Proliferative Response Of Splenocytes Research Articles

Major royal-jelly proteins intake modulates immune functions and gut microbiota in mice

In this study, we investigated the effects of major royal jelly proteins (MRJPs) on the estrogen, gut microbiota, and immunological responses in mice. Mice given 250 or 500 mg/kg, not 125 mg/kg of MRJPs, enhanced the proliferation of splenocytes in response to mitogens. The splenocytes and mesenteric lymphocytes activated by T-cell mitogens (ConA and anti-CD3/CD28 antibodies) released high levels of IL-2 but low levels of IFN-γ and IL-17A. The release of IL-4 was unaffected by MRJPs. Additionally, splenocytes and mesenteric lymphocytes activated by LPS were prevented by MRJPs at the same dose as that required for producing IL-1β and IL-6, two pro-inflammatory cytokines. The production of IL-1β, IL-6, and IFN-γ was negatively associated with estrogen levels, which were higher in the MRJP-treated animals than in the control group. Analysis of the gut microbiota revealed that feeding mice 250 mg/kg of MRJPs maintained the stability of the natural intestinal microflora of mice. Additionally, the LEfSe analysis identified biomarkers in the MRJP-treated mice, including Prevotella, Bacillales, Enterobacteriales, Gammaproteobacteria, Candidatus_Arthromitus, and Shigella. Our results showed that MRJPs are important components of royal jelly that modulate host immunity and hormone levels and help maintain gut microbiota stability.

Immunization with Tp0954, an adhesin of Treponema pallidum, provides protective efficacy in the rabbit model of experimental syphilis.

Syphilis, a chronic multisystemic disease caused by spirochete Treponema pallidum subspecies pallidum infection, continues to be a serious global health problem and congenital syphilis remains a major cause of adverse outcomes in pregnancy in developing countries. The development of an effective vaccine is the most cost-effective way to eliminate syphilis, but so far has been elusive. Here, we evaluated the immunogenicity and protective efficacy of Tp0954, a T. pallidum placental adhesin, as a potential vaccine candidate in a New Zealand White rabbit model of experimental syphilis. Animals immunized with recombinant Tp0954 (rTp0954) produced high titers of Tp0954-specific serum IgG, high levels of IFN-γ from splenocytes and specific splenocyte proliferation response when compared to control animals immunized with PBS and Freund's adjuvant (FA). Furthermore, rTp0954 immunization significantly delayed the development of cutaneous lesions, promoted inflammatory cellular infiltration at the primary lesion sites, as well as inhibited T. pallidum dissemination to distal tissues or organs when compared with that of the control animals. In addition, the naïve rabbits receiving popliteal lymph nodes from Tp0954-immunized, T. pallidum-challenged animals were not infected by T. pallidum, confirming sterile immunity. These findings suggest that Tp0954 is a potential vaccine candidate against syphilis.

Immunomodulatory Effects of Probiotic Lactobacillus casei On GM-CSF-adjuvanted Influenza Dna Vaccine

Aim: This study investigates the protective efficacy of influenza DNA vaccine combined with a granulocyte macrophage-colony stimulating factor (GM-CSF) adjuvant and probiotic Lactobacillus casei, an oral immunomodulator, in a BALB/c mice. Materials & methods: The mice were immunized with HA1 DNA vaccine along with GM-CSF and probiotic twice within a one-week interval. Results: The results showed that both adjuvants exert a synergistic effect in enhancing the humoral and cellular immune responses of the DNA vaccine. This combination also deceased IL-6 and IL-17A levels in the lung homogenates. The protection patterns were closely associated with influenza virus-specific splenocyte proliferative and serum IgG antibody (Ab) responses. Conclusion: The Findings demonstrate L. casei modulate balanced Th1/Th2 immune responses toward HA1 DNA vaccine adjuvanted by GM-CSF.

Preliminary safety assessment of CIGB-210, an investigational peptide for HIV infection.

