Role of higher levels of post-challenge antibodies in protective vaccination against Leishmania tropica infection of BALB/c mice

Objective: To study the role of antibodies in protection against Leishmania tropica (L. tropica) infection in the experimental model of BALB/c mice. Methods: BALB/c mice were vaccinated against L. tropica by soluble Leishmania antigen or recombinant L. tropica stress-inducible protein-1 (LtSTI1) of L. tropica, and against Leishmania major (L. major) by soluble Leishmania antigen. Monophosphoryl lipid A was used as an adjuvant. The L. tropica- or L. major-vaccinated mice were challenged by L. tropica or L. major, respectively. The levels of anti-Leishmania antibodies (IgG1 and IgG2a) were determined after vaccination and after challenge. Results: All vaccinated groups caused a higher antibody response in comparison with the control group. The L. major-vaccinated group showed lower IgG1 response than the control group after the challenge. Conversely, in L. tropica-vaccinated mice, the levels of antibodies were higher than the control group. Moreover, the group receiving rLtSTI1 and monophosphoryl lipid A showed higher levels of antibodies than those of the rLtSTI1 group. In vaccinated mice, antibody responses against L. tropica remained high until 16 weeks after the challenge. Conclusions: The higher levels of post-challenge antibodies are associated with protective vaccination against L. tropica infection of BALB/c mice. Our findings provide new insight into the association of antibody with vaccine-induced protective immunity against L. tropica infection. More studies are needed to clarify the role of antibody in protection against L. tropica.

Role of higher levels of post-challenge antibodies in protective vaccination against Leishmania tropica infection of BALB/c mice

Objective: To study the role of antibodies in protection against Leishmania tropica (L. tropica) infection in the experimental model of BALB/c mice.Methods: BALB/c mice were vaccinated against L. tropica by soluble Leishmania antigen or recombinant L. tropica stress-inducible protein-1 (LtSTI1) of L. tropica, and against Leishmania major (L. major) by soluble Leishmania antigen. Monophosphoryl lipid A was used as an adjuvant. The L. tropica- or L. major-vaccinated mice were challenged by L. tropica or L. major, respectively. The levels of anti-Leishmania antibodies (IgG1 and IgG2a) were determined after vaccination and after challenge. Results: All vaccinated groups caused a higher antibody response in comparison with the control group. The L. major-vaccinated group showed lower IgG1 response than the control group after the challenge. Conversely, in L. tropica-vaccinated mice, the levels of antibodies were higher than the control group. Moreover, the group receiving rLtSTI1 and monophosphoryl lipid A showed higher levels of antibodies than those of the rLtSTI1 group. In vaccinated mice, antibody responses against L. tropica remained high until 16 weeks after the challenge. Conclusions: The higher levels of post-challenge antibodies are associated with protective vaccination against L. tropica infection of BALB/c mice. Our findings provide new insight into the association of antibody with vaccine-induced protective immunity against L. tropica infection. More studies are needed to clarify the role of antibody in protection against L. tropica.

Toll-like Receptor 1/2 Agonist Pam3CSK4 Suppresses Lipopolysaccharide-driven IgG1 Production while Enhancing IgG2a Production by B Cells

Interaction between pathogen-associated molecular patterns and pattern recognition receptors triggers innate and adaptive immune responses. Several studies have reported that toll-like receptors (TLRs) are involved in B cell proliferation, differentiation, and Ig class switch recombination (CSR). However, roles of TLRs in B cell activation and differentiation are not completely understood. In this study, we investigated the direct effect of stimulation of TLR1/2 agonist Pam3CSK4 on mouse B cell viability, proliferation, activation, Ig production, and Ig CSR in vitro. Treatment with 0.5 µg/ml of Pam3CSK4 only barely induced IgG1 production although it enhanced B cell viability. In addition, high-dosage Pam3CSK4 diminished IgG1 production in a dose-dependent manner, whereas the production of other Igs, cell viability, and proliferation increased. Pam3CSK4 additively increased TLR4 agonist lipopolysaccharide (LPS)-induced mouse B cell growth and activation. However, interestingly, Pam3CSK4 abrogated LPS-induced IgG1 production but enhanced LPS-induced IgG2a production. Further, Pam3CSK4 decreased LPS-induced germline γ1 transcripts (GLTγ1)/GLTε expression but increased GLTγ2a expression. On the other hand, Pam3CSK4 had no effect on LPS-induced plasma cell differentiation. Taken together, these results suggest that TLR1/2 agonist Pam3CSK4 acts as a potent mouse B cell mitogen in combination with TLR4 agonist LPS, but these 2 different TLR agonists play diverse roles in regulating the Ig CSR of each isotype, particularly IgG1/IgE and IgG2a.

