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VSA-2 is a recently developed semisynthetic saponin immunostimulant. It is prepared by incorporating a terminal-functionalized side chain to the branched trisaccharide domain at the C3 position of Momordicasaponin II (MS II) isolated from the seeds of perennial Momordica cochinchinensisSpreng. Direct comparison of VSA-2 and the clinically proven saponin adjuvant QS-21 shows that VSA-2 is comparable to QS-21 in enhancing humoral and cellular immune responses. Structure–activity relationship studies show that structural changes in the side chain have a significant impact on saponins’ adjuvant activity. However, with the VSA-2 molecular framework intact, the new VSA-2 analogues with various substitution(s) at the terminal benzyl group of the side chain retain the ability of potentiating antigen-specific humoral and cellular responses.

We have synthesized a number of Quillaja saponariaMolina (QS) saponin analogues with a different C28 sugar unit, which features either 3,4-diacetyl groups or a 3,4-cyclic carbonate group at the reducing end fucoside to mimic the naturally occurring saponin adjuvant QS-7. Immunological evaluations of these analogues in BALB/c mice indicate that truncating the C28 oligosaccharide of the natural product to the tetrasaccharide (as in 5d(β)) could retain the adjuvant’s activity in enhancing IgG1 and IgG2a productions, albeit the activity is lower than that of QS-21. Further truncation or changing stereochemistry of glycosidic linkage between the tetrasaccharide and the triterpenoid quillaic acid (QA) core or within the tetrasaccharide eliminated the saponins’ adjuvant activity in terms of IgG production. On the other hand, increasing resemblance to QS-7 increased adjuvant activity and led to saponin 3’s similar IgG1 and IgG2a activities to QS-21’s, indicating that the unique adjuvant activities of QS saponins are determined by their specific structures.

Bone destruction in inflammatory osteolytic diseases including periodontitis is related to excessive activity of osteoclasts (OC), which originate from precursor cells of the myeloid lineage, termed osteoclast precursors (OCP). In contrast to ample knowledge that we currently have on mature OC, little is known about OCP and their regulation during bacterial infection. Therefore, this study aimed to identify and characterize OCP following chronic infection with a periodontal bacteria Porphyromonas gingivalis(Pg). We used a microosmotic pump to continually release Pgsubcutaneously in a murine model. Two weeks after Pginfection, the frequency of CD11b+c‐fms+Ly6Chipopulation is significantly elevated within the bone marrow, spleen, and peripheral blood. In vitro and in vivo studies identified these cells as the OCP‐containing population and Pginfection significantly enhanced the osteoclastogenic activity of these cells. Furthermore, mRNA sequencing analysis indicated a unique gene and pathway profile in CD11b+c‐fms+Ly6Chipopulation following Pginfection, with changes in genes and pathways related to OC differentiation, cell proliferation and apoptosis, inflammatory response, phagocytosis, and immunity, as well as antigen processing and presentation. Moreover, using IL‐6 knockout mice, we found that IL‐6 is important for Pg‐induced accumulation of CD11b+c‐fms+Ly6Chipopulation from the bone marrow and periphery. Our results provide new insight into the characterization and regulation of OCP following a chronic bacterial infection. This knowledge is relevant to the understanding of the pathogenesis of bacteria‐induced bone loss, and to the identification of potential therapeutic targets of bone loss diseases. Chronic Porphyromonas gingivalisinfection promotes CD11b+c‐fms+Ly6ChiOCP accumulation in BM and periphery through elevated serum IL‐6 and enhances their osteoclastogenic potential through changed gene signatures

We have prepared a number of saponin-based vaccine adjuvant candidates. These unnatural saponins have a different terminal-functionalized side chain incorporated into the glucuronic acid unit that is attached to a triterpenoid core at its C3 position. The semisynthetic saponin adjuvants have shown significantly different immunostimulatory activities, suggesting that the structure of the side chain, triterpenoid core, and oligosaccharide domain together orchestrate saponin adjuvant’s potentiation of immune responses. Among these new adjuvant candidates, VSA-2 (5b), a derivative of Momordicasaponin (MS) II, showed consistent enhancement of immunoglobulin G2a (IgG2a) production when it was in formulation with either ovalbumin or recombinant hemagglutinin B (rHagB) antigen. With rHagB antigen, it induced a significantly higher IgG2a response than the positive control GPI-0100, a well-studied semisynthetic saponin adjuvant mixture derived from Quillaja saponariaMolina saponins, known for its ability to induce a balanced Th1/Th2 immunity. These results confirm that Momordicasaponins are a viable natural source to provide potent saponin adjuvants after simple chemical derivatization and identify VSA-2 (5b) as another MS-based promising immunostimulant lead owing to its distinctive ability in potentiating the IgG2a response.

We have derivatized Momordicasaponins (MS) I and II through their coupling at C3 glucuronic acid site with dodecylamine. The derivatives show significantly different immunostimulant activity profiles from their respective natural parent saponins. In particular, adjuvant VSA-1 (5), the derivative of MS I, potentiates a significantly higher IgG2a responose than the corresponding natural product. Its IgG1 and IgG2a production is similar to that of GPI-0100, indicating a potential mixed and antigen-specific Th1/Th2 immune response, which is different from the Th2 immunity induced by the natural saponin MS I. In addition, toxicity evaluations show that adjuvant VSA-1 (5) is much less toxic than the widely used natural saponin mixture Quil A. These results prove that derivatizing Momordicasaponins can be a viable way for easy access to structurally defined saponin immunostimulants with favorable adjvuant activity and low toxicity.

We have synthesized a QS-17/18 analogue (7) and evaluated its adjuvant activity in the formulation with rHagB antigen. Compound 7and QS-21 analogues 5and 6are presumably the major components of GPI-0100, a widely used complex mixture of semisynthetic derivatives of Quillaja saponaria(QS) Molina saponins. The QS-17/18 analogue 7shows an adjuvant activity profile similar to that of GPI-0100, potentiating mixed Th-1/Th-2 immune responses, which is different from those of QS-21 analogues 5and 6that probably only induce a Th2-like immunity. The combination of QS-17/18 and QS-21 analogues does not show a synergistic effect. These results suggest that QS-17/18 analogue 7might be the active component of GPI-0100 responsible for its immunostimulant property. Therefore, compound 7can not only be a structurally defined alternative to GPI-0100 but also provide a valuable clue for rational design of new QS-based vaccine adjuvants with better adjuvant properties.

ABSTRACTCopper (Cu) ions are likely the most important immunological metal-related toxin utilized in controlling bacterial infections. Impairment of bacterial Cu resistance reduces viability within the host. Thus, pharmacological enhancement of Cu-mediated antibacterial toxicity may lead to novel strategies in drug discovery and development. Screening for Cu toxicity-enhancing antibacterial molecules identified 8-hydroxyquinoline (8HQ) to be a potent Cu-dependent bactericidal inhibitor of Mycobacterium tuberculosis. The MIC of 8HQ in the presence of Cu was 0.16 μM for replicating and nonreplicating M. tuberculosiscells. We found 8HQ's activity to be dependent on the presence of extracellular Cu and to be related to an increase in cell-associated labile Cu ions. Both findings are consistent with 8HQ acting as a Cu ionophore. Accordingly, we identified the 1:1 complex of 8HQ and Cu to be its active form, with Zn, Fe, or Mn neither enhancing nor reducing its Cu-specific action. This is remarkable, considering that the respective metal complexes have nearly identical structures and geometries. Finally, we found 8HQ to kill M. tuberculosisselectively within infected primary macrophages. Given the stark Cu-dependent nature of 8HQ activity, this is the first piece of evidence that Cu ions within macrophages may bestow antibacterial properties to a Cu-dependent inhibitor of M. tuberculosis. In conclusion, our findings highlight the metal-binding ability of the 8-hydroxyquinoline scaffold to be a potential focus for future medicinal chemistry and highlight the potential of innate immunity-inspired screening platforms to reveal molecules with novel modes of action against M. tuberculosis.