Current human immunodeficiency virus treatments need to be periodically administered lifelong. In this study we assess the effect of repeated doses of an anti-HIV peptide drug candidate in C57BL6 strain. Two schemes of up to 15 administrations and one of 30, daily dosing for 5days per week, all by the subcutaneous route were evaluated. Different dose concentrations of the peptide were assayed. CIGB-210 treated animals showed no symptoms or abnormal behavior as compared with placebo. All the animals gained weight during the study. Macroscopic evaluation showed no alterations in any of the organs studied. Microscopic analysis of the tissues did not show morphological changes in thymus, stomach, small and large intestines, kidney, brain, or cerebellum. The proliferative response of splenocytes and their capacity to secrete gamma interferon were not compromised by the repeated administration of CIGB-210. There were not statistically significant differences for any of the parameters evaluated during the study among treated and non-treated groups. We can conclude that CIGB-210 is well tolerated in C57BL6 mice in the dose concentration range explored and merits subsequent toxicological studies.

Abstract 1787: YH29407, a novel IDO1 inhibitor, enhances the anti-tumor effects through increased tumor-reactive T cell functions in solid tumor

Abstract Background: The indoleamine 2,3-dioxygenase (IDO), is an enzyme responsible for catabolizing tryptophan (Trp) to kynurenine (Kyn). The depletion of tryptophan and the increase in kynurenine exert important immunosuppressive functions by activating T regulatory cells and myeloid-derived suppressor cells. Therefore, overexpression of IDO is associated with poor clinical outcome. In this study, we evaluated pharmacokinetic and pharmacodynamic profiles and anti-cancer immune responses of YH29407, a novel IDO1 inhibitor. Methods: MC38 murine tumor models were established for pharmacokinetic and pharmacodynamic profiling. The kynurenine levels in plasma and tumor of YH29407, epacadostat and BMS-986205 following 3 day treatment were detected. The antitumor effects of YH29407 100 mg/kg QD/BID, epacadostat 100 mg/kg BID and BMS-986205 100 mg/kg QD for 10 day administration were evaluated in MC38 models. Various immunologic profiles were evaluated after 5 day treatment of individual inhibitors. Results: The AUC of kynurenine in plasma was decreased by 81.4% with YH29407 100 mg/kg BID, 62.7% with epacadostat 100 mg/kg BID, and 19.8% with BMS-986205 100 mg/kg QD treatment, respectively, compared to control group. The intratumoral concentration of kynurenine was also reduced by 99.4%, 89.9% and 1.1% with 3 day treatment of YH29407 100 mg/kg BID, epacadostat 100 mg/kg BID, and BMS-986205 100 mg/kg QD treatment, respectively, compared to control group. The intratumoral concentration of YH29407 BID, epacadostat BID and BMS-986205 QD at 24 hours after 3 day treatment was 9.697 µg/g, 5.478 µg/g and 0.025 µg/g, respectively. These results indicate favorable pharmacokinetic property of YH29407. Compared to epacadostat and BMS-986205, YH29407 100 mg/kg BID treatment exhibited significant proliferative response of splenocytes (P <0.05). IFN-γ secretion levels were also significantly increased by YH29407 treatment, compared to epacadostat and BMS-986205 treatment (P <0.05). Profiling of serum cytokine levels revealed significant increase of Granzyme B, IFN-γ, IL-12, and MMP2 and decrease of FASL, compared to the vehicle group. Upregulation of IFN-γ in CD3+ tumor-infiltrating lymphocytes was significantly induced in the YH29407 treated group compared to epacadostat and BMS-986205 treated groups (P <0.05). Treatment with YH29407 100 mg/kg BID induced significant tumor growth inhibition, compared to epacadostat 100 mg/kg BID and BMS-986205 100 mg/kg QD. Conclusion: The novel IDO1 inhibitor, YH29407, with improved pharmacokinetic and pharmacodynamic profiles, showed antitumor inflammatory reprogramming leading to enhanced antitumor efficacy. Citation Format: Kyoung-Ho Pyo, Ho-Woong Kang, Sun Min Lim, Do Hee Kim, Dong Kwon Kim, Gyu-Jin Lee, Jong-Suk Park, Se-Woong Oh, Byoung Chul Cho. YH29407, a novel IDO1 inhibitor, enhances the anti-tumor effects through increased tumor-reactive T cell functions in solid tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1787.

