Effect of Quadrat and Core Sizes on Determining the Spatial Pattern of Criconemella sphaerocephalus

An unmanaged pasture was sampled on four occasions (A, B, C, D) with five different quadrat sizes for Criconemella sphaerocephalus by removing a constant soil core volume of 75 cm³ (A) and 300 cm³ (C) from increasing quadrat areas of 0.5-8 m², and removing soil core volumes of increasing size - 75-1,200 cm³ (B) and 300-4,800 cm³ (D) - proportionally with an increase in quadrat area (0.5-8 m²). Frequency counts of C. sphaerocephalus were fitted to six probability distributions. The index of aggregation (b) for Taylor''s power law and Morisita''s index of dispersion were also calculated where appropriate. Twelve of nineteen of the sampling combinations were best described by negative binomial distribution (P = 0.05). Criconemella sphaerocephalus appeared more highly aggregated when sampled with constant soil core volumes (A and C) than from increasing soil core volumes (B and D) based on Taylor''s index of aggregation (b). Morisita''s index of dispersion indicated aggregation at the smallest quadrat area (0.5 m²) for all sampling occasions (A, B, C, D).     

Gestation and breastfeeding in schistosomotic mothers differently modulate the immune response of adult offspring to postnatal Schistosoma mansoni infection

Schistosoma mansoni antigens in the early life alter homologous and heterologous immunity during postnatal infections. We evaluate the immunity to parasite antigens and ovalbumin (OA) in adult mice born/suckled by schistosomotic mothers. Newborns were divided into: born (BIM), suckled (SIM) or born/suckled (BSIM) in schistosomotic mothers, and animals from noninfected mothers (control). When adults, the mice were infected and compared the hepatic granuloma size and cellularity. Some animals were OA + adjuvant immunised. We evaluated hypersensitivity reactions (HR), antibodies levels (IgG1/IgG2a) anti-soluble egg antigen and anti-soluble worm antigen preparation, and anti-OA, cytokine production, and CD4⁺FoxP3⁺T-cells by splenocytes. Compared to control group, BIM mice showed a greater quantity of granulomas and collagen deposition, whereas SIM and BSIM presented smaller granulomas. BSIM group exhibited the lowest levels of anti-parasite antibodies. For anti-OA immunity, immediate HR was suppressed in all groups, with greater intensity in SIM mice accompanied of the remarkable level of basal CD4⁺FoxP3⁺T-cells. BIM and SIM groups produced less interleukin (IL)-4 and interferon (IFN)-g. In BSIM, there was higher production of IL-10 and IFN-g, but lower levels of IL-4 and CD4⁺FoxP3⁺T-cells. Thus, pregnancy in schistosomotic mothers intensified hepatic fibrosis, whereas breastfeeding diminished granulomas in descendants. Separately, pregnancy and breastfeeding could suppress heterologous immunity; however, when combined, the responses could be partially restored in infected descendants.     

Adjuvant Effects of L. acidophilus LW1 on Immune Responses to the Foot-and-Mouth Disease Virus DNA Vaccine in Mice

The adjuvant effects of Lactobacillus acidophilus on DNA vaccination are not fully understood. It has been hypothesized that swine-derived Lactobacillus acidophilus SW1 (LASW1) could function as an immune adjuvant to enhance antigen-specific immune responses after foot-and-mouth disease (FMD) DNA vaccination in mice. To evaluate the effect of oral LASW1 on the immune response to a DNA vaccine (pRC/CMV-vp1) harboring FMD VP1 gene, anti-FMDV antibody and its isotypes, T-cell proliferation, and cytokine detection were investigated. The results showed that LASW1 was able to enhance FMDV-specific antibody levels and FMDV-neutralizing antibodies. After a booster vaccine, the anti-FMDV antibody titers and FMDV-neutralizing antibodies levels induced by pRC/CMV-vp1 were higher in mice treated with LSAW1 than in the group immunized with pRC/CMV-vp1 alone (the control). Using T-cell proliferation, the stimulation index of the LASW1 group was significantly higher in response to ConA and 146S antigen (P<0.05) than in the control group. Importantly, higher concentrations of IFN-γ and IFN-γ-producing cells were also observed in splenocytes isolated from the experimental LASW1 mice, indicating that INF-γ secretion is important to the immune response to LASW1. The results indicate that LASW1 is a promising immune adjuvant in DNA vaccination against FMD when administrated orally.     

