Fusion of pseudorabies virus glycoproteins to IgG Fc enhances protective immunity against pseudorabies virus.

Molecular adjuvants are vaccine delivery vehicle to increase specific antigens effectiveness. Herein, we concentrated on IgG Fc, an effective molecular adjuvant, to develop novel pseudorabies virus (PRV) subunit vaccines. Two major protective antigen genes of PRV were constructed and linked into the mouse IgG Fc fragment. The gD, gD-IgG2aFc, gB and gB-IgG2aFc proteins were expressed using a baculovirus system. Mice intranasally immunized with gD-IgG2aFc or gB-IgG2aFc subunit vaccine exhibited significantly higher PRV-specific antibodies, neutralizing antibodies and intracellular cytokines than the mice intranasally immunized with gD or gB subunit vaccine. Moreover, no histopathological lesions were observed in mice immunized with gB-IgG2aFc subunit vaccine via histopathology examination. Further, the gB-IgG2aFc subunit vaccine was efficient for PRV infection compared with live attenuated vaccine. Overall, these results suggest that IgG2a Fc fragment, as a potential molecular adjuvant, fused with PRV antigen might be a promising and efficient PRV vaccine candidate.

Relation between hyaluronan synthesis and cell morphology in ovarian clear cell carcinomas.

Ovarian clear cell carcinomas often show a spherule-like mucoid stroma. In ascitic fluid, they form spheroids with a hollow acellular space. In spite of the absence of stromal cells, both the mucoid stroma and hollow spheroids contain abundant extracellular matrix, and one of the major components is hyaluronan. It has been suggested that tumor-derived hyaluronan plays a significant role in the formation of these structures. To clarify this, a hyaluronan inhibition assay was performed on HAC-2, a clear cell carcinoma cell line, in vitro. When hyaluronan synthesis was inhibited by 4-methylumbelliferone, HAC-2 failed to show the spherule-like accumulation of hyaluronan or hollow spheroids. Inhibition of hyaluronan synthesis was associated with the reduction of cell growth. Analysis of 28 archival ascites cytology specimens showed that clear cell carcinomas expressed hyaluronan more frequently than serous carcinomas (11 of 14 vs 3 of 14, respectively, P < 0.05). All of these facts indicate that tumor-derived hyaluronan is essential for the formation of the mucoid stroma or hollow spheroids, and that hyaluronan is also involved in the regulation of cell growth in ovarian clear cell carcinomas. The inhibition of hyaluronan synthesis could be a potential adjunctive therapy for refractory clear cell carcinomas outside the ovary.

Interaction between antimicrobial peptides and mycobacteria.

Mycobacteria can cause different severe health problems, including tuberculosis (TB). The treatment of TB with conventional antibiotics is successful, however, the number of multi-drug and extensively-drug resistant Mycobacterium tuberculosis strains increases. Moreover, many classical antimycobacterial antibiotics have severe side effects. Therefore, antimicrobial peptides (AMPs) seem to be good candidates for new therapeutic strategies. On the one hand AMPs can be used as a single drug or in combination with conventional antibiotics to directly kill mycobacteria, or on the other hand to act as immunstimulatory agents. This review summarizes the findings on the role of endogenous human AMPs being involved in TB, the antimycobacterial activity of various AMPs, and the molecular modes of action. Most active AMPs interact with the mycobacterial cell envelope and in particular with the mycomembrane and the plasma membrane. The mycomembrane is a very rigid membrane probably leading to a lower activity of the AMPs against mycobacteria as compared to other Gram-negative or Gram-positive bacteria. For some AMPs also other targets have been identified. Because of the complex environment of intracellular mycobacteria being trapped in the phagosome, within the macrophage, within the granuloma, within the lung, the external administration of AMPs in the latent phase of TB is a challenge. However, in the acute phase the AMPs can attack mycobacteria in a direct way. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

Plant-derived recombinant immune complexes as self-adjuvanting TB immunogens for mucosal boosting of BCG.