We designed and synthesized analogues of a previously identified biofilm inhibitor IIIC5to improve solubility, retain inhibitory activities, and to facilitate encapsulation into pH-responsive hydrogel microparticles. The optimized lead compound HA5showed improved solubility of 120.09 μg/mL, inhibited Streptococcus mutansbiofilm with an IC50value of 6.42 μM, and did not affect the growth of oral commensal species up to a 15-fold higher concentration. The cocrystal structure of HA5with GtfB catalytic domain determined at 2.35 Å resolution revealed its active site interactions. The ability of HA5to inhibit S. mutansGtfs and to reduce glucan production has been demonstrated. The hydrogel-encapsulated biofilm inhibitor (HEBI), generated by encapsulating HA5in hydrogel, selectively inhibited S. mutansbiofilms like HA5. Treatment of S. mutans-infected rats with HA5or HEBIresulted in a significant reduction in buccal, sulcal, and proximal dental caries compared to untreated, infected rats.

ABSTRACTFrancisella tularensisis a Gram-negative immune-evasive coccobacillus that causes tularemia in humans and animals. A safe and efficacious vaccine that is protective against multiple F. tularensisstrains has yet to be developed. In this study, we tested a novel vaccine approach using artificial pathogens, synthetic nanoparticles made from catanionic surfactant vesicles that are functionalized by the incorporation of either F. tularensistype B live vaccine strain (F. tularensisLVS [LVS-V]) or F. tularensistype A Schu S4 strain (F. tularensisSchu S4 [Schu S4-V]) components. The immunization of C57BL/6 mice with “bare” vesicles, which did not express F. tularensiscomponents, partially protected against F. tularensisLVS, presumably through activation of the innate immune response, and yet it failed to protect against the F. tularensisSchu S4 strain. In contrast, immunization with LVS-V fully protected mice against intraperitoneal (i.p.) F. tularensisLVS challenge, while immunization of mice with either LVS-V or Schu S4-V partially protected C57BL/6 mice against an intranasal (i.n.) F. tularensisSchu S4 challenge and significantly increased the mean time to death for nonsurvivors, particularly following the i.n. and heterologous (i.e., i.p./i.n.) routes of immunization. LVS-V immunization, but not immunization with empty vesicles, elicited high levels of IgG against nonlipopolysaccharide (non-LPS) epitopes that were increased after F. tularensisLVS challenge and significantly increased early cytokine production. Antisera from LVS-V-immunized mice conferred passive protection against challenge with F. tularensisLVS. Together, these data indicate that functionalized catanionic surfactant vesicles represent an important and novel tool for the development of a safe and effective F. tularensissubunit vaccine and may be applicable for use with other pathogens.

IL‐10 produced by T cells and CD11b+cells utilizes TLR2 signaling and FimA antigen to inhibit early IFN‐γ T cell responses to Porphyromonas gingivalis. P.g., a Gram‐negative bacterium, is one of the main etiological agents of the chronic inflammatory disease, periodontitis. Disease progression is thought to occur as a result of an inadequate immune response, which although happens locally, can also occur distally as a result of the dissemination of P.g. into the circulation. As IL‐10 and TLR2 are pivotal molecules in the immune response that P.g. elicits, we hypothesized that TLR2‐mediated IL‐10 production, following the initial systemic exposure to P.g., inhibits the IFN‐γ T cell response. To address this hypothesis, mice were primed with P.g., and the types of cells producing IL‐10 and the capacity of T cells to produce IFN‐γ following blocking or neutralization of IL‐10 were assessed. Our results showed that upon initial encounter with P.g., splenic T cells and CD11b+cells produce IL‐10, which when neutralized, resulted in a substantial increase in IFN‐γ production by T cells. Furthermore, IL‐10 production was dependent on TLR2/1 signaling, partly in response to the major surface protein, FimA of P.g. In addition, P.g. stimulation resulted in the up‐regulation of PD‐1 and its ligand PD‐L1 on CD4 T cells and CD11b+cells, respectively. Up‐regulation of PD‐1 was partially dependent on IL‐10 but independent of TLR2 or FimA. These results highlight the role of IL‐10 in inhibiting T cell responses to the initial systemic P.g. exposure and suggest multiple inhibitory mechanisms potentially used by P.g. to evade the hostˈs immune response, thus allowing its persistence in the host.

ABSTRACTA Salmonellavector vaccine expressing the saliva-binding region (SBR) of the adhesin AgI/II of Streptococcus mutanshas been shown to induce a mixed Th1/Th2 anti-SBR immune response in mice and to require Toll-like receptor 2 (TLR2), TLR4, and MyD88 signaling for the induction of mucosal anti-SBR antibody responses. Since dendritic cells (DC) are critical in innate and adaptive immunity, the present study assessed the role of SBR expression by the vector vaccine in DC activation. Bone marrow-derived DC from wild-type and TLR2, TLR4, and MyD88 knockout mice were stimulated with Salmonellavector BRD509, the SBR-expressing Salmonellavector vaccine BRD509(pSBRT7), or SBR protein, and the DC responses to different stimuli were compared by assessing costimulatory molecule expression, cytokine production, and signaling pathways. The DC response to both BRD509(pSBRT7) and BRD509 was dependent mainly on TLR4. BRD509(pSBRT7) and BRD509 induced upregulation of CD80, CD86, CD40, and major histocompatibility complex class II (MHC II) expression. Lower levels of interleukin-10 (IL-10) and IL-12p40 were produced by BRD509(pSBRT7)-stimulated DC than by BRD509-stimulated DC. Furthermore, BRD509(pSBRT7)-stimulated DC showed decreased p38 phosphorylation compared to that induced by DC stimulated with BRD509. However, BRD509(pSBRT7)-treated DC produced a higher level of IL-6 than BRD509-stimulated cells. The low IL-12p40 and high IL-6 cytokine profile expressed by BRD509(pSBRT7)-stimulated DC may represent a shift toward a Th2 response, as suggested by the increased expression in Jagged-1. These results provide novel evidence that a heterologous protein expressed by a Salmonellavector vaccine can differentially affect DC activation.

A Salmonella vector vaccine expressing the saliva-binding region (SBR) of the adhesin AgI/II of Streptococcus mutans has been shown to induce a mixed Th1/Th2 anti-SBR immune response in mice and to require Toll-like receptor 2 (TLR2), TLR4, and MyD88 signaling for the induction of mucosal anti-SBR antibody responses. Since dendritic cells (DC) are critical in innate and adaptive immunity, the present study assessed the role of SBR expression by the vector vaccine in DC activation. Bone marrow-derived DC from wild-type and TLR2, TLR4, and MyD88 knockout mice were stimulated with Salmonella vector BRD509, the SBR-expressing Salmonella vector vaccine BRD509(pSBRT7), or SBR protein, and the DC responses to different stimuli were compared by assessing costimulatory molecule expression, cytokine production, and signaling pathways. The DC response to both BRD509(pSBRT7) and BRD509 was dependent mainly on TLR4. BRD509(pSBRT7) and BRD509 induced upregulation of CD80, CD86, CD40, and major histocompatibility complex class II (MHC II) expression. Lower levels of interleukin-10 (IL-10) and IL-12p40 were produced by BRD509(pSBRT7)-stimulated DC than by BRD509-stimulated DC. Furthermore, BRD509(pSBRT7)-stimulated DC showed decreased p38 phosphorylation compared to that induced by DC stimulated with BRD509. However, BRD509(pSBRT7)-treated DC produced a higher level of IL-6 than BRD509-stimulated cells. The low IL-12p40 and high IL-6 cytokine profile expressed by BRD509(pSBRT7)-stimulated DC may represent a shift toward a Th2 response, as suggested by the increased expression in Jagged-1. These results provide novel evidence that a heterologous protein expressed by a Salmonella vector vaccine can differentially affect DC activation.

Francisella tularensisis the causative agent of tularemia, a severe, debilitating disease of humans and other mammals. As this microorganism is also classified as a “category‐A pathogen” and a potential biowarfare agent, there is a need for an effective vaccine. Several antigens of F. tularensis, including the heat shock protein DnaK, have been proposed for use in a potential subunit vaccine. In this study, we characterized the innate immune response of murine bone marrow‐derived dendritic cells (DC) to F. tularensisDnaK. Recombinant DnaK was produced using a bacterial expression system and purified using affinity, ion‐exchange, and size‐exclusion chromatography. DnaK induced the activation of MAPKs and NF‐κB in DC and the production of the proinflammatory cytokines IL‐6, TNF‐α, and IL‐12 p40, as well as low levels of IL‐10. DnaK induced phenotypic maturation of DC, as demonstrated by an up‐regulation of costimulatory molecules CD40, CD80, and CD86. DnaK stimulated DC through TLR4 and the adapters MyD88 and Toll/IL‐1R domain‐containing adaptor‐inducing IFN‐β (TRIF) that mediated differential responses. DnaK induced activation of MAPKs and NF‐κB in a MyD88‐ or TRIF‐dependent manner. However, the presence of MyD88‐ and TRIF‐dependent signaling pathways was essential for an optimal, DnaK‐induced cytokine response in DC. In contrast, DnaK induced DC maturation in a TRIF‐dependent, MyD88‐independent manner. These results provide insight about the molecular interactions between an immunodominant antigen of F. tularensisand host immune cells, which is crucial for the rational design and development of a safe and efficacious vaccine against tularemia.