M tuberculosis in the Adjuvant Modulates Time of Appearance of CNS-Specific Effector T Cells in the Spleen through a Polymorphic Site of TLR2

DC deliver information regulating trafficking of effector T cells along T-cell priming. However, the role of pathogen-derived motives in the regulation of movement of T cells has not been studied. We hereinafter report that amount of M tuberculosis in the adjuvant modulates relocation of PLP139-151 specific T cells. In the presence of a low dose of M tuberculosis in the adjuvant, T cells (detected by CDR3 BV-BJ spectratyping, the so-called “immunoscope”) mostly reach the spleen by day 28 after immunization (“late relocation”) in the SJL strain, whereas T cells reach the spleen by d 14 with a high dose of M tuberculosis (“early relocation”). The C57Bl/6 background confers a dominant “early relocation” phenotype to F1 (SJL×C57Bl/6) mice, allowing early relocation of T cells in the presence of low dose M tuberculosis. A single non-synonymous polymorphism of TLR2 is responsible for “early/late” relocation phenotype. Egress of T lymphocytes is regulated by TLR2 expressed on T cells. Thus, pathogens engaging TLR2 on T cells regulate directly T-cell trafficking, and polymorphisms of TLR2 condition T-cell trafficking upon a limiting concentration of ligand.     

Toxoplasma gondii Protein Disulfide Isomerase (TgPDI) Is a Novel Vaccine Candidate against Toxoplasmosis

Toxoplasma gondii is a ubiquitous protozoan parasite that can infect all warm-blooded animals, including both mammals and birds. Protein disulfide isomerase (PDI) localises to the surface of T. gondii tachyzoites and modulates the interactions between parasite and host cells. In this study, the protective efficacy of recombinant T. gondii PDI (rTgPDI) as a vaccine candidate against T. gondii infection in BALB/c mice was evaluated. rTgPDI was expressed and purified from Escherichia coli. Five groups of animals (10 animals/group) were immunised with 10, 20, 30, 40 μg of rTgPDI per mouse or with PBS as a control group. All immunisations were performed via the nasal route at 1, 14 and 21 days. Two weeks after the last immunisation, the immune responses were evaluated by lymphoproliferative assays and by cytokine and antibody measurements. The immunised mice were challenged with tachyzoites of the virulent T. gondii RH strain on the 14th day after the last immunisation. Following the challenge, the tachyzoite loads in tissues were assessed, and animal survival time was recorded. Our results showed that the group immunised with 30 μg rTgPDI showed significantly higher levels of specific antibodies against the recombinant protein, a strong lymphoproliferative response and significantly higher levels of IgG2a, IFN-gamma (IFN-γ), IL-2 and IL-4 production compared with other doses and control groups. While no changes in IL-10 levels were detected. After being challenged with T. gondii tachyzoites, the numbers of tachyzoites in brain and liver tissues from the rTgPDI group were significantly reduced compared with those of the control group, and the survival time of the mice in the rTgPDI group was longer than that of mice in the control group. Our results showed that immunisation with rTgPDI elicited a protective immune reaction and suggested that rTgPDI might represent a promising vaccine candidate for combating toxoplasmosis.     

Identification of B- and T-cell epitopes on HtrA protein of Orientia tsutsugamushi

