) inactivated F. tularensis, when targeted to FcRs, enhances protection against the highly virulent SchuS4 strain of F. tularensis, a category A biothreat agent. In summary, these studies show for the first time the use of FcRs as a highly effective vaccination strategy against a highly virulent mucosal intracellular pathogen.

T-Cell-independent humoral immunity is sufficient for protection against fatal intracellular ehrlichia infection.

Although humoral immunity has been shown to contribute to host defense during intracellular bacterial infections, its role has generally been ancillary. Instead, CD4 T cells are often considered to play the dominant role in protective immunity via their production of type I cytokines. Our studies of highly pathogenic Ehrlichia bacteria isolated from Ixodes ovatus (IOE) reveal, however, that this paradigm is not always correct. Immunity to IOE infection can be induced by infection with a closely related weakly pathogenic ehrlichia, Ehrlichia muris. Type I cytokines (i.e., gamma interferon, tumor necrosis factor alpha, and interleukin-12) were not necessary for E. muris-induced immunity. In contrast, humoral immunity was essential, as shown by the fact that E. muris-infected B-cell-deficient mice were not protected from IOE challenge and because E. muris immunization was effective in CD4-, CD8-, and major histocompatibility complex (MHC) class II-deficient mice. Immunity was unlikely due to nonspecific inflammation, as prior infection with Listeria monocytogenes did not induce immunity to IOE. Antisera from both wild-type and MHC-II-deficient mice provided at least partial resistance to challenge infection, and protection could also be achieved following transfer of total, but not B-cell-depleted, splenocytes obtained from E. muris-immunized mice. The titers of class-switched antibodies in immunized CD4 T-cell- and MHC class II-deficient mice, although lower than those observed in immunized wild-type mice, were significant, indicating that E. muris can induce class switch recombination in the absence of classical T-cell-mediated help. These studies highlight a major protective role for classical T-cell-independent humoral immunity during an intracellular bacterial infection.