To define the role of lipoteichoic acid (LTA) in innate immunity to gram-positive bacteria, we investigated the production of tumor necrosis factor alpha (TNF-) by macrophages stimulated with gram-positive bacterial culture supernatants (GPCSs) after their LTA was removed or inactivated. GPCSs were obtained from three gram-positive species (pneumococci, staphylococci, and group B streptococci) during the exponential growth phase (designated early GPCSs) or at the senescent stage (designated late GPCSs). LTA was removed using an anti-LTA antibody or was inactivated by alkaline hydrolysis or platelet-activating factor acetylhydrolase (PAF-AH) treatment. Both early and late GPCSs from the three gram-positive bacteria stimulated macrophages to produce TNF- primarily via Toll-like receptor 2 (TLR2), although late pneumococcal supernatant could stimulate macrophages via TLR4 as well. Following LTA inactivation by both methods, early GPCS lost about 85 to 100% of its activity and late GPCS lost about 50 to 90%. Both early and late culture supernatants from Escherichia coli could be inactivated by alkali hydrolysis but not by PAF-AH. In addition, removal of LTA from an early staphylococcal culture supernatant with a monoclonal antibody reduced about 70 to 85% of its potency. Reconstitution of inactivated early GPCS with a highly purified LTA restored its inflammatory activity, but the restored GPCS had higher activity than the pure LTA alone. These findings indicate that LTA is the primary TLR2 ligand in the early phase of gram-positive bacterial infection and remains a major ligand in the late phase when another TLR2 and TLR4 ligand(s) appears. In addition, our findings suggest that another gram-positive bacterial factor(s) synergizes with LTA in inducing inflammatory responses.

ABSTRACTTo define the role of lipoteichoic acid (LTA) in innate immunity to gram-positive bacteria, we investigated the production of tumor necrosis factor alpha (TNF-α) by macrophages stimulated with gram-positive bacterial culture supernatants (GPCSs) after their LTA was removed or inactivated. GPCSs were obtained from three gram-positive species (pneumococci, staphylococci, and group B streptococci) during the exponential growth phase (designated early GPCSs) or at the senescent stage (designated late GPCSs). LTA was removed using an anti-LTA antibody or was inactivated by alkaline hydrolysis or platelet-activating factor acetylhydrolase (PAF-AH) treatment. Both early and late GPCSs from the three gram-positive bacteria stimulated macrophages to produce TNF-α primarily via Toll-like receptor 2 (TLR2), although late pneumococcal supernatant could stimulate macrophages via TLR4 as well. Following LTA inactivation by both methods, early GPCS lost about 85 to 100% of its activity and late GPCS lost about 50 to 90%. Both early and late culture supernatants from Escherichia colicould be inactivated by alkali hydrolysis but not by PAF-AH. In addition, removal of LTA from an early staphylococcal culture supernatant with a monoclonal antibody reduced about 70 to 85% of its potency. Reconstitution of inactivated early GPCS with a highly purified LTA restored its inflammatory activity, but the restored GPCS had higher activity than the pure LTA alone. These findings indicate that LTA is the primary TLR2 ligand in the early phase of gram-positive bacterial infection and remains a major ligand in the late phase when another TLR2 and TLR4 ligand(s) appears. In addition, our findings suggest that another gram-positive bacterial factor(s) synergizes with LTA in inducing inflammatory responses.

Francisella tularensis, an aerobic, non-spore-forming, gram-negative coccobacillus, is the causative agent of tularemia. We reported previously that F. tularensis live vaccine strain (LVS) elicited strong, dose-dependent NF-B reporter activity in Toll-like receptor 2 (TLR2)-expressing HEK293T cells and proinflammatory gene expression in primary murine macrophages. Herein, we report that F. tularensis LVS-induced murine macrophage proinflammatory cytokine gene and protein expression are overwhelmingly TLR2 dependent, as evidenced by the abrogated responses of TLR2–/–macrophages. F. tularensis LVS infection also increased expression of TLR2 both in vitro, in mouse macrophages, and in vivo, in livers from F. tularensis LVS-infected mice. Colocalization of intracellular F. tularensis LVS, TLR2, and MyD88 was visualized by confocal microscopy. Signaling was abrogated if the F. tularensis LVS organisms were heat or formalin killed or treated with chloramphenicol, indicating that the TLR2 agonist activity is dependent on new bacterial protein synthesis. F. tularensis LVS replicates in macrophages; however, bacterial replication was not required for TLR2 signaling because LVSguaA, an F. tularensis LVS guanine auxotroph that fails to replicate in the absence of exogenous guanine, activated NF-B in TLR2-transfected HEK293T cells and induced cytokine expression in wild-type macrophages comparably to wild-type F. tularensis LVS. Collectively, these data indicate that the primary macrophage response to F. tularensis LVS is overwhelmingly TLR2 dependent, requires de novo bacterial protein synthesis, and is independent of intracellular F. tularensis replication.

ABSTRACTFrancisella tularensis, an aerobic, non-spore-forming, gram-negative coccobacillus, is the causative agent of tularemia. We reported previously that F. tularensislive vaccine strain (LVS) elicited strong, dose-dependent NF-κB reporter activity in Toll-like receptor 2 (TLR2)-expressing HEK293T cells and proinflammatory gene expression in primary murine macrophages. Herein, we report that F. tularensisLVS-induced murine macrophage proinflammatory cytokine gene and protein expression are overwhelmingly TLR2 dependent, as evidenced by the abrogated responses of TLR2−/−macrophages. F. tularensisLVS infection also increased expression of TLR2 both in vitro, in mouse macrophages, and in vivo, in livers from F. tularensisLVS-infected mice. Colocalization of intracellular F. tularensisLVS, TLR2, and MyD88 was visualized by confocal microscopy. Signaling was abrogated if the F. tularensisLVS organisms were heat or formalin killed or treated with chloramphenicol, indicating that the TLR2 agonist activity is dependent on new bacterial protein synthesis. F. tularensisLVS replicates in macrophages; however, bacterial replication was not required for TLR2 signaling because LVSΔguaA, an F. tularensis LVSguanine auxotroph that fails to replicate in the absence of exogenous guanine, activated NF-κB in TLR2-transfected HEK293T cells and induced cytokine expression in wild-type macrophages comparably to wild-type F. tularensisLVS. Collectively, these data indicate that the primary macrophage response to F. tularensisLVS is overwhelmingly TLR2 dependent, requires de novo bacterial protein synthesis, and is independent of intracellular F. tularensisreplication.

Francisella tularensis, a gram-negative bacterium, is the etiologic agent of tularemia and has recently been classified as a category A bioterrorism agent. Infections with F. tularensis result in an inflammatory response that plays an important role in the pathogenesis of the disease; however, the cellular mechanisms mediating this response have not been completely elucidated. In the present study, we determined the role of Toll-like receptors (TLRs) in mediating inflammatory responses to F. tularensis LVS, and the role of NF-κB in regulating these responses. Stimulation of bone marrow-derived dendritic cells from C57BL/6 wild-type (wt) and TLR4–/– but not TLR2–/– mice, with live F. tularensis LVS elicited a dose-dependent increase in the production of tumor necrosis factor alpha. F. tularensis LVS also induced in a dose-dependent manner an up-regulation in the expression of the costimulatory molecules CD80 and CD86 and of CD40 and the major histocompatibility complex class II molecules on dendritic cells from wt and TLR4–/– but not TLR2–/– mice. TLR6, not TLR1, was shown to be involved in mediating the inflammatory response to F. tularensis LVS, indicating that the functional heterodimer is TLR2/TLR6. Stimulation of dendritic cells with F. tularensis resulted in the activation of NF-κB, which resulted in a differential effect on the production of pro- and anti-inflammatory cytokines. Taken together, our results demonstrate the role of TLR2/TLR6 in the host's inflammatory response to F. tularensis LVS in vitro and the regulatory function of NF-κB in modulating the inflammatory response.