Progress with protein-based tuberculosis (TB) vaccines has been limited by poor availability of adjuvants suitable for human application. Here, we developed and tested a novel approach to molecular engineering of adjuvanticity that circumvents the need for exogenous adjuvants. Thus, we generated and expressed in transgenic tobacco plants the recombinant immune complexes (RICs) incorporating the early secreted Ag85B and the latency-associated Acr antigen of Mycobacterium tuberculosis, genetically fused as a single polypeptide to the heavy chain of a monoclonal antibody to Acr. The RICs were formed by virtue of the antibody binding to Acr from adjacent molecules, thus allowing self-polymerization of the complexes. TB-RICs were purified from the plant extracts and shown to be biologically active by demonstrating that they could bind to C1q component of the complement and also to the surface of antigen-presenting cells. Mice immunized with BCG and then boosted with two intranasal immunizations with TB-RICs developed antigen-specific serum IgG antibody responses with mean end-point titres of 1 : 8100 (Acr) and 1 : 24 300 (Ag85B) and their splenocytes responded to in vitro stimulation by producing interferon gamma. 25% of CD4+ proliferating cells simultaneously produced IFN-γ, IL-2 and TNF-α, a phenotype that has been linked with protective immune responses in TB. Importantly, mucosal boosting of BCG-immunized mice with TB-RICs led to a reduced M. tuberculosis infection in their lungs from log10 mean = 5.69 ± 0.1 to 5.04 ± 0.2, which was statistically significant. We therefore propose that the plant-expressed TB-RICs represent a novel molecular platform for developing self-adjuvanting mucosal vaccines.

HIV-1 adenoviral vector vaccines expressing multi-trimeric BAFF and 4-1BBL enhance T cell mediated anti-viral immunity.

Adenoviral vectored vaccines have shown considerable promise but could be improved by molecular adjuvants. Ligands in the TNF superfamily (TNFSF) are potential adjuvants for adenoviral vector (Ad5) vaccines based on their central role in adaptive immunity. Many TNFSF ligands require aggregation beyond the trimeric state (multi-trimerization) for optimal biological function. Here we describe Ad5 vaccines for HIV-1 Gag antigen (Ad5-Gag) adjuvanted with the TNFSF ligands 4-1BBL, BAFF, GITRL and CD27L constructed as soluble multi-trimeric proteins via fusion to Surfactant Protein D (SP-D) as a multimerization scaffold. Mice were vaccinated with Ad5-Gag combined with Ad5 expressing one of the SP-D-TNFSF constructs or single-chain IL-12p70 as adjuvant. To evaluate vaccine-induced protection, mice were challenged with vaccinia virus expressing Gag (vaccinia-Gag) which is known to target the female genital tract, a major route of sexually acquired HIV-1 infection. In this system, SP-D-4-1BBL or SP-D-BAFF led to significantly reduced vaccinia-Gag replication when compared to Ad5-Gag alone. In contrast, IL-12p70, SP-D-CD27L and SP-D-GITRL were not protective. Histological examination following vaccinia-Gag challenge showed a dramatic lymphocytic infiltration into the uterus and ovaries of SP-D-4-1BBL and SP-D-BAFF-treated animals. By day 5 post challenge, proinflammatory cytokines in the tissue were reduced, consistent with the enhanced control over viral replication. Splenocytes had no specific immune markers that correlated with protection induced by SP-D-4-1BBL and SP-D-BAFF versus other groups. IL-12p70, despite lack of anti-viral efficacy, increased the total numbers of splenic dextramer positive CD8+ T cells, effector memory T cells, and effector Gag-specific CD8+ T cells, suggesting that these markers are poor predictors of anti-viral immunity in this model. In conclusion, soluble multi-trimeric 4-1BBL and BAFF adjuvants led to strong protection from vaccinia-Gag challenge, but the protection was independent of standard immune markers. Soluble multi-trimeric SP-D-4-1BBL and SP-D-BAFF provide a novel technology to enhance adenoviral vector vaccines against HIV-1.

Separation and characterization of the immunostimulatory components in unpolished rice black vinegar (kurozu).