Orientia tsutsugamushi, a cause of scrub typhus is emerging as an important pathogen in several parts of the tropics. The control of this infection relies on rapid diagnosis, specific treatment, and prevention through vector control. Development of a vaccine for human use would be very important as a public health measure. Antibody and T-cell response have been found to be important in the protection against scrub typhus. This study was undertaken to predict the peptide vaccine that elicits both B- and T-cell immunity. The outer-membrane protein, 47-kDa high-temperature requirement A was used as the target protein for the identification of protective antigen(s). Using BepiPred2 program, the potential B-cell epitope PNSSWGRYGLKMGLR with high conservation among O. tsutsugamushi and the maximum surface exposed residues was identified. Using IEDB, NetMHCpan, and NetCTL programs, T-cell epitopes MLNELTPEL and VTNGIISSK were identified. These peptides were found to have promiscuous class-I major histocompatibility complex (MHC) binding affinity to MHC supertypes and high proteasomal cleavage, transporter associated with antigen processing prediction, and antigenicity scores. In the I-TASSER generated model, the C-score was −0.69 and the estimated TM-score was 0.63 ± 0.14. The location of the epitope in the 3D model was external. Therefore, an antibody to this outer-membrane protein epitope could opsonize the bacterium for clearance by the reticuloendothelial system. The T-cell epitopes would generate T-helper function. The B-cell epitope(s) identified could be evaluated as antigen(s) in immunodiagnostic assays. This cocktail of three peptides would elicit both B- and T-cell immune response with a suitable adjuvant and serve as a vaccine candidate.     

Partial Protective Effect of Intranasal Immunization with Recombinant Toxoplasma gondii Rhoptry Protein 17 against Toxoplasmosis in Mice

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that infects a variety of mammals, including humans. An effective vaccine for this parasite is therefore needed. In this study, RH strain T. gondii rhoptry protein 17 was expressed in bacteria as a fusion with glutathione S-transferase (GST) and the recombinant proteins (rTgROP17) were purified via GST-affinity chromatography. BALB/c mice were nasally immunised with rTgROP17, and induction of immune responses and protection against chronic and lethal T. gondii infections were investigated. The results revealed that mice immunised with rTgROP17 produced high levels of specific anti-rTgROP17 IgGs and a mixed IgG1/IgG2a response of IgG2a predominance. The systemic immune response was associated with increased production of Th1 (IFN-γand IL-2) and Th2 (IL-4) cytokines, and enhanced lymphoproliferation (stimulation index, SI) in the mice immunised with rTgROP17. Strong mucosal immune responses with increased secretion of TgROP17-specific secretory IgA (SIgA) in nasal, vaginal and intestinal washes were also observed in these mice. The vaccinated mice displayed apparent protection against chronic RH strain infection as evidenced by their lower liver and brain parasite burdens (59.17% and 49.08%, respectively) than those of the controls. The vaccinated mice also exhibited significant protection against lethal infection of the virulent RH strain (survival increased by 50%) compared to the controls. Our data demonstrate that rTgROP17 can trigger strong systemic and mucosal immune responses against T. gondii and that ROP17 is a promising candidate vaccine for toxoplasmosis.     

Silica Vesicle Nanovaccine Formulations Stimulate Long-Term Immune Responses to the Bovine Viral Diarrhoea Virus E2 Protein

Bovine Viral Diarrhoea Virus (BVDV) is one of the most serious pathogen, which causes tremendous economic loss to the cattle industry worldwide, meriting the development of improved subunit vaccines. Structural glycoprotein E2 is reported to be a major immunogenic determinant of BVDV virion. We have developed a novel hollow silica vesicles (SV) based platform to administer BVDV-1 Escherichia coli-expressed optimised E2 (oE2) antigen as a nanovaccine formulation. The SV-140 vesicles (diameter 50 nm, wall thickness 6 nm, perforated by pores of entrance size 16 nm and total pore volume of 0.934 cm³g^-1) have proven to be ideal candidates to load oE2 antigen and generate immune response. The current study for the first time demonstrates the ability of freeze-dried (FD) as well as non-FD oE2/SV140 nanovaccine formulation to induce long-term balanced antibody and cell mediated memory responses for at least 6 months with a shortened dosing regimen of two doses in small animal model. The in vivo ability of oE2 (100 μg)/SV-140 (500 μg) and FD oE2 (100 μg)/SV-140 (500 μg) to induce long-term immunity was compared to immunisation with oE2 (100 μg) together with the conventional adjuvant Quil-A from the Quillaja saponira (10 μg) in mice. The oE2/SV-140 as well as the FD oE2/SV-140 nanovaccine generated oE2-specific antibody and cell mediated responses for up to six months post the final second immunisation. Significantly, the cell-mediated responses were consistently high in mice immunised with oE2/SV-140 (1,500 SFU/million cells) at the six-month time point. Histopathology studies showed no morphological changes at the site of injection or in the different organs harvested from the mice immunised with 500 μg SV-140 nanovaccine compared to the unimmunised control. The platform has the potential for developing single dose vaccines without the requirement of cold chain storage for veterinary and human applications.     