ABSTRACTFrancisella tularensis, a gram-negative bacterium, is the etiologic agent of tularemia and has recently been classified as a category A bioterrorism agent. Infections with F. tularensisresult in an inflammatory response that plays an important role in the pathogenesis of the disease; however, the cellular mechanisms mediating this response have not been completely elucidated. In the present study, we determined the role of Toll-like receptors (TLRs) in mediating inflammatory responses to F. tularensisLVS, and the role of NF-κB in regulating these responses. Stimulation of bone marrow-derived dendritic cells from C57BL/6 wild-type (wt) and TLR4−/−but not TLR2−/−mice, with live F. tularensisLVS elicited a dose-dependent increase in the production of tumor necrosis factor alpha. F. tularensisLVS also induced in a dose-dependent manner an up-regulation in the expression of the costimulatory molecules CD80 and CD86 and of CD40 and the major histocompatibility complex class II molecules on dendritic cells from wt and TLR4−/−but not TLR2−/−mice. TLR6, not TLR1, was shown to be involved in mediating the inflammatory response to F. tularensisLVS, indicating that the functional heterodimer is TLR2/TLR6. Stimulation of dendritic cells with F. tularensisresulted in the activation of NF-κB, which resulted in a differential effect on the production of pro- and anti-inflammatory cytokines. Taken together, our results demonstrate the role of TLR2/TLR6 in the host's inflammatory response to F. tularensisLVS in vitro and the regulatory function of NF-κB in modulating the inflammatory response.

An expanding area of interest is the utilization of microbe-based components to augment mucosal and systemic immune responses to target antigens. Thus, the aim of the present study was to assess if the flagellin component FljB from Salmonella enterica serovar Typhimurium could act as a mucosal adjuvant and then to determine the cellular mechanism(s) by which FljB mediates its adjuvant properties. To determine if FljB could act as a mucosal adjuvant, mice were immunized by the intranasal (i.n.) route with antigen alone or in conjunction with FljB. Additionally, we assessed how FljB affected the levels of the costimulatory molecules B7-1 and B7-2 on dendritic cells by flow cytometry and determined the functional role these costimulatory molecules played in the adjuvant properties of FljB in vivo. Mice immunized by the i.n. route with antigen and FljB exhibited significantly elevated levels of mucosal and systemic antibody and CD4+-T-cell responses compared to mice given antigen only. Stimulation of dendritic cells in vitro with FljB resulted in a pronounced increase in the surface expression of B7-1 and B7-2. The percentage of dendritic cells expressing B7-2 but not B7-1 increased significantly when stimulated with FljB over a concentration range of 10 to 10,000 ng/ml. Immunization of wild-type and B7-1, B7-2, and B7-1/2 knockout mice by the i.n. route revealed that the ability of FljB to increase B7-2 expression is largely responsible for its adjuvant effect in vivo. These findings demonstrate that FljB can act as an effective mucosal adjuvant and that its ability to enhance the level of B7-2 expression is predominantly responsible for its adjuvant properties.

ABSTRACTAn expanding area of interest is the utilization of microbe-based components to augment mucosal and systemic immune responses to target antigens. Thus, the aim of the present study was to assess if the flagellin component FljB from Salmonella entericaserovar Typhimurium could act as a mucosal adjuvant and then to determine the cellular mechanism(s) by which FljB mediates its adjuvant properties. To determine if FljB could act as a mucosal adjuvant, mice were immunized by the intranasal (i.n.) route with antigen alone or in conjunction with FljB. Additionally, we assessed how FljB affected the levels of the costimulatory molecules B7-1 and B7-2 on dendritic cells by flow cytometry and determined the functional role these costimulatory molecules played in the adjuvant properties of FljB in vivo. Mice immunized by the i.n. route with antigen and FljB exhibited significantly elevated levels of mucosal and systemic antibody and CD4+-T-cell responses compared to mice given antigen only. Stimulation of dendritic cells in vitro with FljB resulted in a pronounced increase in the surface expression of B7-1 and B7-2. The percentage of dendritic cells expressing B7-2 but not B7-1 increased significantly when stimulated with FljB over a concentration range of 10 to 10,000 ng/ml. Immunization of wild-type and B7-1, B7-2, and B7-1/2 knockout mice by the i.n. route revealed that the ability of FljB to increase B7-2 expression is largely responsible for its adjuvant effect in vivo. These findings demonstrate that FljB can act as an effective mucosal adjuvant and that its ability to enhance the level of B7-2 expression is predominantly responsible for its adjuvant properties.

Hemagglutinin B (HagB) is a nonfimbrial adhesin expressed on the surface of Porphyromonas gingivalis and has been implicated as a potential virulence factor involved in mediating the attachment of the bacteria to host cells. However, the molecular mechanisms underlying host responses to HagB and their roles in pathogenesis have yet to be elucidated. Mitogen-activated protein kinases (MAPKs) are activated following engagement of a variety of cell surface receptors via dual tyrosine and threonine phosphorylation and are thought to be involved in various cellular responses. The purpose of this study was to determine the role of intracellular signaling pathways including the MAPKs and NF-κB in regulating the production of proinflammatory and anti-inflammatory cytokines following stimulation of murine macrophages with recombinant HagB (rHagB). Stimulation of peritoneal macrophages with rHagB resulted in the production of the proinflammatory cytokines interleukin-12p40 (IL-12p40), gamma interferon (IFN-γ), and tumor necrosis factor alpha, as well as the anti-inflammatory cytokine IL-10. We also demonstrated the activation of extracellular signal-related kinase (ERK), c-Jun NH2-terminal protein kinase (JNK), and p38 MAPKs by rHagB-stimulated macrophages. Furthermore, blocking of the ERK and p38 signaling pathways by using specific inhibitors revealed differential regulatory roles in the rHagB-mediated production of proinflammatory and anti-inflammatory cytokines. ERK and p38 were important in down-regulation of IL-12p40 and IFN-γ production and up-regulation of IL-10 production. The enhanced levels of IL-12p40 in rHagB-stimulated macrophages by inhibition of ERK or p38 activity were partially attributable to the inhibition of IL-10 production. Moreover, NF-κB was found to be critical for up-regulation of IL-12p40 and down-regulation of IL-10 production in rHagB-stimulated macrophages. Taken together, our results demonstrate a role for the p38 and ERK pathways and the transcription factor NF-κB in modulating key immunoregulatory cytokines involved in the development of immune responses to P. gingivalis HagB.

ABSTRACTHemagglutinin B (HagB) is a nonfimbrial adhesin expressed on the surface of Porphyromonas gingivalisand has been implicated as a potential virulence factor involved in mediating the attachment of the bacteria to host cells. However, the molecular mechanisms underlying host responses to HagB and their roles in pathogenesis have yet to be elucidated. Mitogen-activated protein kinases (MAPKs) are activated following engagement of a variety of cell surface receptors via dual tyrosine and threonine phosphorylation and are thought to be involved in various cellular responses. The purpose of this study was to determine the role of intracellular signaling pathways including the MAPKs and NF-κB in regulating the production of proinflammatory and anti-inflammatory cytokines following stimulation of murine macrophages with recombinant HagB (rHagB). Stimulation of peritoneal macrophages with rHagB resulted in the production of the proinflammatory cytokines interleukin-12p40 (IL-12p40), gamma interferon (IFN-γ), and tumor necrosis factor alpha, as well as the anti-inflammatory cytokine IL-10. We also demonstrated the activation of extracellular signal-related kinase (ERK), c-Jun NH2-terminal protein kinase (JNK), and p38 MAPKs by rHagB-stimulated macrophages. Furthermore, blocking of the ERK and p38 signaling pathways by using specific inhibitors revealed differential regulatory roles in the rHagB-mediated production of proinflammatory and anti-inflammatory cytokines. ERK and p38 were important in down-regulation of IL-12p40 and IFN-γ production and up-regulation of IL-10 production. The enhanced levels of IL-12p40 in rHagB-stimulated macrophages by inhibition of ERK or p38 activity were partially attributable to the inhibition of IL-10 production. Moreover, NF-κB was found to be critical for up-regulation of IL-12p40 and down-regulation of IL-10 production in rHagB-stimulated macrophages. Taken together, our results demonstrate a role for the p38 and ERK pathways and the transcription factor NF-κB in modulating key immunoregulatory cytokines involved in the development of immune responses to P. gingivalisHagB.