Baicalein, wogonin, and Scutellaria baicalensis ethanol extract alleviate ovalbumin-induced allergic airway inflammation and mast cell-mediated anaphylactic shock by regulation of Th1/Th2 imbalance and histamine release / 대한해부학회지

Asthma is characterized by chronic inflammation, goblet cell hyperplasia, the aberrant production of the Th2 cytokines, and eosinophil infiltration into the lungs. In this study, we examined the effects of baicalein, wogonin, and Scutellaria baicalensis ethanol extract on ovalbumin (OVA)-induced asthma by evaluating Th1/Th2 cytokine levels, histopathologic analysis, and compound 48/80-induced systemic anaphylaxis and mast cell activation, focusing on the histamine release from rat peritoneal mast cells. Baicalein, wogonin, and S. baicalensis ethanol extract also decreased the number of inflammatory cells especially eosinophils and downregulated peribronchial and perivascular inflammation in the lungs of mice challenged by OVA. Baicalein, wogonin, and S. baicalensis ethanol extract significantly reduced the levels of tumor necrosis factor α, interleukin (IL)-1β, IL-4, IL-5 and the production of OVA-specific IgE and IgG1, and upregulated the level of interferon-γ and OVA-specific IgG2a. In addition, oral administration of baicalein, wogonin, and S. baicalensis ethanol extract inhibited compound 48/80-induced systemic anaphylaxis and plasma histamine release in mice. Moreover, baicalein, wogonin, and S. baicalensis ethanol extract suppressed compound 48/80-induced mast cell degranulation and histamine release from rat peritoneal mast cells. Conclusively, baicalein and wogonin as major flavonoids of S. baicalensis may have therapeutic potential for allergic asthma through modulation of Th1/Th2 cytokine imbalance and histamine release from mast cells.

Role of chitin in controlling atopic dermatitis (AD)---study on a murine AD model / 中国免疫学杂志

Objective:To investigate the effects of chitin on atopic dermatitis in an OVA induced AD murine model.Methods:Twenty-eight BALB/c mice were randomly divided into three groups:the normal control group (N)(8),the chitin group(E) (10) and the AD model group(M)(10).The murine model of atopic dermatitis was established through intraperitoneal injection of OVA followed by repeated epicutaneous application of OVA on mice back skin( AD model group).During the set up of AD murine model,mice of the chitin group were given intragastric gavage of 3 mg/d for 4 weeks.At the end of the experiment, the mice were sacrificed and skin lesions were biopsied for histological study.HE and O-toluidine stained paraffin sections were observed under microscope.The spleen cells were cultured and challenged with OVA and chitin,respectively,the supernatant was obtained for cytokine determination.Serum levels of total and OVA-specific IgE and total IgG2a were determined with ELISA.Results:Chitin significantly inhibited skin inflammation induced by OVA.Compared with the AD model group,the thickness of the epidermis and dermis in the chitin group were obviously decreased.The numbers of dermal infiltrated inflammatory cells,eosinophils and mast cells were significantly decreased in the chitin group compared with the AD model group ( P<0.05-0.001 ).The serum level of total IgE and OVA-specific IgE were significantly lower in the chitin group than in the AD model group(P<0.05-0.001),while the serum level of IgG2a in the chitin group was significantly higher than that of the AD model group( P<0.001).The cultured spleen cells of the chitin group produced significantly higher levels of IL-12 and IFN-γ,but lower level of IL-4 compared with those of the AD model group after OVA challenge (P<0.05).Conclusion:Chitin can inhibit the inflammation and decease the seum level of IgE in the murine AD model.The antiallergic effect of chitin might be associated with the induced production of Th1 type cytokines by mice spleen cells.