Unpolished rice black vinegar (kurozu), a traditional Japanese vinegar, is considered to have beneficial health effects. Kurozu is produced via a static fermentation process involving the saccharification of rice by Aspergillus oryzae, alcohol fermentation by Saccharomyces cerevisiae, and the oxidation of ethanol to acetic acid by acetic acid bacteria such as Acetobacter pasteurianus. Since this process requires about 6 months' fermentation and then over a year of aging, most of these organisms die during the production process and so microbial components, which might stimulate the innate immune system, are expected to be present in the vinegar. In this study, we investigated whether microbial components are present in kurozu, and after confirming this we characterized their immunostimulatory activities. Lyophilized kurozu stimulated murine spleen cells to produce tumor necrosis factor (TNF)-α, at least in part, via Toll-like receptor (TLR) 2 and the Nod-like receptors NOD1 and 2. The active components associated with TLR2 activation were concentrated by Triton X-114-water phase partitioning and hydrophobic interaction chromatography on Octyl Sepharose. TLR4-activating components were also enriched by these methods. The concentrated preparation stimulated murine spleen cells to produce TNF-α and interferon (IFN)-γ. These results indicate that long-term fermented kurozu contains immunostimulatory components and that the TLR2 and TLR4-activating immunostimulatory components of kurozu are hydrophobic. These components might be responsible for the beneficial health effects of kurozu.

Production of recombinant immunotherapeutics for anticancer treatment: the role of bioengineering.

Cancer is one of the most important health problems because many cases are difficult to prevent. Cancer still has unknown mechanisms of pathogenesis, and its capacity to produce temporary or permanent damage, besides death, is very high. Although many anticancer therapies are available, finding a cure for cancer continues to be a difficult task. Thus, many efforts have been made to develop more effective treatments, such as immunotherapy based on a new class of tumor-specific products that are produced using recombinant DNA technology. These recombinant products are used with the main objectives of killing the tumor and stimulating immune cells to respond to the cancer cells. The principal recombinant products in anticancer therapy are immunostimulants, vaccines, antibodies, immunotoxins and fusion proteins. This review focuses on the general aspects of these genetically engineered products, their clinical performance, current advances and future prospects for this type of anticancer therapy.

Suppression of hyaluronan synthesis alleviates inflammatory responses in murine arthritis and in human rheumatoid synovial fibroblasts.

OBJECTIVE: To clarify the roles of hyaluronan (HA) in joint inflammation and the process of joint destruction, using 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis, in a mouse model of collagen-induced arthritis (CIA) and in a monolayer culture of fibroblast-like synoviocytes (FLS) derived from patients with rheumatoid arthritis. METHODS: DAB/1J mice were immunized with type II collagen. The effects of 4-MU were evaluated by the physiologic arthritis score, paw swelling, the histologic arthritis score, and expression of matrix metalloproteinase 3 (MMP-3) and MMP-13 in chondrocytes and synovial tissue. In vitro, the effect of 4-MU on messenger RNA and protein expression of MMP-1 and MMP-3 was determined. The effects of 4-MU on HA deposition and on serum/medium concentrations of HA were analyzed using biotinylated HA binding protein staining and an HA binding assay, respectively. RESULTS: Treatment with 4-MU in mice with CIA dramatically decreased the severity of arthritis (based on the arthritis score), paw thickness, and histopathologic changes. MMP-3 and MMP-13 expression in chondrocytes and synovial cells was significantly inhibited by 4-MU in vivo. Treatment with 4-MU also inhibited MMP-1 and MMP-3 expression in tumor necrosis factor α-stimulated FLS, in a dose-dependent manner. The 4-MU-induced decreases in the serum HA concentration in mice with CIA and in "medium" and "pericellular" HA concentrations in cultured FLS support the contention that the inhibitory mechanism of 4-MU is mediated by HA suppression. CONCLUSION: Reduced disease activity induced by 4-MU in mice with CIA revealed HA to be a crucial regulator in the course of arthritis. Therefore, 4-MU is a potential therapeutic agent in arthritis, and its inhibitory mechanism is possibly mediated by suppression of HA synthesis.

Salmonella synthesizing 1-dephosphorylated [corrected] lipopolysaccharide exhibits low endotoxic activity while retaining its immunogenicity.