ABSTRACTThe purpose of the present study was to evaluate the ability of an attenuated Salmonella entericaserovar Typhimurium vaccine strain to up-regulate B7-1 and B7-2 on antigen-presenting cells and to examine the functional roles these costimulatory molecules play in mediating immune responses to Salmonellaand to an expressed cloned antigen, the saliva-binding region (SBR) of antigen I/II. In vitro stimulation of B cells (B220+), macrophages (CD11b+), and dendritic cells (CD11c+) with S. entericaserovar Typhimurium induced an up-regulation of B7-2 and, especially, B7-1 expression. The in vivo functional roles of B7-1, B7-2, and B7-1/2 were evaluated in BALB/c wild-type and B7-1, B7-2, and B7-1/2 knockout (KO) mice following intranasal immunization with the Salmonellaexpressing the cloned SBR. Differential requirements for B7-1 and B7-2 were observed upon primary and secondary immunizations. Compared to wild-type controls, B7-1 and B7-2 KO mice had reduced mucosal and systemic anti-Salmonellaantibody responses after a single immunization, while only B7-1 KO mice exhibited suppressed anti-Salmonellaantibody responses following the second immunization. Mucosal and systemic antibody responses to SBR were reduced following the primary immunization, whereas a compensatory role for either B7-1 or B7-2 was observed after the second immunization. B7-1/2 double KO mice failed to induce detectable levels of mucosal or systemic immunoglobulin A (IgA) or IgG antibody responses to either Salmonellaor SBR. These findings demonstrate that B7-1 and B7-2 can play distinct as well as redundant roles for mediating mucosal and systemic antibody responses, which are likely dependent upon the nature of the antigen.

In addition to antigen-specific signals mediated through the T-cell receptor, T cells also require antigen nonspecific costimulation for activation. The B7 family of molecules on antigen-presenting cells, which include B7-1 (CD80) and B7-2 (CD86), play important roles in providing costimulatory signals required for development of antigen-specific immune responses. Hemagglutinin B (HagB) is a nonfimbrial adhesin of the periodontopathic microorganism Porphyromonas gingivalis and is thought to be involved in the attachment of the bacterium to host tissues. However, the immune mechanisms involved in responses to HagB and their roles in pathogenesis have yet to be elucidated. Therefore, the purpose of this study was to determine the role of B7 costimulatory molecules on T-helper-cell differentiation for the induction of immune responses to HagB. Mice deficient in either or both of the costimulatory molecules B7-1 and B7-2 were used to explore their role in immune responses to HagB after subcutaneous immunization. B7-1(-/-) mice had levels of immunoglobulin G (IgG) anti-HagB antibody activity in serum similar to those of wild-type mice, whereas lower serum IgG anti-HagB antibody responses were seen in B7-2(-/-) mice. Moreover, significantly lower numbers of IgG antibody-secreting cells and lower levels of CD4(+)-T-cell proliferation were observed in B7-2(-/-) mice compared to wild-type mice. No serum IgG response to HagB was detected in B7-1/B7-2(-/-) mice. Analysis of the subclass of the serum IgG responses and the cytokines induced in response to HagB revealed that B7-2(-/-) mice had significantly lower IgG1 and higher IgG2a anti-HagB antibody responses compared to wild-type mice. The B7-2(-/-) mice also had significantly reduced levels of interleukin-4 (IL-4) and IL-5 and enhanced level of gamma interferon. Furthermore, assessment of B7-1 and B7-2 expression on B cells and macrophages derived from wild-type BALB/c mice after in vitro stimulation with HagB revealed a predominant upregulation in the expression of the B7-2 costimulatory molecule on B cells and macrophages. Essentially no change was seen in the expression of B7-1. Taken together, these results suggest a critical role for B7, especially B7-2, for the preferential induction of a Th2-like response to HagB.

ABSTRACTIn addition to antigen-specific signals mediated through the T-cell receptor, T cells also require antigen nonspecific costimulation for activation. The B7 family of molecules on antigen-presenting cells, which include B7-1 (CD80) and B7-2 (CD86), play important roles in providing costimulatory signals required for development of antigen-specific immune responses. Hemagglutinin B (HagB) is a nonfimbrial adhesin of the periodontopathic microorganism Porphyromonas gingivalisand is thought to be involved in the attachment of the bacterium to host tissues. However, the immune mechanisms involved in responses to HagB and their roles in pathogenesis have yet to be elucidated. Therefore, the purpose of this study was to determine the role of B7 costimulatory molecules on T-helper-cell differentiation for the induction of immune responses to HagB. Mice deficient in either or both of the costimulatory molecules B7-1 and B7-2 were used to explore their role in immune responses to HagB after subcutaneous immunization. B7-1−/−mice had levels of immunoglobulin G (IgG) anti-HagB antibody activity in serum similar to those of wild-type mice, whereas lower serum IgG anti-HagB antibody responses were seen in B7-2−/−mice. Moreover, significantly lower numbers of IgG antibody-secreting cells and lower levels of CD4+-T-cell proliferation were observed in B7-2−/−mice compared to wild-type mice. No serum IgG response to HagB was detected in B7-1/B7-2−/−mice. Analysis of the subclass of the serum IgG responses and the cytokines induced in response to HagB revealed that B7-2−/−mice had significantly lower IgG1 and higher IgG2a anti-HagB antibody responses compared to wild-type mice. The B7-2−/−mice also had significantly reduced levels of interleukin-4 (IL-4) and IL-5 and enhanced level of gamma interferon. Furthermore, assessment of B7-1 and B7-2 expression on B cells and macrophages derived from wild-type BALB/c mice after in vitro stimulation with HagB revealed a predominant upregulation in the expression of the B7-2 costimulatory molecule on B cells and macrophages. Essentially no change was seen in the expression of B7-1. Taken together, these results suggest a critical role for B7, especially B7-2, for the preferential induction of a Th2-like response to HagB.

Streptococcus pneumoniae is a leading cause of gram-positive sepsis, and lipoteichoic acid (LTA) may be important in causing gram-positive bacterial septic shock. Even though pneumococcal LTA is structurally distinct from the LTA of other gram-positive bacteria, the immunological properties of pneumococcal LTA have not been well characterized. We have investigated the ability of LTAs to stimulate human monocytes by using highly pure and structurally intact preparations of pneumococcal LTA and its two structural variants. The variants were pneumococcal LTA with only one acyl chain (LTA-1) and completely deacylated LTA (LTA-0). The target cells used in the study were peripheral blood mononuclear cells (PBMCs) and two model cell lines (CHO/CD14/TLR2 and CHO/CD14/TLR4) that express human CD25 protein in response to Toll-like receptor 2 (TLR2) and TLR4 stimulation, respectively. Intact pneumococcal LTA and LTA-1 stimulated PBMC and CHO/CD14/TLR2 cells in a dose-dependent manner but did not stimulate CHO/CD14/TLR4 cells. Pneumococcal LTA was about 100-fold less potent than Staphylococcus aureus LTA in stimulating the CHO/CD14/TLR2 cells and PBMCs. LTA-0 (or pneumococcal teichoic acid) stimulated neither CHO/CD14/TLR2 nor CHO/CD14/TLR4 cells even at high concentrations. Excess teichoic acid, LTA-0, antibodies to phosphocholine, or antibodies to TLR4 did not inhibit the LTA-induced TLR2 stimulation. However, antibodies to CD14, TLR1, or TLR2 suppressed tumor necrosis factor alpha (TNF-alpha) production by PBMCs in response to LTA or LTA-1. These results suggest that pneumococcal LTA with one or both acyl chains stimulates PBMCs primarily via TLR2 with the help of CD14 and TLR1.