Inhibitory effects of TLR7/8 ligand R-848 on IgE production of murine in vivo / 重庆医学

Objective To investigate whether R-848 could inhibit IgE production of mice immunized with OVA plus ALUM in vivo.Methods BALB/c mice were immunized s.c on the back with PBS,OVA,OVA plus R-848,OVA plus CpG,OVA plus ALUM,and OVA plus ALUM in R-848 or CpG every two weeks for three times.The blood from the mice was harvested after the last immunization,and the concentration of OVA specific or total IgE,IgG1 and IgG2a in the sera was determined;the splenocytes from the immunized mice were harvested and cultured with or without OVA for 3 days,and the concentration of IL-4 and IFNγ in the supernatants was determined.Results Firstly,we investigated that R-848 as Th1 adjuvant could enhance the production of OVA specific IgG2a in mice immunized with OVA.Secondly,further study showed that R-848 could inhibit the production of OVA specific IgE and total IgE,and promote the production of OVA specific IgG2a in the sera of mice immunized with OVA plus ALUM.Moreover,the results of ELISA showed that R-848 inhibiting IgE production was related to its reducing IL-4 production and enhancing IFNγ production.Conclusion R-848 could inhibit IgE production of mice immunized with OVA plus ALUM in vivo.

Changes of cytokine mRNA expression and IgG responses in rats infected with Capillaria hepatica

The mRNA expression of several cytokines was evaluated in splenocytes and mesenteric lymph node (MLN) cells of rats infected with Capillaria hepatica by reverse-transcription (RT)-PCR until week 12 after infection. IgG1 and IgG2a, which are associated with Th1 and Th2 response, respectively, were also assessed by ELISA. The results indicated that the majority of cytokines, including the Th1 (IL-2 and IFN-gamma) and Th2 cytokines (IL-4, IL-5 and IL-10) were expressed at maximal levels during the early stage of infection (after week 1-2), and the ELISA data also evidenced a similar pattern of changes in IgG1 and IgG2a. Th1 and Th2 cytokines responded in a similar fashion in this rat model. The expression of cytokines in splenocytes was significantly higher than that in MLN cells, thereby indicating that cytokine production is controlled more by spleen than by MLN. In addition, the observation that IFN-gamma expression increased unexpectedly at the time of maximal egg production (6 weeks after infection) indicated that IFN-gamma is a cytokine reacting against egg production. However, increased IL-5 expression occurring in tandem with worm activity indicated that the activity of C. hepatica might be controlled by IL-5 expression.

Purification and biochemical characterization of two novel antigens from Leishmania major promastigotes

The identification and characterization of antigens that elicit human T cell responses is an important step toward understanding of Leishmania major infection and ultimately in the development of a vaccine. Micropreparative SDS-PAGE followed by electrotransfer to a PVDF membrane and elution of proteins from the PVDF, was used to separate 2 novel proteins from L. major promastigotes, which can induce antibodies of the IgG2a isotype in mice and also are recognized by antisera of recovered human cutaneous leishmaniasis subjects. Fractionation of the crude extract of L. major revealed that all detectable proteins of interest were present within the soluble Leishmania antigens (SLA). Quantitation of these proteins showed that their expression in promastigotes is relatively very low. Considering the molecular weight, immunoreactivity, chromatographic and electrophoretic behavior in reducing and non-reducing conditions, these proteins are probably 2 isoforms of a single protein. A digest of these proteins was resolved on Tricine-SDS-PAGE and immunoreactive fragments were identified by human sera. Two immunoreactive fragments (36.4 and 34.8 kDa) were only generated by endoproteinase Glu-C treatment. These immunoreactive fragments or their parent molecules may be ideal candidates for incorporation in a cocktail vaccine against cutaneous leishmaniasis.