The development of safe live, attenuated Salmonella vaccines may be facilitated by detoxification of its LPS. Recent characterization of the lipid A 1-phosphatase, LpxE, from Francisella tularensis allowed us to construct recombinant, plasmid-free strains of Salmonella that produce predominantly 1-dephosphorylated lipid A, similar to the adjuvant approved for human use. Complete lipid A 1-dephosphorylation was also confirmed under low pH, low Mg(2+) culture conditions, which induce lipid A modifications. LpxE expression in Salmonella reduced its virulence in mice by five orders of magnitude. Moreover, mice inoculated with these detoxified strains were protected against wild-type challenge. Candidate Salmonella vaccine strains synthesizing pneumococcal surface protein A (PspA) were also confirmed to possess nearly complete lipid A 1-dephosphorylation. After inoculation by the LpxE/PspA strains, mice produced robust levels of anti-PspA Abs and showed significantly improved survival against challenge with wild-type Streptococcus pneumoniae WU2 compared with vector-only-immunized mice, validating Salmonella synthesizing 1-dephosphorylated lipid A as an Ag-delivery system.

Constitutive CD40L expression on B cells prematurely terminates germinal center response and leads to augmented plasma cell production in T cell areas.

CD40/CD40L engagement is essential to T cell-dependent B cell proliferation and differentiation. However, the precise role of CD40 signaling through cognate T-B interaction in the generation of germinal center and memory B cells is still incompletely understood. To address this issue, a B cell-specific CD40L transgene (CD40LBTg) was introduced into mice with B cell-restricted MHC class II deficiency. Using this mouse model, we show that constitutive CD40L expression on B cells alone could not induce germinal center differentiation of MHC class II-deficient B cells after immunization with T cell-dependent Ag. Thus, some other MHC class II-dependent T cell-derived signals are essential for the generation of germinal center B cells in response to T cell-dependent Ag. In fact, CD40LBTg mice generated a complex Ag-specific IgG1 response, which was greatly enhanced in early, but reduced in late, primary response compared with control mice. We also found that the frequency of Ag-specific germinal center B cells in CD40LBTg mice was abruptly reduced 1 wk after immunization. As a result, the numbers of Ag-specific IgG1 long-lived plasma cells and memory B cells were reduced. By histology, large numbers of Ag-specific plasma cells were found in T cell areas adjacent to Ag-specific germinal centers of CD40LBTg mice, temporarily during the second week of primary response. These results indicate that CD40L expression on B cells prematurely terminated their ongoing germinal center response and produced plasma cells. Our results support the notion that CD40 signaling is an active termination signal for germinal center reaction.

[Immunoadjuvant effect of Hsp70L1 in tumor vaccine].

AIM: To explore the immune enhancement of Hsp70L1 in the tumor cell vaccines. METHODS: TRP2(153-243) and Hsp70L1 genes were obtained by RT-PCR from B16 cells in murine melanoma and from spleens of C57BL/6 mice and then were inserted into pcDNA3.1/V5-His eukaryotic expression vectors respectively. The recombinants of pTRP2, pHSP70L1 or pTRP2-Hsp fusion gene were obtained and transfected into B16 cells respectively. TRP2(153-243), HSP70L1 or TRP2-Hsp fusion gene-expressing B16 cells were then induced to necrosis by freezing-thawing or to apoptosis by mitomycin C. C57BL/6 mice were immunized with the necrotic or apoptotic B16 cells twice, then the live B16 tumor cells were transplanted into the immunized mice and the tumor growth was observed in some tumor-bearing mice. IFN-gamma-producing cells in splenocytes were measured by flow cytometry and the CTL activity of spleno-lymphocyte was detected by LDH release assay. RESULTS: After the normal mice were immunized with the necrotic or apoptotic tumor vaccines modified with TRP2(153-243), Hsp70L1 or TRP2-Hsp fusion genes, CTL lysis activity and IFN-gamma production from the splenic lymphocytes were promoted in the groups of Hsp70L1 and TRP2-Hsp modified tumor vaccines (P<0.05 or P<0.01). Additionally, the tumor growth was inhibited obviously in the groups of mice immunized with necrotic tumor vaccines (P<0.05 or P<0.01). However, no marked inhibition of tumor growth was observed in the groups of mice immunized with apoptotic tumor vaccines (P>0.05). CONCLUSION: Hsp70L1 remarkably improves the immunogenicity of B16 tumor vaccines, especially that of necrotic tumor vaccines.