ABSTRACTStreptococcus pneumoniaeis a leading cause of gram-positive sepsis, and lipoteichoic acid (LTA) may be important in causing gram-positive bacterial septic shock. Even though pneumococcal LTA is structurally distinct from the LTA of other gram-positive bacteria, the immunological properties of pneumococcal LTA have not been well characterized. We have investigated the ability of LTAs to stimulate human monocytes by using highly pure and structurally intact preparations of pneumococcal LTA and its two structural variants. The variants were pneumococcal LTA with only one acyl chain (LTA-1) and completely deacylated LTA (LTA-0). The target cells used in the study were peripheral blood mononuclear cells (PBMCs) and two model cell lines (CHO/CD14/TLR2 and CHO/CD14/TLR4) that express human CD25 protein in response to Toll-like receptor 2 (TLR2) and TLR4 stimulation, respectively. Intact pneumococcal LTA and LTA-1 stimulated PBMC and CHO/CD14/TLR2 cells in a dose-dependent manner but did not stimulate CHO/CD14/TLR4 cells. Pneumococcal LTA was about 100-fold less potent than Staphylococcus aureusLTA in stimulating the CHO/CD14/TLR2 cells and PBMCs. LTA-0 (or pneumococcal teichoic acid) stimulated neither CHO/CD14/TLR2 nor CHO/CD14/TLR4 cells even at high concentrations. Excess teichoic acid, LTA-0, antibodies to phosphocholine, or antibodies to TLR4 did not inhibit the LTA-induced TLR2 stimulation. However, antibodies to CD14, TLR1, or TLR2 suppressed tumor necrosis factor alpha (TNF-α) production by PBMCs in response to LTA or LTA-1. These results suggest that pneumococcal LTA with one or both acyl chains stimulates PBMCs primarily via TLR2 with the help of CD14 and TLR1.

Monophosphoryl lipid A (MPL) is a nontoxic derivative of lipopolysaccharide (LPS) that exhibits adjuvant properties similar to those of the parent LPS molecule. However, the mechanism by which MPL initiates its immunostimulatory properties remains unclear. Due to the involvement of Toll-like receptors in recognizing and transducing intracellular signals in response to LPS, the aim of the present study was to determine the ability of MPL to utilize the Toll-like receptor 2 (TLR2) and TLR4. We provide evidence that MPL differentially utilizes TLR2 and TLR4 for the induction of tumor necrosis factor alpha, interleukin 10 (IL-10), and IL-12 by purified human monocytes as well as by human peripheral blood mononuclear cells. Assessment of NF-kappa B activity demonstrated that MPL utilized TLR2 and especially TLR4 for the activation of NF-kappa B p65 by human monocytes. In addition, stimulation of human monocytes by MPL led to an up-regulation of the costimulatory molecules CD80 and CD86, an effect that could be reduced by pretreatment of cells with a monoclonal antibody to TLR2 or TLR4. Analysis of MPL-induced activation of the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinases revealed that MPL utilized both TLR2 and TLR4 for the phosphorylation of ERK1/2, while TLR4 was the predominant receptor involved in the ability of MPL to phosphorylate p38. Moreover, using selective inhibitors for MAP kinase kinase (PD98059) and p38 (SB203580), we show that ERK1/2 exhibited differential effects on production of TNF-alpha and IL-12 p40 by human monocytes, whereas MPL-induced activation of p38 appeared to be predominantly involved in production of IL-10 and IL-12 p40 by MPL-stimulated monocytes. Taken together, these findings aid in understanding the cellular mechanisms by which MPL induces host cell activation and subsequent adjuvant properties.

ABSTRACTMonophosphoryl lipid A (MPL) is a nontoxic derivative of lipopolysaccharide (LPS) that exhibits adjuvant properties similar to those of the parent LPS molecule. However, the mechanism by which MPL initiates its immunostimulatory properties remains unclear. Due to the involvement of Toll-like receptors in recognizing and transducing intracellular signals in response to LPS, the aim of the present study was to determine the ability of MPL to utilize the Toll-like receptor 2 (TLR2) and TLR4. We provide evidence that MPL differentially utilizes TLR2 and TLR4 for the induction of tumor necrosis factor alpha, interleukin 10 (IL-10), and IL-12 by purified human monocytes as well as by human peripheral blood mononuclear cells. Assessment of NF-κB activity demonstrated that MPL utilized TLR2 and especially TLR4 for the activation of NF-κB p65 by human monocytes. In addition, stimulation of human monocytes by MPL led to an up-regulation of the costimulatory molecules CD80 and CD86, an effect that could be reduced by pretreatment of cells with a monoclonal antibody to TLR2 or TLR4. Analysis of MPL-induced activation of the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinases revealed that MPL utilized both TLR2 and TLR4 for the phosphorylation of ERK1/2, while TLR4 was the predominant receptor involved in the ability of MPL to phosphorylate p38. Moreover, using selective inhibitors for MAP kinase kinase (PD98059) and p38 (SB203580), we show that ERK1/2 exhibited differential effects on production of TNF-α and IL-12 p40 by human monocytes, whereas MPL-induced activation of p38 appeared to be predominantly involved in production of IL-10 and IL-12 p40 by MPL-stimulated monocytes. Taken together, these findings aid in understanding the cellular mechanisms by which MPL induces host cell activation and subsequent adjuvant properties.

The saliva-binding region (SBR) of the cell surface antigen I/II (AgI/II) and the glucan-binding region (GLU) of the glucosyltransferase enzyme of Streptococcus mutans have been implicated in the initial adherence of S. mutans to saliva-coated tooth surfaces and the subsequent sucrose-dependent accumulation of S. mutans, respectively. Here, we describe the construction and characterization of a genetic chimeric protein consisting of the two virulence determinants SBR and GLU (SBR-GLU). The effectiveness of this construct in inducing mucosal and systemic immune responses to each virulence determinant following intranasal immunization was compared to that of each antigen alone or an equal mixture of SBR and GLU (SBR+GLU) in a mouse model. Furthermore, the ability of antibodies induced to SBR-GLU to protect against S. mutans infection was also investigated. Immunization of mice with the chimeric protein SBR-GLU resulted in significantly enhanced (P < 0.001) levels of serum immunoglobulin G (IgG) anti-SBR antibody activity compared to those in the SBR and SBR+GLU groups. The SBR-GLU-immunized mice also demonstrated a significant (P < 0.05) increase in salivary and vaginal IgA antibody responses to SBR and GLU. Analysis of the serum IgG subclass responses to SBR in mice immunized with SBR alone indicated a mixed IgG1 and IgG2a response. A preferential IgG1 response compared to an IgG2a anti-GLU response was induced in mice immunized with GLU alone. Similarly, a preferential IgG1 response was also induced to SBR when GLU was present in either a mixed or conjugated form. Finally, a significant reduction (P < 0.05) in S. mutans colonization was observed only in mice immunized with the SBR-GLU chimeric protein. Taken together, our results indicate that the chimeric protein SBR-GLU significantly enhanced mucosal immune responses to SBR and GLU and systemic immune responses to SBR. The ability of SBR-GLU to induce responses effective in protection against colonization of S. mutans suggests its potential as a vaccine antigen for dental caries.

ABSTRACTThe saliva-binding region (SBR) of the cell surface antigen I/II (AgI/II) and the glucan-binding region (GLU) of the glucosyltransferase enzyme of Streptococcus mutanshave been implicated in the initial adherence of S. mutansto saliva-coated tooth surfaces and the subsequent sucrose-dependent accumulation of S. mutans, respectively. Here, we describe the construction and characterization of a genetic chimeric protein consisting of the two virulence determinants SBR and GLU (SBR-GLU). The effectiveness of this construct in inducing mucosal and systemic immune responses to each virulence determinant following intranasal immunization was compared to that of each antigen alone or an equal mixture of SBR and GLU (SBR+GLU) in a mouse model. Furthermore, the ability of antibodies induced to SBR-GLU to protect against S. mutansinfection was also investigated. Immunization of mice with the chimeric protein SBR-GLU resulted in significantly enhanced (P< 0.001) levels of serum immunoglobulin G (IgG) anti-SBR antibody activity compared to those in the SBR and SBR+GLU groups. The SBR-GLU-immunized mice also demonstrated a significant (P< 0.05) increase in salivary and vaginal IgA antibody responses to SBR and GLU. Analysis of the serum IgG subclass responses to SBR in mice immunized with SBR alone indicated a mixed IgG1 and IgG2a response. A preferential IgG1 response compared to an IgG2a anti-GLU response was induced in mice immunized with GLU alone. Similarly, a preferential IgG1 response was also induced to SBR when GLU was present in either a mixed or conjugated form. Finally, a significant reduction (P< 0.05) in S. mutanscolonization was observed only in mice immunized with the SBR-GLU chimeric protein. Taken together, our results indicate that the chimeric protein SBR-GLU significantly enhanced mucosal immune responses to SBR and GLU and systemic immune responses to SBR. The ability of SBR-GLU to induce responses effective in protection against colonization of S. mutanssuggests its potential as a vaccine antigen for dental caries.