Zika Virus Envelope Domain III Recombinant Protein Delivered With Saponin-Based Nanoadjuvant From Quillaja brasiliensis Enhances Anti-Zika Immune Responses, Including Neutralizing Antibodies and Splenocyte Proliferation.

Nanoadjuvants that combine immunostimulatory properties and delivery systems reportedly bestow major improvements on the efficacy of recombinant, protein-based vaccines. Among these, self-assembled micellar formulations named ISCOMs (immune stimulating complexes) show a great ability to trigger powerful immunological responses against infectious pathogens. Here, a nanoadjuvant preparation, based on saponins from Quillaja brasiliensis, was evaluated together with an experimental Zika virus (ZIKV) vaccine (IQB80-zEDIII) and compared to an equivalent vaccine with alum as the standard adjuvant. The preparations were administered to mice in two doses (on days zero and 14) and immune responses were evaluated on day 28 post-priming. Serum levels of anti-Zika virus IgG, IgG1, IgG2b, IgG2c, IgG3 were significantly increased by the nanoadjuvant vaccine, compared to the mice that received the alum-adjuvanted vaccine or the unadjuvanted vaccine. In addition, a robust production of neutralizing antibodies and in vitro splenocyte proliferative responses were observed in mice immunized with IQB80-zEDIII nanoformulated vaccine. Therefore, the IQB80-zEDIII recombinant preparation seems to be a suitable candidate vaccine for ZIKV. Overall, this study identified saponin-based delivery systems as an adequate adjuvant for recombinant ZIKV vaccines and has important implications for recombinant protein-based vaccine formulations against other flaviviruses and possibly enveloped viruses.

Synergistic Therapeutic Effects of Probiotic Lactobacillus casei TD-2 Consumption on GM-CSF-Induced Immune Responses in a Murine Model of Cervical Cancer

We perceive the potential of combined immunotherapy for the synergistic treatment of human papillomavirus (HPV)-associated tumors. So, the tumor inhibiting effects of combination of L. casei TD2a and GM-CSF on the TC-1 growth were evaluated In Vivo using lymphocyte proliferation, lymphocyte cytotoxicity, splenocyte, and tumor cytokine assays. The results showed that tumor inhibition in transplanted mice in the GM-CSF combined with probiotic L. casei group was significantly higher than that observed in the other groups excluding GM-CSF group whose tumor inhibition effect was considerable. The findings also indicated that the combined group could generate tumor-specific cytolytic and splenocyte proliferative responses. The levels of IFN–γ, IL-4, and IL-12 after treating with GM-CSF combined with probiotic L. casei were significantly higher than those of other groups. The intratumoral Tumor Necrosis Factor Related Apoptosis-Inducing Ligand (TRAIL) was also significantly increased in the combined group. Tumor analysis further showed that the combined group decreased the accumulation of IL-10 in the tumor microenvironment of treated mice. Furthermore, tumor volume analysis demonstrated that combination group and even GM-CSF suppress tumor growth. Our findings showed that the combination of GM-CSF and probiotic results in improved tumor suppression against HPV-associated tumors and stimulates enhancement of specific antitumor immune responses.

Lactococcus lactis NZ9000 Prevents Asthmatic Airway Inflammation and Remodelling in Rats through the Improvement of Intestinal Barrier Function and Systemic TGF-β Production

Introduction: The use of probiotics has been broadly popularized due to positive effects in the attenuation of aberrant immune responses such as asthma. Allergic asthma is a chronic respiratory disease characterized by airway inflammation and remodelling. Objective: This study was aimed to evaluate the effect of oral administration of Lactococcus lactis NZ9000 on asthmatic airway inflammation and lung tissue remodelling in rats and its relation to the maintenance of an adequate intestinal barrier. Methods: Wistar rats were ovalbumin (OVA) sensitized and challenged and orally treated with L. lactis. Lung inflammatory infiltrates and cytokines were measured, and remodelling was evaluated. Serum OVA-specific immunoglobulin (Ig) E levels were assessed. We also evaluated changes on intestinal environment and on systemic immune response. Results: L. lactis diminished the infiltration of proinflammatory leucocytes, mainly eosinophils, in the bronchoalveolar compartment, decreased lung IL-4 and IL-5 expression, and reduced the level of serum allergen-specific IgE. Furthermore, L. lactis prevented eosinophil influx, collagen deposition, and goblet cell hyperplasia in lung tissue. In the intestine, L. lactis-treated asthmatic rats increased Peyer’s patch and goblet cell quantity and mRNA expression of IgA, MUC-2, and claudin. Additionally, intestinal morphological alterations were normalized by L. lactis administration. Splenocyte proliferative response to OVA was abolished, and serum levels of transforming growth factor (TGF)-β were increased by L. lactis treatment. Conclusions: These findings suggest that L. lactis is a potential candidate for asthma prevention, and the effect is mediated by the improvement of intestinal barrier function and systemic TGF-β production.