Signal-transducing adaptor protein-2 regulates stromal cell-derived factor-1 alpha-induced chemotaxis in T cells.

Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains pleckstrin and Src homology 2-like domains, as well as a YXXQ motif in its C-terminal region. Our previous studies revealed that STAP-2 regulates integrin-mediated T cell adhesion. In the present study, we find that STAP-2 expression affects Jurkat T cell migration after stromal cell-derived factor-1alpha (SDF-1alpha)-treatment. Furthermore, STAP-2-deficient T cells exhibit reduced cell migration after SDF-1alpha-treatment. Importantly, overexpression of STAP-2 in Jurkat T cells induces activation of small guanine triphosphatases, such as Rac1 and Cdc42. Regarding the mechanism for this effect, we found that STAP-2 associates with Vav1, the guanine-nucleotide exchanging factor for Rac1, and enhances downstream Vav1/Rac1 signaling. These results reveal a novel STAP-2-mediated mechanism for the regulation of SDF-1alpha-induced chemotaxis of T cells via activation of Vav1/Rac1 signaling.

Attenuated expression of A20 markedly increases the efficacy of double-stranded RNA-activated dendritic cells as an anti-cancer vaccine.

A20 is a zinc finger protein with ubiquitin-modifying activity. A20 has been described as negatively regulating signaling induced by the TNF receptor and TLR family in a number of cell types, including mouse bone marrow-derived dendritic cells (DCs). However, the expression and effect of A20 in activated human monocyte-derived DCs have not been previously evaluated. We report that DCs activated with the TLR3 ligand poly(I:C) up-regulate A20. Down-regulating A20 demonstrated its role in the functional activation of DCs. A20 down-regulated DCs showed higher activation of the transcription factors NF-kappaB and activator protein-1, which resulted in increased and sustained production of IL-6, IL-10, and IL-12p70. We additionally silenced the immunosuppressive cytokine IL-10 and demonstrated that IL-10 inhibits T cell proliferation. We further demonstrated that A20 down-regulated DCs skew naive CD4+ T cells toward IFN-gamma producing Th1 cells, a process which is dependent on IL-12p70 and which is unaffected by IL-10. Furthermore, A20 and/or IL-10 down-regulated DCs had an enhanced capacity to prime Melan-A/MART-1 specific CD8+ T cells. Finally, we demonstrated that potent T cell stimulatory DCs are generated by the simultaneous delivery of poly(I:C12U), A20, or A20/IL-10 small interfering RNA and Ag-encoding mRNA, introducing a one step approach to improve DC-based vaccines. Together these findings demonstrate that A20 negatively regulates NF-kappaB and activator protein-1 in DCs and that down-regulation of A20 results in DCs with enhanced T cell stimulatory capacity.

CCL3-CCR5 axis regulates intratumoral accumulation of leukocytes and fibroblasts and promotes angiogenesis in murine lung metastasis process.

Metastasis proceeds through interaction between cancer cells and resident cells such as leukocytes and fibroblasts. An i.v. injection of a mouse renal cell carcinoma, Renca, into wild-type mice resulted in multiple metastasis foci in lungs and was associated with intratumoral accumulation of macrophages, granulocytes, and fibroblasts. A chemokine, CCL3, was detected in infiltrating cells and, to a lesser degree, tumor cells, together with an infiltration of leukocytes expressing CCR5, a specific receptor for CCL3. A deficiency of the CCL3 or CCR5 gene markedly reduced the number of metastasis foci in the lung, and the analysis using bone marrow chimeric mice revealed that both bone marrow- and non-bone marrow-derived cells contributed to metastasis formation. CCL3- and CCR5-deficient mice exhibited a reduction in intratumoral accumulation of macrophages, granulocytes, and fibroblasts. Moreover, intratumoral neovascularization, an indispensable process for metastasis, was attenuated in these gene-deficient mice. Intrapulmonary expression of matrix metalloproteinase (MMP)-9 and hepatocyte growth factor (HGF) was enhanced in wild-type mice, and the increases were markedly diminished in CCL3- and CCR5-deficient mice. Furthermore, MMP-9 protein was detected in macrophages and granulocytes, the cells that also express CCR5 and in vitro stimulation by CCL3-induced macrophages to express MMP-9. Intratumoral fibroblasts expressed CCR5 and HGF protein. In vitro CCL3 stimulated fibroblasts to express HGF. Collectively, the CCL3-CCR5 axis appears to regulate intratumoral trafficking of leukocytes and fibroblasts, as well as MMP-9 and HGF expression, and as a consequence to accelerate neovascularization and subsequent metastasis formation.