Immunogenic Burkholderia pseudomallei Outer Membrane Proteins as Potential Candidate Vaccine Targets

Background

Burkholderia pseudomallei is the causative agent of melioidosis, a disease of significant morbidity and mortality in both human and animals in endemic areas. There is no vaccine towards the bacterium available in the market, and the efficacy of many of the bacterium''s surface and secreted proteins are currently being evaluated as vaccine candidates.

Methodology/Principal Findings

With the availability of the B. pseudomallei whole genome sequence, we undertook to identify genes encoding the known immunogenic outer membrane protein A (OmpA). Twelve OmpA domains were identified and ORFs containing these domains were fully annotated. Of the 12 ORFs, two of these OmpAs, Omp3 and Omp7, were successfully cloned, expressed as soluble protein and purified. Both proteins were recognised by antibodies in melioidosis patients'' sera by Western blot analysis. Purified soluble fractions of Omp3 and Omp7 were assessed for their ability to protect BALB/c mice against B. pseudomallei infection. Mice were immunised with either Omp3 or Omp7, subsequently challenged with 1×10⁶ colony forming units (cfu) of B. pseudomallei via the intraperitoneal route, and examined daily for 21 days post-challenge. This pilot study has demonstrated that whilst all control unimmunised mice died by day 9 post-challenge, two mice (out of 4) from both immunised groups survived beyond 21 days post-infection.

Conclusions/Significance

We have demonstrated that B. pseudomallei OmpA proteins are immunogenic in mice as well as melioidosis patients and should be further assessed as potential vaccine candidates against B. pseudomallei infection.     

Differential clinical efficacy of anti-CD4 monoclonal antibodies in rat adjuvant arthritis is paralleled by differential influence on NF-κB binding activity and TNF-α secretion of T cells

The aim of this study was to analyze the differential effects of three anti-CD4 monoclonal antibodies (mAbs) (with distinct epitope specifities) in the treatment of rat adjuvant arthritis (AA) and on T-cell function and signal transduction. Rat AA was preventively treated by intraperitoneal injection of the anti-CD4 mAbs W3/25, OX35, and RIB5/2 (on days -1, 0, 3, and 6, i.e. 1 day before AA induction, on the day of induction [day 0], and thereafter). The effects on T-cell reactivity in vivo (delayed-type hypersensitivity), ex vivo (ConA-induced proliferation), and in vitro (mixed lymphocyte culture) were assessed. The in vitro effects of anti-CD4 preincubation on T-cell receptor (TCR)/CD3-induced cytokine production and signal transduction were also analyzed. While preventive treatment with OX35 and W3/25 significantly ameliorated AA from the onset, treatment with RIB5/2 even accelerated the onset of AA by approximately 2 days (day 10), and ameliorated the arthritis only in the late phase (day 27). Differential clinical effects at the onset of AA were paralleled by a differential influence of the mAbs on T-cell functions, i.e. in comparison with OX35 and W3/25, the 'accelerating' mAb RIB5/2 failed to increase the delayed-type hypersentivity (DTH) to Mycobacterium tuberculosis, increased the in vitro tumor necrosis factor (TNF)-α secretion, and more strongly induced NF-κB binding activity after anti-CD4 preincubation and subsequent TCR/CD3-stimulation. Depending on their epitope specificity, different anti-CD4 mAbs differentially influence individual proinflammatory functions of T cells. This fine regulation may explain the differential efficacy in the treatment of AA and may contribute to the understanding of such treatments in other immunopathologies.     