Porphyromonas gingivalis, a gram-negative, black-pigmented anaerobe, is among the microorganisms implicated in the etiology of adult periodontal disease. This bacterium possesses a number of factors, including hemagglutinins, of potential importance in virulence. Our laboratory has shown the induction of protection to P. gingivalis infection after subcutaneous immunization with recombinant hemagglutinin B (rHagB). The purpose of this study was to determine if humoral antibody responses are induced after intranasal (i.n.) immunization of rHagB and if monophosphoryl lipid A (MPL), a nontoxic derivative of the lipid A region of lipopolysaccharide, acts as a mucosal adjuvant and potentiates responses to rHagB. Further, the effects of MPL on the nature of the response to HagB and on the costimulatory molecules B7-1 and B7-2 on different antigen-presenting cells (APC) were evaluated. Groups of BALB/c mice were immunized three times (2-week intervals) by the i.n. route with HagB (20 microg) alone or with MPL (25 microg). A group of nonimmunized mice served as control. Serum and saliva samples were collected prior to immunization and at approximately 2-week intervals and evaluated for serum immunoglobulin G (IgG) and IgG subclass and for salivary IgA antibody activity by enzyme-linked immunosorbent assay. Mice immunized with rHagB plus MPL had significantly higher salivary IgA (P < 0.05) and serum IgG (P < 0.05) anti-HagB responses than mice immunized with rHagB alone. The IgG1 and IgG2a subclass responses seen in mice immunized with rHagB plus MPL were significantly higher (P < 0.05) than those seen in mice immunized with rHagB only. Further, the IgG2a/IgG1 ratio in the latter group was approximately 1, whereas in mice immunized with rHagB plus MPL the ratio was <1. These results provide evidence for the participation of T helper (Th) 1 and Th2 cells in responses to rHagB and that MPL potentiates a type 2 response to HagB. MPL was also shown to preferentially up-regulate B7-2 expression on B cells, whereas a preferential increase in B7-1 costimulatory molecule was seen on macrophages and dendritic cells. These results provide evidence that MPL exerts a differential regulation in the expression of costimulatory molecules on APC.

ABSTRACTPorphyromonas gingivalis, a gram-negative, black-pigmented anaerobe, is among the microorganisms implicated in the etiology of adult periodontal disease. This bacterium possesses a number of factors, including hemagglutinins, of potential importance in virulence. Our laboratory has shown the induction of protection to P. gingivalisinfection after subcutaneous immunization with recombinant hemagglutinin B (rHagB). The purpose of this study was to determine if humoral antibody responses are induced after intranasal (i.n.) immunization of rHagB and if monophosphoryl lipid A (MPL), a nontoxic derivative of the lipid A region of lipopolysaccharide, acts as a mucosal adjuvant and potentiates responses to rHagB. Further, the effects of MPL on the nature of the response to HagB and on the costimulatory molecules B7-1 and B7-2 on different antigen-presenting cells (APC) were evaluated. Groups of BALB/c mice were immunized three times (2-week intervals) by the i.n. route with HagB (20 μg) alone or with MPL (25 μg). A group of nonimmunized mice served as control. Serum and saliva samples were collected prior to immunization and at approximately 2-week intervals and evaluated for serum immunoglobulin G (IgG) and IgG subclass and for salivary IgA antibody activity by enzyme-linked immunosorbent assay. Mice immunized with rHagB plus MPL had significantly higher salivary IgA (P< 0.05) and serum IgG (P< 0.05) anti-HagB responses than mice immunized with rHagB alone. The IgG1 and IgG2a subclass responses seen in mice immunized with rHagB plus MPL were significantly higher (P< 0.05) than those seen in mice immunized with rHagB only. Further, the IgG2a/IgG1 ratio in the latter group was ≈1, whereas in mice immunized with rHagB plus MPL the ratio was <1. These results provide evidence for the participation of T helper (Th) 1 and Th2 cells in responses to rHagB and that MPL potentiates a type 2 response to HagB. MPL was also shown to preferentially up-regulate B7-2 expression on B cells, whereas a preferential increase in B7-1 costimulatory molecule was seen on macrophages and dendritic cells. These results provide evidence that MPL exerts a differential regulation in the expression of costimulatory molecules on APC.

ABSTRACTPorphyromonas gingivalishas been implicated as an etiologic agent of adult periodontitis. We have previously shown that P. gingivaliscan degrade the epithelial cell-cell junction complexes, thus suggesting that this bacterium can invade the underlying connective tissues via a paracellular pathway. However, the precise mechanism(s) involved in this process has not been elucidated. The purpose of this study was to determine if the arginine- and lysine-specific gingipains of P. gingivalis(i.e., HRgpA and RgpB, and Kgp, respectively) were responsible for the degradation of E-cadherin, the cell-cell adhesion protein in the adherens junctions. In addition, we compared the degradative abilities of the whole gingipains HRgpA and Kgp to those of their catalytic domains alone. In these studies, immunoprecipitated E-cadherin as well as monolayers of polarized Madin-Darby canine kidney (MDCK) epithelial cell cultures were incubated with the gingipains and hydrolysis of E-cadherin was assessed by Western blot analysis. Incubation of P. gingivaliscells with immunoprecipitated E-cadherin resulted in degradation, whereas prior exposure of P. gingivaliscells to leupeptin and especially acetyl-Leu-Val-Lys-aldehyde (which are arginine- and lysine-specific inhibitors, respectively) reduced this activity. Furthermore, incubation of E-cadherin immunoprecipitates with the different gingipains resulted in an effective and similar hydrolysis of the protein. However, when monolayers of MDCK cells were exposed to the gingipains, Kgp was most effective in hydrolyzing the E-cadherin molecules in the adherens junction. Kgp was more effective than its catalytic domain in degrading E-cadherin at 500 nM but not at a lower concentration (250 nM). These results suggest that the hemagglutinin domain of Kgp plays a role in degradation and that there is a critical threshold concentration for this activity. Taken together, these results provide evidence that the gingipains, especially Kgp, are involved in the degradation of the adherens junction of epithelial cells, which may be important in the invasion of periodontal connective tissue by P. gingivalis.

Porphyromonas gingivalis has been implicated as an etiologic agent of adult periodontitis. We have previously shown that P. gingivalis can degrade the epithelial cell-cell junction complexes, thus suggesting that this bacterium can invade the underlying connective tissues via a paracellular pathway. However, the precise mechanism(s) involved in this process has not been elucidated. The purpose of this study was to determine if the arginine- and lysine-specific gingipains of P. gingivalis (i.e., HRgpA and RgpB, and Kgp, respectively) were responsible for the degradation of E-cadherin, the cell-cell adhesion protein in the adherens junctions. In addition, we compared the degradative abilities of the whole gingipains HRgpA and Kgp to those of their catalytic domains alone. In these studies, immunoprecipitated E-cadherin as well as monolayers of polarized Madin-Darby canine kidney (MDCK) epithelial cell cultures were incubated with the gingipains and hydrolysis of E-cadherin was assessed by Western blot analysis. Incubation of P. gingivalis cells with immunoprecipitated E-cadherin resulted in degradation, whereas prior exposure of P. gingivalis cells to leupeptin and especially acetyl-Leu-Val-Lys-aldehyde (which are arginine- and lysine-specific inhibitors, respectively) reduced this activity. Furthermore, incubation of E-cadherin immunoprecipitates with the different gingipains resulted in an effective and similar hydrolysis of the protein. However, when monolayers of MDCK cells were exposed to the gingipains, Kgp was most effective in hydrolyzing the E-cadherin molecules in the adherens junction. Kgp was more effective than its catalytic domain in degrading E-cadherin at 500 nM but not at a lower concentration (250 nM). These results suggest that the hemagglutinin domain of Kgp plays a role in degradation and that there is a critical threshold concentration for this activity. Taken together, these results provide evidence that the gingipains, especially Kgp, are involved in the degradation of the adherens junction of epithelial cells, which may be important in the invasion of periodontal connective tissue by P. gingivalis.