New Brucella abortus S19 Mutant to Improve Distinction Between Infected and Vaccinated Animals.

Background: Using Brucella abortus Strain 19 (S19) to control bovine brucellosis is restricted due to induce antibodies to the O-side chain of the smooth lipopolysaccharide (LPS) which may be difficult to differentiate vaccinated and infected animals. Furthermore, it is virulent for humans and can induce abortion to cattle.Objectives: The aim of this study was to employ gene knockout B. abortus S19 for the first time to eliminate diagnostic defects and obtain the attenuated mutant strain.Material and Methods: The wbkA gene, which is one of the LPS O-chain coding genes, was knocked out in vaccinal Brucella abortus S19. The proliferative response and immunoglobulin M production were analyzed in wbkA deletion strain-infected BALB/c mice.Results: The loss of wbkA gene function resulted in induction of the splenocyte proliferative response in mice infected by the mutant S19 strain compare to those induced by parental S19 and RB51 strains. Moreover, wbkA mutant did not induce any IgM antibody response using the enzyme-linked immunosorbent assay.Conclusions: As a result, the new mutant S19 strain had deficiency in its LPS O-chain structure, besides cannot induce IgM response then, reduce mistakes to discriminate between vaccinated and infected animal, and also can be considered as a new vaccine candidate.

Attenuated Salmonella secreting Brucella protective antigens confer dual-faceted protection against brucellosis and salmonellosis in a mouse model

We demonstrated the use of attenuated Salmonella strains secreting Brucella antigens SodC, Omp19, BLS, and PrpA as live vaccine candidates against Brucella abortus infection and presented their cross-protection against Salmonella infections using a BALB/c mice model. Here, a single immunization with each individual strain was capable of establishing significantly high (p < 0.05) Brucella-specific systemic immunoglobulin (Ig)G and secretory IgA (sIgA) responses compared to control mice. Upon stimulation of the splenocytes harvested from immunized mice with the respective antigens SodC, Omp19, BLS, and PrpA, significant increases in splenocyte proliferative responses against all four antigens versus PBS and vector controls were observed (p < 0.05). Additionally, interferon-γ and interleukin-4 secretion clearly demonstrated an uplift of these cytokines in all four strains upon immunization compared to the control groups. However, a significantly high response was noted in the mice groups immunized with Salmonella secreting SodC and Omp19 only. Upon virulent Brucella abortus 544 challenge, all four antigens presented a significantly high protection index (PI) in the spleen, as follows: 0.85 for SodC; 0.96 for Omp19; 0.6 for BLS; and 0.66 for PrpA. In contrast, in the liver, the same antigens resulted in PI values of 1.37, 1.14, 1.12, and 1.81, respectively. Immunological profiling of immunized mice against Salmonella-specific immune responses also showed significant elicitation of both humoral and cell-mediated immune responses as measured by IgG, sIgA, splenocyte proliferation, and cytokine induction. In addition, full protection against virulent Salmonella challenge was shown with no mortality in immunized mice, whereas 100% (8/8) mortality was observed in control mice over a two-week post-Salmonella challenge. In conclusion, we show that the live attenuated Salmonella delivering Brucella protective antigens may efficiently confer dual protection against both brucellosis and salmonellosis in immunized mice.