In Situ Patrolling of Regulatory T Cells Is Essential for Protecting Autoimmune Exocrinopathy

Background

Migration of T cells, including regulatory T (Treg) cells, into the secondary lymph organs is critically controlled by chemokines and adhesion molecules. However, the mechanisms by which Treg cells regulate organ-specific autoimmunity via these molecules remain unclear. Although we previously reported autoimmune exocrinopathy resembling Sjögren''s syndrome (SS) in the lacrimal and salivary glands from C-C chemokine receptor 7 (CCR7)-deficient mice, it is still unclear whether CCR7 signaling might specifically affect the dynamics and functions of Treg cells in vivo. We therefore investigated the cellular mechanism for suppressive function of Treg cells via CCR7 in autoimmunity using mouse models and human samples.

Methods and Findings

Patrolling Treg cells were detected in the exocrine organs such as lacrimal and salivary glands from normal mice that tend to be targets for autoimmunity while the Treg cells were almost undetectable in the exocrine glands of CCR7 ^−/− mice. In addition, we found the significantly increased retention of CD4⁺CD25⁺Foxp3⁺ Treg cells in the lymph nodes of CCR7 ^−/− mice with aging. Although Treg cell egress requires sphingosine 1-phosphate (S1P), chemotactic function to S1P of CCR7−/− Treg cells was impaired compared with that of WT Treg cells. Moreover, the in vivo suppression activity was remarkably diminished in CCR7 ^−/− Treg cells in the model where Treg cells were co-transferred with CCR7 ^−/− CD25^-CD4⁺ T cells into Rag2 ^−/− mice. Finally, confocal analysis showed that CCR7⁺Treg cells were detectable in normal salivary glands while the number of CCR7⁺Treg cells was extremely decreased in the tissues from patients with Sjögren''s syndrome.

Conclusions

These results indicate that CCR7 essentially governs the patrolling functions of Treg cells by controlling the traffic to the exocrine organs for protecting autoimmunity. Characterization of this cellular mechanism could have clinical implications by supporting development of new diagnosis or treatments for the organ-specific autoimmune diseases such as Sjögren''s syndrome and clarifying how the local immune system regulates autoimmunity.     

Cancer vaccines: single-epitope anti-idiotype vaccine versus multiple-epitope antigen vaccine

In this study, we compared the immunogenicity and tumor-protective activity of anti-idiotypic antibodies mimicking a single tumor-associated epitope and tumor-associated antigen expressing multiple potentially immunogenic epitopes. We focused our study on the colorectal-carcinoma(CRC)-associated antigen GA733 (also known as CO17-1A/KS1-4/KSA/EpCAM). Monoclonal anti-idiotypic antibody (Ab2) BR3E4 was produced against murine anti-CRC mAb CO17-1A (Ab1) in rats. Full-length native GA733 protein was isolated from human tumor cells, and the extracellular domain protein (GA733-2E) was isolated from supernatants of recombinant baculovirus-infected insect cells by immunoafffinity chromatography. The immunomodulatory activity of the Ab2 was compared with that of the antigen, both in rabbits and in mice. Mice, like humans but not rabbits, express a GA733 antigen homologue on some of their normal tissues. Thus, these in vivo models allow the comparison of the immunogenicity of Ab2 and antigen in the presence (mice) and absence (rabbits) of normal tissue expression and immunological tolerance of the GA733 antigen homologue. In rabbits, aluminum-hydroxide(alum)-precipitated native GA733 antigen was superior to alum-precipitated Ab2 in inducing specific humoral immunity. In mice, alum-precipitated recombinant GA733-2E antigen, but not alum-precipitated Ab2, induced specific humoral immunity. However, when the Ab2 was administered to mice in Freund's complete adjuvant, specific humoral immune responses were elicited. Ab2 in complete Freund's adjuvant and GA733-2E in alum were compared for their capacity to induce antigen-specific cellular immunity in mice. Whereas lymphoproliferative responses were obtained with the recombinant antigen only, delayed-type hypersensitivity responses were obtained with both recombinant antigen and Ab2, although these responses were lower than after antigen immunization. The recombinant antigen in alum did not protect mice against challenge with antigen-positive syngeneic murine CRC cells. Similar studies with Ab2 BR3E4 mimicking the CO17-1A epitope were not possible because the tumor cells do not express this epitope after transfection with the human GA733-2 cDNA. However, similar studies with Ab2 mimicking the epitope defined by mAb GA733, which is expressed by the transfected tumor cells, indicated a lack of tumor-protective activity of this Ab2. In contrast, the full-length antigen expressed by recombinant adenovirus inhibited the growth of established tumors in mice. In conclusion, soluble antigen is a more potent modulator of humoral and cellular immune responses than Ab2, both administered in adjuvant. However, for induction of protective immunity, the immunogenicity of the antigen must be further enhanced, e.g., by expression of the antigen in a viral vector. Received: 27 December 1999 / Accepted: 27 January 2000     