Saliva contains an array of nonimmunoglobulin defense factors which are thought to contribute to the protection of the hard and soft tissue surfaces of the oral cavity by modulating microbial colonization and metabolism. Here we report the discovery of a putative innate defense factor in human saliva that inhibits the glucosyltransferase (GTF) of Streptococcus mutans, a virulence enzyme involved in oral colonization by this pathogen. The GTF-inhibiting factor (GIF) was initially identified as a nonimmunoglobulin salivary component that interfered with detection of antibodies to the glucan-binding region (GLU) of GTF by an enzyme-linked immunosorbent assay. This inhibitory activity was present in whole saliva and submandibular-sublingual saliva, but it was essentially absent from parotid saliva. GIF inhibited the recognition of S. mutans cell surface-associated GTF by specific antibodies but had no effect on antibodies to other cell surface antigens, suggesting that GIF specifically binds to GTF on S. mutans. GIF purified by size exclusion or affinity chromatography was used for biochemical and functional characterization. Analysis of GIF by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a high-molecular-weight glycoprotein after staining with Coomassie blue or Schiff's reagent. Heating and reduction with 2-mercaptoethanol of GIF resulted in the release of a approximately 58-kDa protein that was identified as alpha-amylase by Western blotting using anti-alpha-amylase antibodies. GLU bound blotted alpha-amylase, suggesting that the latter molecule is the GLU-binding component of the GIF complex. The ability of GTF to synthesize extracellular glucans was inhibited by GIF but not by uncomplexed alpha-amylase or an unrelated high-molecular-weight glycoprotein. In conclusion, our findings demonstrate that in human saliva, there is a high-molecular-weight glycoprotein-alpha-amylase complex which is capable of inhibiting GTF and may contribute to control of S. mutans colonization in the oral cavity.

ABSTRACTSaliva contains an array of nonimmunoglobulin defense factors which are thought to contribute to the protection of the hard and soft tissue surfaces of the oral cavity by modulating microbial colonization and metabolism. Here we report the discovery of a putative innate defense factor in human saliva that inhibits the glucosyltransferase (GTF) of Streptococcus mutans, a virulence enzyme involved in oral colonization by this pathogen. The GTF-inhibiting factor (GIF) was initially identified as a nonimmunoglobulin salivary component that interfered with detection of antibodies to the glucan-binding region (GLU) of GTF by an enzyme-linked immunosorbent assay. This inhibitory activity was present in whole saliva and submandibular-sublingual saliva, but it was essentially absent from parotid saliva. GIF inhibited the recognition of S. mutanscell surface-associated GTF by specific antibodies but had no effect on antibodies to other cell surface antigens, suggesting that GIF specifically binds to GTF on S. mutans.GIF purified by size exclusion or affinity chromatography was used for biochemical and functional characterization. Analysis of GIF by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a high-molecular-weight glycoprotein after staining with Coomassie blue or Schiff's reagent. Heating and reduction with 2-mercaptoethanol of GIF resulted in the release of a ∼58-kDa protein that was identified as α-amylase by Western blotting using anti-α-amylase antibodies. GLU bound blotted α-amylase, suggesting that the latter molecule is the GLU-binding component of the GIF complex. The ability of GTF to synthesize extracellular glucans was inhibited by GIF but not by uncomplexed α-amylase or an unrelated high-molecular-weight glycoprotein. In conclusion, our findings demonstrate that in human saliva, there is a high-molecular-weight glycoprotein-α-amylase complex which is capable of inhibiting GTF and may contribute to control of S. mutanscolonization in the oral cavity.

ABSTRACTAttenuated Salmonella entericaserovar Typhimurium has been used for targeted delivery of recombinant antigens to gut- and nose-associated lymphoid tissues. Contradictory reports have described the effect of preexisting immunity to the antigen delivery vehicle. We decided to examine this discrepancy by studying the effect of immunizing mice by the intranasal (i.n.) route withSalmonellaexpressing an insoluble protein and to study the ability to augment recall responses by boosting with eitherSalmonella-expressed protein or purified soluble protein alone. The glucan-binding domain (GLU) of the enzyme glucosyltransferase (GTF), which is an important virulence factor ofStreptococcus mutans, was recombinantly expressed in the insoluble phase in S. entericaserovar Typhimurium, and the immunogenicity of this construct was studied in mice. We examined the induction of primary immune responses by insoluble GLU polypeptide delivered in Salmonellaat week 1 (groups 1 and 2) and recall responses after a week 15 boost with eitherSalmonellaexpressing GLU (group 1) or purified GLU polypeptide (groups 2 and 3). Group 4 served as the control and received phosphate-buffered saline alone by the i.n. route. Significant anti-GLU serum immunoglobulin G (IgG) levels were seen in groups 1, 2, and 3 at week 18 (P< 0.001), i.e., 3 weeks after the booster immunization. Mice in group 2, who receivedSalmonellafollowed by GLU, had the highest GLU-specific IgG levels among all groups. The serum IgG levels persisted in all responding groups for at least 7 weeks after the boost (week 22). The IgG2a/IgG1 subclass ratio of serum anti-GLU antibodies in group 1 significantly increased after the boost. These results support the induction of a type 1-like immune response to GLU after primary and booster immunizations with Salmonellaexpressing GLU. On the other hand, group 2 mice, which received Salmonellaexpressing GLU as the primary dose and soluble protein as the booster dose, exhibited a shift from a type 1-like to a more type 2-like immune response to GLU following the boost. These results indicate thatS. entericaserovar Typhimurium is an excellent delivery vehicle for the insoluble and recombinantly expressed GLU of GTF and that this construct was especially effective in priming the host for a secondary response to soluble GLU polypeptide.

ABSTRACTCholera toxin (CT) and the type II heat-labile enterotoxins (HLT) LT-IIa and LT-IIb act as potent systemic and mucosal adjuvants and induce distinct T-helper (Th)-cell cytokine profiles. In the present study, CT and the type II HLT were found to differentially affect cytokine production by anti-CD3-stimulated human peripheral blood mononuclear cells (PBMC), and the cellular mechanisms responsible were investigated. CT suppressed interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-α), and IL-12 production by PBMC cultures more than either LT-IIa or LT-IIb. CT but not LT-IIa or LT-IIb reduced the expression of CD4+T-cell surface activation markers (CD25 and CD69) and subsequent proliferative responses of anti-CD3-stimulated T cells. CT but not LT-IIa or LT-IIb significantly reduced the expression of CD40 ligand (CD40L) on CD4+T cells. In a coculture system, CT-treated CD4+T cells induced significantly less TNF-α and IL-12 p70 production by both autologous monocytes and monocyte-derived dendritic cells than either LT-IIa- or LT-IIb-treated CD4+T cells. These findings demonstrate that CT, LT-IIa, and LT-IIb differentially affect CD40-CD40L interactions between antigen-presenting cells and T cells and help explain the distinct cytokine profiles observed with type I and type II HLT when used as mucosal adjuvants.

ABSTRACTThe purpose of the present study was to evaluate the effectiveness of an attenuated Salmonella entericaserovar Typhimurium vaccine strain expressing the saliva-binding region (SBR) of theStreptococcus mutansantigen I/II adhesin, either alone or linked with the mucosal adjuvant cholera toxin A2 and B subunits (CTA2/B) and under the control of the anaerobically induciblenirBpromoter, in inducing a protective immune response against S. mutansinfection. BALB/c mice were immunized by either the intranasal or the intragastric route with a single dose of 109or 1010SalmonellaCFU, respectively. The Salmonellavaccine strain expressing an unrelated antigen (fragment C of tetanus toxin [TetC]) was also used for immunization as a control. Samples of serum and secretion (saliva and vaginal washes) were collected prior to and following immunization and assessed for antibody activity by enzyme-linked immunosorbent assay. Anti-SBR antibodies were detected in the serum and saliva of experimental animals by week 3 after immunization. A booster immunization at week 17 after the initial immunization resulted in enhanced immune responses to the SBR. The serum immunoglobulin G subclass profiles were indicative of T helper type 1 responses against both the vector and the SBR antigen. To determine the effectiveness of these responses on the protection against S. mutansinfection, mice were challenged after the second immunization with a virulent strain of S. mutanswhich was resistant to tetracycline and erythromycin. Prior to the challenge, mice were treated for 5 days with tetracycline, erythromycin, and penicillin.S. mutanswas initially recovered from all of the challenged mice. This bacterium persisted at high levels for at least 5 weeks in control TetC-immunized or nonimmunized mice despite the reappearance of indigenous oral organisms. However, mice immunized withSalmonellaclones expressing SBR or SBR-CTA2/B demonstrated a significant reduction in the number of S. mutanspresent in plaque compared to the control groups. These results provide evidence for the effectiveness of the Salmonellavector in delivering the SBR antigen for the induction of mucosal and systemic immune responses to SBR. Furthermore, the induction of a salivary anti-SBR response corresponded with protection against S. mutanscolonization of tooth surfaces.