MiR-155 influences cell-mediated immunity in Balb/c mice treated with aflatoxin M1

Aflatoxin M1 (AFM1) is a 4-hydroxylated metabolite of aflatoxin B1 (AFB1). It induces various toxicological effects including immunotoxicity. In the present study, we investigated the effects of AFM1 on immune system and its modulation by MicroRNA (miR)-155. AFM1 was administered intraperitoneally at doses of 25 and 50 µg/kg for 28 days to Balb/c mice and different immune system parameters were analyzed. The levels of miR-155 and targeted proteins were evaluated in isolated T cells from spleens of mice. Spleen weight was reduced in mice exposed to AFM1 compared to negative control. Proliferation of splenocytes in response to phytohemagglutinin-A was reduced in mice exposed to AFM1. IFN-γ was decreased in mice exposed to AFM1, whereas IL-10 was increased. Concentration of IL-4 did not change different in mice exposed to AFM1 compared to negative control. Exposure to AFM1 reduced the expression of miR-155. Significant upregulation of phosphatidylinositol-3, 4, 5-trisphosphate 5-phosphatase 1 (Ship1) and suppressor of cytokine signaling 1 (Socs1) was observed in isolated T cells from spleens of mice treated with AFM1, but the transcription factor Maf (c-MAF) was not affected. These results suggest that miR-155 and targeted proteins might be involved in the immunotoxicity observed in mice exposed to AFM1.

Mouse strain and sex as determinants of immune response to trivalent influenza vaccine

AimsThe study examined the influence of sex and mouse strain on germinal center (GC) reaction and antibody responses to seasonal split trivalent influenza vaccine (TIV). Main methodsC57BL/6 and BALB/c mice of both sexes were immunized with TIV and examined for specific antibody response by ELISA. Splenic T follicular regulatory (Tfr), T follicular helper (Tfh) and GC B cells are detected by flow cytometry. The proliferative response of splenocytes, and concentrations of IFN-γ and IL-4 upon restimulation with vaccine antigens were examined by 7-AAD staining and ELISA, respectively. Key findingsBALB/c mice developed more robust IgG responses to vaccine type A antigens than their sex-matched C57BL/6 counterparts, while that to B antigen did not differ between strains. In both strains IgG responses against type A vaccine antigens were greater in females than in males. The greater IgG responses correlated with lower splenic Tfr/Tfh and Tfr/GC B cell ratios and greater vaccine antigen-specific proliferative responses of CD4+ and B cells in splenocyte cultures. In both mouse strains IgG2a(c)/IgG1 ratios were comparable between sexes, but lower in BALB/c than in C57BL/6 mice indicating a shift in Th1/Th2 balance towards Th2 response in BALB/c ones. Consistently, splenocytes from BALB/c mice produced more IL-4 and less IFN-γ than those from C57BL/6 mice. SignificanceThe study indicated that magnitude of humoral response to influenza type A haemagglutinins depends on mouse strain and sex, and thereby set background for the vaccination strategies taking into account biological sex, and in a longterm perspective individual differences in immune reactivity.

Oral recombinant Lactobacillus vaccine targeting the intestinal microfold cells and dendritic cells for delivering the core neutralizing epitope of porcine epidemic diarrhea virus

BackgroundPorcine epidemic diarrhea caused by porcine epidemic diarrhea virus (PEDV) has led to serious economic losses to the swine industry worldwide. In this study, an oral recombinant Lactobacillus casei vaccine against PEDV infection targeting the intestinal microfold (M) cells and dendritic cells (DCs) for delivering the core neutralizing epitope (COE) of PEDV spike protein was developed with M cell-targeting peptide (Col) and dendritic cell-targeting peptide (DCpep). The immunogenicity of the orally administered recombinant strains was evaluated.ResultsAfter immunization, significantly higher levels of anti-PEDV specific IgG antibodies with PEDV neutralizing activity in the sera and mucosal sIgA antibodies in the tractus genitalis, intestinal mucus, and stools were detected in mice orally administered with the recombinant strain pPG-COE-Col-DCpep/L393, which expressed DCpep and Col targeting ligands fused with the PEDV COE antigen, compared to mice orally immunized with the recombinant strain pPG-COE/L393 without the DCpep and Col targeting ligands. Moreover, in response to restimulation with the PEDV COE antigen in vitro, a significant difference in splenocyte proliferation response and Th2-associated cytokine IL-4 level was observed in the group of mice orally immunized with pPG-COE-Col-DCpep/L393 (p < 0.05) compared to the groups of mice that received pPG-COE-Col/L393 and pPG-COE-DCpep/L393, respectively.ConclusionsThe intestinal M cells- and DCs-targeting oral delivery of genetically engineered Lactobacillus expressing the COE antigen of PEDV can efficiently induce anti-PEDV mucosal, humoral, and cellular immune responses via oral administration, suggesting a promising vaccine strategy against PEDV infection.