In Vitro and In Vivo Studies for Assessing the Immune Response and Protection-Inducing Ability Conferred by Fasciola hepatica-Derived Synthetic Peptides Containing B- and T-Cell Epitopes

Fasciolosis is considered the most widespread trematode disease affecting grazing animals around the world; it is currently recognised by the World Health Organisation as an emergent human pathogen. Triclabendazole is still the most effective drug against this disease; however, resistant strains have appeared and developing an effective vaccine against this disease has increasingly become a priority. Several bioinformatics tools were here used for predicting B- and T-cell epitopes according to the available data for Fasciola hepatica protein amino acid sequences. BALB/c mice were immunised with the synthetic peptides by using the ADAD vaccination system and several immune response parameters were measured (antibody titres, cytokine levels, T-cell populations) to evaluate their ability to elicit an immune response. Based on the immunogenicity results so obtained, seven peptides were selected to assess their protection-inducing ability against experimental infection with F. hepatica metacercariae. Twenty-four B- or T-epitope-containing peptides were predicted and chemically synthesised. Immunisation of mice with peptides so-called B1, B2, B5, B6, T14, T15 and T16 induced high levels of total IgG, IgG1 and IgG2a (p<0.05) and a mixed Th1/Th2/Th17/Treg immune response, according to IFN-γ, IL-4, IL-17 and IL-10 levels, accompanied by increased CD62L⁺ T-cell populations. A high level of protection was obtained in mice vaccinated with peptides B2, B5, B6 and T15 formulated in the ADAD vaccination system with the AA0029 immunomodulator. The bioinformatics approach used in the present study led to the identification of seven peptides as vaccine candidates against the infection caused by Fasciola hepatica (a liver-fluke trematode). However, vaccine efficacy must be evaluated in other host species, including those having veterinary importance.     

Mannan-Binding Lectin Deficiency Limits Inflammation-induced Myeloid-Derived Suppressor Cells Expansion via Modulating Tumor Necrosis Factor Alpha-triggered Apoptosis

Background: Mannan-binding lectin (MBL), a soluble pattern recognition molecule in the innate immune system, is reported to be associated with the function of immune cells. Myeloid-derived suppressor cells (MDSCs) are mainly characterized by immunosuppressive activities involving several inflammatory diseases such as cancer, infection, and arthritis. Some of the factors inducing their apoptosis are known, however, mechanisms have not been identified. The underlying impact of MBL on the MDSCs especially under inflammatory conditions remains unknown. This study was designed to investigate whether MBL affects MDSCs survival during inflammation conditions.Methods: WT and MBL-deficient (MBL^-/-) mice were induced on day 0 of the experiment by subcutaneous injection of complete Freund''s adjuvant and then injected with incomplete Freund''s adjuvant into the knee joint space under general anesthesia on day 14 to induce inflammatory arthritis. The proportions of MDSCs in the spleen and blood and the serum level of the inflammatory cytokines were measured. In vitro study, MDSCs were isolated from the bone marrow of WT and MBL^-/- mice and cultured in the presence of interleukin-6 (IL-6) and granulocyte-macrophage colony-stimulating factor (GM-CSF) for 5 days with or without tumor necrosis factor-alpha (TNF-α).Results: After adjuvant treatment, MBL^-/- mice had a significantly lower frequency of MDSCs as well as elevated serum inflammatory cytokines levels compared to WT mice. MBL deficiency markedly inhibited the MDSCs frequency from mice bone marrow induced by IL-6 and GM-CSF in the presence of TNF-α in vitro. Mechanistic studies established that MBL inhibited MDSCs apoptosis via down-regulation of TNF-α/tumor necrosis factor-alpha receptor 1 (TNFR1) signaling pathway and subsequent caspase 3-dependent manner.Conclusion: Mannan-binding lectin deficiency inhibits myeloid-derived suppressor cells expansion via modulating TNF-α triggered apoptosis.     