Infection of myeloid dendritic cells with Listeria monocytogenes leads to the suppression of T cell function by multiple inhibitory mechanisms.

Myeloid dendritic cells (DC) and macrophages play an important role in pathogen sensing and antimicrobial defense. In this study we provide evidence that myeloid DC respond to infection with Listeria monocytogenes with simultaneous induction of multiple stimulatory and inhibitory molecules. However, the overall impact of infected DC during T cell encounter results in suppression of T cell activation, indicating that inhibitory pathways functionally predominate. Inhibitory activity of infected DC is effected mainly by IL-10 and cyclooxygenase 2-mediated mechanisms, with soluble CD25 acting as an IL-2 scavenger as well as by the products of tryptophan catabolism. These inhibitory pathways are strictly TNF-dependent. In addition to direct infection, DC bearing this regulatory phenotype can be induced in vitro by a combination of signals including TNF, TLR2, and prostaglandin receptor ligation and by supernatants derived from the infected cells. Both infection-associated DC and other in vitro-induced regulatory DC are characterized by increased resistance to infection and enhanced bactericidal activity. Furthermore, myeloid DC expressing multiple regulatory molecules are identified in vivo in granuloma during listeriosis and tuberculosis. Based on the in vivo findings and the study of in vitro models, we propose that in granulomatous infections regulatory DC may possess dual function evolved to protect the host from disseminating infection via inhibition of granuloma destruction by T cells and control of pathogen spreading.

4-1BB enhances proliferation of beryllium-specific T cells in the lung of subjects with chronic beryllium disease.

In contrast to naive T cells, reactivation of memory cells is less dependent on CD28-mediated costimulation. We have shown that circulating beryllium-specific CD4(+) T cells from chronic beryllium disease patients remain CD28-dependent, while those present in the lung no longer require CD28 for T cell activation. In the present study, we analyzed whether other costimulatory molecules are essential for beryllium-induced T cell function in the lung. Enhanced proliferation of a beryllium-responsive, HLA-DP2-restricted T cell line was seen after the induction of 4-1BB ligand expression on the surface of HLA-DP2-expressing fibroblasts. Following beryllium exposure, CD4(+) T cells from blood and bronchoalveolar lavage of chronic beryllium disease patients up-regulate 4-1BB expression, and the majority of beryllium-responsive, IFN-gamma-producing CD4(+) T cells in blood coexpress CD28 and 4-1BB. Conversely, a significant fraction of IFN-gamma-producing bronchoalveolar lavage (BAL) T cells express 4-1BB in the absence of CD28. In contrast to blood, inhibition of the 4-1BB ligand-4-1BB interaction partially blocked beryllium-induced proliferation of BAL CD4(+) T cells, and a lack of 4-1BB expression on BAL T cells was associated with increased beryllium-induced cell death. Taken together, these findings suggest an important role of 4-1BB in the costimulation of beryllium-responsive CD4(+) T cells in the target organ.

Changes in adrenal steroidogenesis in spontaneous bacterial peritonitis and sterile ascites.

Adrenal androgens, particularly dehydroepiandrosterone (DHEA), may have important regulatory effects on the immune system in humans. This study measured the changes in adrenal steroidogenesis in 13 non-infected cirrhosis patients with sterile ascites and 13 patients with spontaneous bacterial peritonitis and the relation with circulating interleukin-6 (IL-6) levels. Comparisons were made with 10 healthy age-matched control subjects. The severity of bacterial peritonitis in liver cirrhosis was significantly associated with enhanced serum IL-6 and cortisol levels, and a decrease in serum DHEA sulfate in relation to serum IL-6 concentrations. Careful, long-term studies on DHEA administered to cirrhosis patients are needed to assess its safety in improving a number of pathological conditions that complicate liver cirrhosis.