Identification of novel Leishmania major antigens that elicit IgG2a response in resistant and susceptible mice

Experimental murine models with high, intermediate and low levels of genetically based susceptibility to Leishmania major infection reproduce almost entire spectrum of clinical manifestations of the human disease. There are increasing non-comparative studies on immune responses against isolated antigens of L. major in different murine strains. The aim of the present study was to find out whether there is an antigen that can induce protective immune response in resistant and susceptible murine strains. To do that, crude antigenic extract of procyclic and metacyclic promastigotes of L. major was prepared and subjected to SDS-PAGE electrophoresis. Western-blotting was used to search for antigen(s) capable of raising high antibody level of IgG2a versus IgG1 in the sera of both infected resistant and susceptible strains. Two novel antigens from metacyclic promastigotes of L. major (140 and 152 kDa) were potentially able to induce specific dominant IgG2a responses in BALB/c and C57BL/6 mice. The 2 antigens also reacted with IgG antibody of cutaneous leishmaniasis patients. We confirm that 140 and 152 kDa proteins of L. major promastigotes are inducing IgG production in mice and humans.

A CTL epitope of respiratory syncytial virus fused with G protein fragment regulates specific immunoresponses / 中国免疫学杂志

Objective: To investigate the regulation of respiratory syncytial virus CTL epitope fused with G protein antigen fragment G1 to the specific immunoresponses. Methods: The recombinant plasmid pET-DsbA-G1 or pET-DsbA-G1F/M2 was transferred into E.coli BL21(DE3) and the fusion protein DsbA-G1F/M2 or DsbA-G1 was expressed.The expressing product was induced and purified by affinity chromatography. The two proteins were used to immunized BALB/c mice i.p, respectively. Serum and spleen cells were collected regularly. RSV-specific CTL responses were measured by MTT, IgG and IgG1 and IgG2a antibodies by ELISA, neutralizing antibodies by plaque reduction assay. Results: The recombinant proteins were expressed successfully and the purity was over 90% after purified by affinity chromatography. The protein DsbA-G1F/M2 induced significant RSV-specific CTL responses, while DsbA-G1 without CTL epitope did not induce detctable CTL responses. Strong IgG antibody responses were elicited,indicated by both. IgG1 and IgG2a titers induced by DsbA-G1F/M2, while only IgG1 was induced by DsbA-G1.The ratio of IgG1/IgG2a was downregulated significantly. Both antigens induced high level of neutralizing antibodies, but the latter was a little lower. Conclusion: DsbA-G1F/M2, the fusion protein of a CTL epitope and G protein fragment G1 can induce both cellular immunity and humoral immunity. The activation of CTLs downregulates the ratio of IgG1/IgG2a.The more balanced immunoresponse is advantageous for improving safety of the candidate vaccine.

The effect of CpG ODN on Th1/Th2 type immune response in mice immunized with rHBsAg / 中国免疫学杂志

Objective:To investigate the Th1 type immune response effects of CpG oligodeoxynucleotide(CpG ODN) immunized with recombinant hepatitis B surface antigen(rHBsAg).Methods:The CpG ODN and rHBsAg were injected into the back leg tibialis anterior(TA) muscle of BALB/c mice for two times.The serum anti-HBs IgG sub-class ratio of IgG2a/IgG1 were detected by ELISA.The level of IFN-?,IL-2 and IL-12 in the supernatant of induced splenocyte with bioactived assay.The serum IL-4 and IL-10 level were detected by ABC-ELISA.Results:The anti-HBs IgG sub-class IgG2a titer of additional CpG ODN groups are obviously higher than those of other groups;The result showed that high level of Th1 type cytokines including IL-2,IFN-? and IL-12 were induced in CpG ODN groups,where the level of Th2 type cytokines such as IL-4 and IL-10 were inhibited obviously.Conclusion:CpG ODN has enhanced efficacy of rHBsAg for anti-HBs IgG sub-class production of IgG2a and induced the expression of Th1 type cytokines wherease inhibited the expression of Th2 type cytokines.