Immune Responses to HBsAg Conjugated to Protein D of Non-Typeable Haemophilus influenzae in Mice

BackgroundHepatitis B vaccine that contains an aluminum hydroxide adjuvant induces apoptotic death of Hepa 1–6 cells. Difficult-to-degrade chemical additives in vaccines effectively enhance vaccine immunogenicity, but also affect the host tissue. Identification of bio-molecules that are readily degraded and compatible in vivo as an adjuvant is important for vaccine research. The hapten–carrier effect suggests that stimulation of helper T (Th) cells by carrier adjuvants is feasible. Protein D (PD) of non-typeable Haemophilus influenzae covalently conjugated to some polysaccharide vaccines has been confirmed to convert T-cell independent (TI) antigens into T-cell dependent (TD) antigens, and elicit strong T-cell responses ultimately. Herein, we would substitube PD for aluminum hydroxide adjuvant in Hepatitis B vaccine.ConclusionsRecombinant truncated PD covalently conjugated to HBsAg antigen enhanced the immunogenicity of the antigen in mice simultaneously by humoral and cellular immune response, which would facilitate therapeutic hepatitis B vaccines.     

The Adjuvant Double Mutant Escherichia coli Heat Labile Toxin Enhances IL-17A Production in Human T Cells Specific for Bacterial Vaccine Antigens

The strong adjuvant activity and low enterotoxicity of the novel mucosal adjuvant double mutant Escherichia coli heat labile toxin, LT(R192G/L211A) or dmLT, demonstrated in mice, makes this molecule a promising adjuvant candidate. However, little is known about the mechanisms responsible for the adjuvant effect of dmLT or whether dmLT also has an adjuvant function in humans.We investigated the effect of dmLT on human T cell responses to different bacterial vaccine antigens: the mycobacterial purified protein derivative (PPD) antigen, tested in individuals previously vaccinated with Bacillus Calmette-Guérin, the LT binding subunit (LTB), evaluated in subjects immunised with oral inactivated whole cell vaccines against enterotoxigenic Escherichia coli, and Streptococcus pneumoniae whole cell vaccine antigens, tested in subjects naturally exposed to pneumococci. We found that dmLT enhanced the production of IL-17A by peripheral blood mononuclear cells in response to all antigens tested. dmLT had comparable effects on IL-17A responses to PPD as the single mutant LT(R192G) adjuvant, which has demonstrated clinical adjuvant activity in humans. Neutralisation of IL-1β and IL-23, but not IL-6, suppressed the IL-17A-enhancing effect of dmLT. Furthermore, CD4+ T cells produced higher levels of IL-17A when stimulated with monocytes pulsed with PPD and dmLT compared to PPD alone, supporting an important role of antigen presenting cells in enhancing IL-17A responses. dmLT also potentiated mitogen-induced IL-17A and IL-13 production. However, dmLT had variable influences on IFN-γ responses to the different stimuli tested.Our demonstration of a potent ability of dmLT to enhance human Th17 type T cell responses to bacterial vaccine antigens encourages further evaluation of the adjuvant function of dmLT in humans.     

Induction of humoral antibody response by soluble polysaccharide of Cryptococcus neoformans

Mice were immunized with purifiedCryptococcus neoformans soluble polysaccharide, or with the polysaccharide coupled to methylated bovine serum albumin or methylated bovine gamma globulin. The polysaccharide-methylated protein complexes were no more immunogenic than the purified polysaccharide when used without Freund's incomplete adjuvant; however, the methylated protein complexes induced higher levels of antibody than purified polysaccharide when emulsified in the adjuvant. Sera from mice were titrated by passive hemagglutination, and maximum antibody titers were observed 14 to 21 days after immunization. Antibody titers declined rapidly after 14 to 21 days in mice immunized with antigen alone; whereas, animals immunized with cryptococcal antigen emulsified in adjuvant remained at peak levels throughout a six week experimental period. All antigens were immunogenic over a wider dosage range when contained in adjuvant. Individual mice immunized with an adjuvant emulsion of purified polysaccharide varied widely in the amount of antibody produced, with some of the animals producing no detectable antibody.     