LFA-1-mediated T cell costimulation through increased localization of TCR/class II complexes to the central supramolecular activation cluster and exclusion of CD45 from the immunological synapse.

T cell activation is associated with a dramatic reorganization of cell surface proteins and associated signaling components into discrete subdomains within the immunological synapse in T cell:APC conjugates. However, the signals that direct the localization of these proteins and the functional significance of this organization have not been established. In this study, we have used wild-type and LFA-1-deficient, DO11.10 TCR transgenic T cells to examine the role of LFA-1 in the formation of the immunological synapse. We found that coengagement of LFA-1 is not required for the formation of the central supramolecular activation cluster (cSMAC) region, but does increase the accumulation of TCR/class II complexes within the cSMAC. In addition, LFA-1 is required for the recruitment and localization of talin into the peripheral supramolecular activation cluster region and exclusion of CD45 from the synapse. The ability of LFA-1 to increase the amount of TCR engaged during synapse formation and segregate the phosphatase, CD45, from the synapse suggests that LFA-1 might enhance proximal TCR signaling. To test this, we combined flow cytometry-based cell adhesion and calcium-signaling assays and found that coengagement of LFA-1 significantly increased the magnitude of the intracellular calcium response following Ag presentation. These data support the idea that in addition to its important role on regulating T cell:APC adhesion, coengagement of LFA-1 can enhance T cell signaling, and suggest that this may be accomplished in part through the organization of proteins within the immunological synapse.

Enhanced protective efficacy and reduced viral load of foot-and-mouth disease DNA vaccine with co-stimulatory molecules as the molecular adjuvants.

To improve efficacy of DNA vaccination, various approaches have been developed, including the use of plasmid expressing co-stimulatory molecules as molecular adjuvants. In this study, we investigated whether co-inoculation of a construct expressing either 4-1BBL or OX40L as the molecular adjuvant with FMDV DNA vaccine, pcD-VP1, can increase immune responses and protective efficacies. Compared to the group immunized with pcD-VP1 alone, the co-inoculation of either molecular adjuvant induced a higher ratio of IgG2a/IgG1, higher levels of expression of IFN-gamma in CD4(+) and CD8(+) T cells and antigen-specific CTL responses, and more importantly provided an enhanced protection against the live FMDV challenge in animals. Concurrently, 4-1BBL as the molecular adjuvant dramatically reduced the viral loads of FMDV in vivo after the challenge. Together, the results demonstrate that co-stimulatory molecules 4-1BBL and OX40L can enhance the antigen-specific cell-mediated responses elicited by VP1 DNA vaccine and provide an enhanced protective efficacy with the reduced viral loads.

A novel Fc gamma receptor ligand augments humoral responses by targeting antigen to Fc gamma receptors.

Generating efficient antibody (Ab) responses against weak antigens remains challenging. Ab responses require antigen (Ag) uptake by antigen-presenting cells (APC), followed by presentation of processed Ag to T cells. Limited uptake of antigenic peptides by APC constrains Ab responses. Here we improve vaccine efficacy by targeting Ag to Fcgamma receptors (FcgammaR) using R4, a recombinant FcgammaR ligand. R4 has four repeats per chain of the hinge region and CH2 domain (HCH2) of human IgG1. HCH2 encompasses the FcgammaR binding site. The repeats are linked to the human IgG1 framework. To test R4 in augmenting Ag uptake, we expressed human serum albumin domain 1 (HSA1) at the N terminus of R4 to produce HSA1R4. HSA1R4 (50 microg) administered to mice in Ribi adjuvant induces up to 1100-fold higher HSA1-specific IgG titers than HSA1 (p<0.001). HSA1R4 (250 ng) induces up to 130 times more anti-HSA1 Ab than HSA1Fc, a protein with HSA1 linked to the IgG1 framework (p<0.001). HSA-reactive T cells proliferate more briskly to HSA1R4 than to HSA1Fc (p<0.008). Immunization with HSA1R4 yields greater T cell reactivity to HSA1 ex vivo than immunization with HSA1Fc (p<0.004). Linking antigenic peptides to linear HCH2 polymers may facilitate vaccine development.