Oral immunization with a Lactobacillus casei-based anti-porcine epidemic diarrhoea virus (PEDV) vaccine expressing microfold cell-targeting peptide Co1 fused with the COE antigen of PEDV.

The aims of this study were to develop an effective M cell-targeting oral vaccine, involving Lactobacillus casei to deliver the porcine epidemic diarrhoea virus (PEDV) core neutralizing epitope (COE) antigen conjugated with M cell-targeting peptide Co1 as an adjuvant, against PEDV infection. Genetically engineered L. casei 393 (L393) strains expressing PEDV COE antigen only (pPG-COE/L393) or fused-expressing COE and M cell-targeting peptide Co1 (pPG-COE-Co1/L393) were constructed, and the immunogenicity upon administration as an oral vaccine was evaluated. The results showed that higher anti-PEDV serum IgG and mucosal SIgA antibody responses were induced in mice orally immunized with strain pPG-COE-Co1/L393 as compared to the mice immunized with strain L393 expressing COE alone or carrying the empty plasmid. In addition, the use of the Co1 ligand elicited a splenocyte proliferative response more effectively in comparison with the COE antigen alone and supported a skewed T helper 2 type of immune response against PEDV. pPG-COE-Co1/L393 can effectively induce mucosal, humoural and Th2-type cellular immune responses against PEDV infection via oral administration. Furthermore, M cell-targeting peptide ligand Co1 is a good mucosal adjuvant. Lactobacillus casei delivering the COE antigen of PEDV conjugated with a M cell-targeting peptide Co1 as an immune adjuvant is a promising oral vaccine candidate for PEDV.

Farnesyltransferase inhibitor FTI-277 inhibits PD-L1 expression on septic spleen lymphocytes and promotes spleen lymphocyte activation.

Farnesyltransferase inhibitors have been tested in clinical trials for the treatment of tumours. In sepsis, the binding of programmed death 1 (PD-1) to programmed death ligand 1 (PD-L1) promotes lymphocyte apoptosis and decreases cytokine expression, thus affecting survival rates. The PD-1/PD-L1 pathway plays an important role in chronic viral infection, bacterial infection and sepsis. However, the precise immunosuppressive and anti-inflammatory functions of this pathway remain poorly understood. In our previous study, the induction of sepsis by caecal ligation and puncture (CLP) resulted in increased farnesyltransferase activity and farnesylated protein levels in the spleen relative to sham treatment. However, the effect of inhibition of farnesyltransferase activity on overall survival rates in patients with sepsis and the specific signalling pathway involved remain to be investigated. In this study, mice with CLP-induced sepsis were treated with farnesyltransferase inhibitor (FTI-277), and PD-L1 expression on septic spleen lymphocytes was examined. Flow cytometric analysis revealed that PD-L1 is expressed constitutively on lymphocytes and that PD-L1 protein expression was up-regulated strongly following CLP. FTI-277 down-regulated PD-L1 mRNA and protein expression on septic spleen lymphocytes in a dose-dependent manner. This effect was associated closely with nuclear factor kappa B (NF-κB). In addition, the significant damping effect of FTI-277 on the PD-L1 signal promoted interferon (IFN)-γ secretion, interleukin (IL)-2 production and splenocyte proliferation in response to anti-CD3+ CD28+ antibodies in mice. Furthermore, FTI-277 reduced spleen lymphocyte apoptosis in septic mice. Therefore, FTI-277 regulates spleen lymphocyte activity via the PD-L1 signalling pathway, with significant anti-inflammatory effects attributable to suppression of the NF-κB pathway. Farnesyltransferase represents a valuable therapeutic target for the treatment of sepsis.