Idiotypic analysis of anti-I-Ak monoclonal antibodies. III. T- and B-cell responses to anti-Ia idiotopes are not modulated by syngeneic anti-idiotypic monoclonal antibodies

To investigate whether anti-idiotypic (anti-Id) antibodies activate T cells either directly or indirectly, we examined the ability of syngeneic anti-Id monoclonal antibodies (mAbs) to regulate idiotype (Id) expression, antigen-binding antibody production, and T-cell reactivity to antigen. Our idiotypic system consists of an anti-I-A mAb that carries an infrequently expressed Id. Using three syngeneic anti-Id mAbs (Ab2), we previously defined the idiotype of the 11-5.2.1.9 (11-5) anti-I-Ak mAb. Two of these mAbs, IIID1 and IA2, recognize the same or closely related epitopes on 11-5 and cross react with two additional anti-I-Ak mAbs, 8B and 39J; the third anti-Id mAb, VC6, recognizes a distinct epitope shared by 11-5 and 8B. In the present study, BALB/c (H-2d) mice were primed with varying doses of these anti-Ids and were then boosted with C3H (H-2k) spleen cells. Among 130 such primed mice, the syngeneic anti-Ids when tested at priming doses between 10 ng and 10 micrograms were unable to induce Id production. The priming anti-Id mAbs persisted in the serum of the mice and were detectable as late as 40 days after priming. Ab1 expression was not modulated in BALB/c mice immunized with KLH-coupled Ab2, however, this immunization elicited the production of Ab3 which shared idiotypes with 11-5, 8B, and 39J. BALB/c anti-C3H alloreactive T-cell clones were also not induced by anti-Id priming, nor could they be shown to bind directly to the three Ab2 used. Nevertheless, the proliferative response of one anti-I-Ak specific T-cell clone that recognizes the same epitope as 11-5, 8B, and 39J, was inhibited by the IIID1 and IA2 Ab2. Thus, a T cell can express an idiotype shared by a B cell, but the linked recognition of an Id-associated carrier determinant(s) by an alloreactive T cell is required to elicit an anti-Id antibody response. These results favor the possibility that the activation of T cells is not dependent upon their ability to bind to anti-Id, but rather on their capacity to respond to epitopes of Id-anti-Id antigen-antibody complexes formed on B cells.     

Identification of Conserved and HLA Promiscuous DENV3 T-Cell Epitopes

Anti-dengue T-cell responses have been implicated in both protection and immunopathology. However, most of the T-cell studies for dengue include few epitopes, with limited knowledge of their inter-serotype variation and the breadth of their human leukocyte antigen (HLA) affinity. In order to expand our knowledge of HLA-restricted dengue epitopes, we screened T-cell responses against 477 overlapping peptides derived from structural and non-structural proteins of the dengue virus serotype 3 (DENV3) by use of HLA class I and II transgenic mice (TgM): A2, A24, B7, DR2, DR3 and DR4. TgM were inoculated with peptides pools and the T-cell immunogenic peptides were identified by ELISPOT. Nine HLA class I and 97 HLA class II novel DENV3 epitopes were identified based on immunogenicity in TgM and their HLA affinity was further confirmed by binding assays analysis. A subset of these epitopes activated memory T-cells from DENV3 immune volunteers and was also capable of priming naïve T-cells, ex vivo, from dengue IgG negative individuals. Analysis of inter- and intra-serotype variation of such an epitope (A02-restricted) allowed us to identify altered peptide ligands not only in DENV3 but also in other DENV serotypes. These studies also characterized the HLA promiscuity of 23 HLA class II epitopes bearing highly conserved sequences, six of which could bind to more than 10 different HLA molecules representing a large percentage of the global population. These epitope data are invaluable to investigate the role of T-cells in dengue immunity/pathogenesis and vaccine design.