Brucella lipopolysaccharide reinforced Salmonella delivering Brucella immunogens protects mice against virulent challenge

Intracellular pathogen Salmonella exhibits natural infection broadly analogous to Brucella, this phenomenon makes Salmonella a pragmatic choice for an anti-Brucella vaccine delivery platform. In this study we developed and formulated a combination of four attenuated Salmonella Typhimurium live vector strains delivering heterologous Brucella antigens (rBs), namely lumazine synthase, proline racemase subunit A, lipoprotein outer membrane protein-19, and Cu-Zn superoxide dismutase. With an aim to develop a cross-protecting vaccine, Brucella pan-species conserved rBs were selected. The present study compared the efficacy of smooth and rough variants of Salmonella delivery vector and also evaluated the inclusion of purified Brucella lipopolysaccharide (LPS) in the formulation. Immunization of SPF-BALB/c mice with the vaccine combinations significantly (P≤0.05) reduced splenic wild-type Brucella abortus 544 colonization as compared to non-immunized mice as well as Salmonella only immunized mice. Increased induction of Brucella specific-IgG, sIgA production, and antigen-specific splenocyte proliferative responses were observed in the mice immunized with the formulations as compared to naïve or vector only immunized mice. Modulatory effects of rB and LPS on production of interleukin (IL)-4, IL-12, and interferon-γ were detected in splenocytes of mice immunized with the formulation. Rough Salmonella variant in combination with LPS could further enhance the efficacy of the delivery when applied intraperitoneally. Taken together, it is compelling that Brucella LPS-augmented Salmonella vector delivering immunogenic Brucella proteins may be more suitable than the current non-ideal live Brucella abortus vaccine. The vaccine system also provides a basis for the development of cross-protecting vaccine capable of preventing multispecies brucellosis.

Recombinant \u03b1-actinin subunit antigens of Trichomonas vaginalis as potential vaccine candidates in protecting against trichomoniasis

BackgroundHuman trichomoniasis caused by Trichomonas vaginalis is one of the most common sexually transmitted diseases with more than 200 million cases worldwide. It has caused a series of health problems to patients. For prevention and control of infectious diseases, vaccines are usually considered as one of the most cost-efficient tools. However, until now, work on the development of T. vaginalis vaccines is still mainly focused on the screening of potential immunogens. Alpha-actinin characterized by high immunogenicity in T. vaginalis was suggested as a promising candidate. Therefore, the purpose of this study was to evaluate the protective potency of recombinant α-actinin against T. vaginalis infection in a mouse intraperitoneal model.MethodsTwo selected coding regions of α-actinin (ACT-F, 14–469 aa and ACT-T, 462–844 aa) amplified from cDNA were cloned into pET-32a (+) expression vector and transfected into BL21 cells. After induction with IPTG and purification with electroelution, the two recombinant fusion proteins were emulsified in Freund’s adjuvant (FA) and used to immunize BALB/C mice. Following intraperitoneal inoculation with T. vaginalis, the survival rate of mice was monitored for the assessment of protective potency. After immunization, the antibody level in mouse serum was assessed by ELISA, splenocyte proliferation response was detected with CCK8 and cytokines in the supernatant of splenocytes were quantified with a cytometric bead-based assay.ResultsWe successfully obtained purified ACT-F (70.33 kDa) and ACT-T (61.7kDa). Both recombinant proteins could provide significant protection against T. vaginalis challenge, especially ACT-T (with 100% protection within one month). Meanwhile, high levels of specific total IgG and subtypes (IgG1 > IgG2a) were detected in sera from the immunized mice. Our results also revealed a statistically significant increase in splenocyte proliferation and related cytokine (IFN-γ, IL-6, IL-17A and IL-10) production after repeated stimulation with the corresponding antigens in vitro.ConclusionsImmunization with both ACT-F and ACT-T could confer partial to complete protection and trigger strong Th1/Th2 mixed humoral and cellular immune responses in the mouse host. This suggested that recombinant α-actinin subunit antigens may be promising vaccine candidates against trichomoniasis.

Ethyl Caffeate Ameliorates Collagen-Induced Arthritis by Suppressing Th1 Immune Response.

The present study was designed to assess the antiarthritic potential of ECF in collagen-induced arthritis (CIA) and explore its underlying mechanism. Methods. In vitro, lymphocyte proliferation assay was measured by CCK-8 kit. In vivo, the therapeutic potential of ECF on CIA was investigated; surface marker, Treg cell, and intracellular cytokines (IL-17A and IFN-γ) were detected by flow cytometry. Th1 cell differentiation assay was performed, and mRNA expression in interferon-γ-related signaling was examined by q-PCR analysis. Results. In vitro, ECF markedly inhibited the proliferation of splenocytes in response to ConA and anti-CD3. In vivo, ECF treatment reduced the severity of CIA, inhibited IFN-γ and IL-6 secretion, and decreased the proportion of CD11b+Gr-1+ splenic neutrophil. Meanwhile, ECF treatment significantly inhibited the IFN-γ expression in CD4+T cell without obviously influencing the development of Th17 cells and T regulatory cells. In vitro, ECF suppressed the differentiation of naive CD4+ T cells into Th1. Furthermore, ECF intensely blocked the transcriptional expression in interferon-γ-related signaling, including IFN-γ, T-bet, STAT1, and STAT4. Conclusion. Our results indicated that ECF exerted antiarthritic potential in collagen-induced arthritis by suppressing Th1 immune response and interferon-γ-related signaling.

Splenic lymphocyte immune response induced by intranasal immunization with recombinant Toxoplasma gondii profilin in mice

To observe the splenocytes immune response elicited by different concentrations of recombinant Toxoplasma gondii profiling (rTgPRF) through the nasal route, and determine the optimal dose. Fifty female BALB/c mice were randomly divided into 5 groups. The immunized groups were intranasally administered with 10, 20, 30 μg or 40 μg of rTgPRF that was separately dissolved in 20 μl of phosphate-buffered saline (PBS) on days 0, 14, and 21 respectively, while the control mice were given PBS solution instead. Two weeks after the last immunization, all mice were killed. Under asceptic conditions, the spleens from the immunized mice were dissected, and then the splenocyte proliferative responses in vitro were tested by CCK-8 kit. The levels of IFN-γ, IL-2, IL-4 and IL-10 of splenocyte culture supernatant were detected by ELISA. Compared to the control group, the splenocytes from the 30 μg and 40 μg groups exhibited a significantly higher proliferative response to rTgPRF (P < 0.05), and SI from the 30 μg rTgPRF group was higher than that from the 40 μg group (P < 0.05). The levels of IFN-γ in all the immunized groups (P < 0.05) and IL-2 in the 20, 30 μg and 40 μg groups were significantly stronger than those in the control (P < 0.05), and the 30 μg group presented the highest concentrations of IFN-γ (P < 0.01) and IL-2 (P < 0.01). There were no statistical differences among the groups in the levels of IL-4 and IL-10. The intranasal immunization with rTgPRF can induce the splenocyteproliferation and Th1-type mediated immunity. The best immunized dose is confirmed as 30 μg.

Cinobufagin enhances the protective efficacy of formalin-inactivated Salmonella typhimurium vaccine through Th1 immune response

Cinobufagin (CBG), one active ingredient isolated from Venenum Bufonis, has been demonstrated to have immunoregulatory effect. The aim of this study was to investigate whether CBG can enhance the protective efficacy of formalin-inactivated Salmonella typhimurium (FIST) in mice. ICR mice were immunized with FIST (106 CFU/mouse) alone or mixed with CBG (10, 20, and 40 μg) or alum (200 μg) on day 1 and day 15. Two weeks after the second immunization, serum and spleen were sampled for measuring FIST-specific antibody levels, cytokine levels, and splenocyte proliferation. The results showed that CBG enhanced FIST-specific IgG and IgG2a, the levels of interferon-gamma (IFNγ) and nitric oxide (NO), and the splenocyte proliferation response induced by concanavalin A, lipopolysaccharide, and FIST. In vivo protection studies showed that CBG significantly decreased the bacterial burdens in the spleen and prolonged the survival time of FIST-immunized mice challenged with live Salmonella typhimurium. In vivo IFNγ neutralization led to a significant reduction in FIST-specific IgG2a and IFNγ levels, and in the protective efficacy in CBG/FIST-immunized mice. In conclusion, CBG enhances the protective efficacy of formalin-inactivated Salmonella typhimurium vaccine by promoting the Th1 immune response.