Th1-type Response Research Articles

A novel vaccine adjuvant based on straight polyacrylate potentiates vaccine-induced humoral and cellular immunity in cynomolgus macaques

ABSTRACT Adjuvants are central to the efficacy of subunit vaccines. Although several new adjuvants have been approved in human vaccines over the last decade, the panel of adjuvants in licensed human vaccines remains small. There is still a need for novel adjuvants that can be safely used in humans, easy to source and to formulate with a wide range of antigens and would be broadly applicable to a wide range of vaccines. In this article, using the Respiratory Syncytial Virus (RSV) nanoparticulate prefusion F model antigen developed by Sanofi, we demonstrate in the macaque model that the polyacrylate (PAA)-based adjuvant SPA09 is well tolerated and increases vaccine antigen-specific humoral immunity (sustained neutralizing antibodies, memory B cells and mucosal immunity) and elicits strong TH1-type responses (based on IFNγ and IL-2 ELISpots) in a dose-dependent manner. These data warrant further development of the SPA09 adjuvant for evaluation in clinical trials.

Dermal immune responses against Psoroptes ovis in two cattle breeds and effects of anti-inflammatory dexamethasone treatment on the development of psoroptic mange

Psoroptic mange is a common disease of livestock, caused by Psoroptes ovis. Compared to Holstein–Friesian (HF) cattle, the Belgian Blue (BB) cattle breed is highly susceptible to the infestation. However, the mechanism for this difference is still unclear. To determine the factors responsible for this breed susceptibility, the immune response to P. ovis was studied in experimentally infested BB and HF cattle, using clinical signs, histology, immunohistochemical profiling and gene expression analysis of skin biopsies. The mite numbers and lesion area of BB cattle were greater than in HF during the whole study period. Significant influxes of eosinophils in the epidermis and dermis were detected in comparison with the pre-infestation samples in both breeds, with significantly higher eosinophils in BB at 6 weeks post infestation (wpi). Mast cell numbers were unaffected at all stages of infestation in HF, but were significantly elevated relative to pre-infestation in BB cattle at 2 and 6 wpi. The more pronounced cutaneous eosinophilia and higher IL-4 levels at 6 wpi in BB cattle suggest that a Th2-type immune response is underlying the higher susceptibility of the BB breed. In naturally infested BB cattle, development of the psoroptic mange lesions and eosinophils and CD3+ T cell areas were severely depressed after anti-inflammatory treatment with dexamethasone. Together, these results suggest that a stronger Th2-type immune response to P. ovis causes the skin lesions in psoroptic mange in BB cattle and that local anti-inflammatory treatment could potentially be an alternative to control the pathology caused by this parasite.

Potential mechanisms of tumor progression associated with postoperative infectious complications.

There is increasing evidence that postoperative infectious complications (PICs) are associated with poor prognosis after potentially curative surgery. However, the role that PICs play in tumor development remains unclear. In this article, we reviewed the literature for novel insights on the mechanisms of cancer progression associated with PICs. The Medline and EMBASE databases were searched for publications regarding the role of suppression of antitumor immunity by PIC in tumor progression and selected 916 manuscripts were selected for this review. In addition, a summary of the authors' own experimental data from this field was set in the context of current knowledge regarding cancer progression under septic conditions. Initially, sepsis/microbial infection dramatically activates the systemic immune system with increases in pro-inflammatory mediators, which results in the development of systemic inflammatory response syndrome; however, when sepsis persists in septic patients, a shift toward an anti-inflammatory immunosuppressive state, characterized by macrophage deactivation, reduced antigen presentation, T cell anergy, and a shift in the T helper cell pattern to a predominantly TH2-type response, occurs. Thus, various cytokine reactions and the immune status dynamically change during microbial infection, including PIC. We proposed three possible mechanisms for the tumor progression associated with PIC: first, a mechanism in which microbes and/or microbial PAMPs may be directly involved in cancer growth; second, a mechanism in which factors released from immunocompetent cells during infections may affect tumor progression; and third, a mechanism in which factors suppress host tumor immunity during infections, which may result in tumor progression. A more detailed understanding by surgeons of the immunological features in cancer patients with PIC can subsequently open new avenues for improving unfavorable long-term oncological outcomes associated with PICs.

Indoleamine 2,3-Dioxygenase in Hematopoietic Stem Cell-Derived Cells Suppresses Rhinovirus-Induced Neutrophilic Airway Inflammation by Regulating Th1- and Th17-Type Responses

Asthma exacerbations are a major cause of intractable morbidity, increases in health care costs, and a greater progressive loss of lung function. Asthma exacerbations are most commonly triggered by respiratory viral infections, particularly with human rhinovirus (hRV). Respiratory viral infections are believed to affect the expression of indoleamine 2,3-dioxygenase (IDO), a limiting enzyme in tryptophan catabolism, which is presumed to alter asthmatic airway inflammation. Here, we explored the detailed role of IDO in the progression of asthma exacerbations using a mouse model for asthma exacerbation caused by hRV infection. Our results reveal that IDO is required to prevent neutrophilic inflammation in the course of asthma exacerbation caused by an hRV infection, as corroborated by markedly enhanced Th17- and Th1-type neutrophilia in the airways of IDO-deficient mice. This neutrophilia was closely associated with disrupted expression of tight junctions and enhanced expression of inflammasome-related molecules and mucin-inducing genes. In addition, IDO ablation enhanced allergen-specific Th17- and Th1-biased CD4+ T-cell responses following hRV infection. The role of IDO in attenuating Th17- and Th1-type neutrophilic airway inflammation became more apparent in chronic asthma exacerbations after repeated allergen exposures and hRV infections. Furthermore, IDO enzymatic induction in leukocytes derived from the hematopoietic stem cell (HSC) lineage appeared to play a dominant role in attenuating Th17- and Th1-type neutrophilic inflammation in the airway following hRV infection. Therefore, IDO activity in HSC-derived leukocytes is required to regulate Th17- and Th1-type neutrophilic inflammation in the airway during asthma exacerbations caused by hRV infections.

Flow Cytometric Evaluation of CD4+ and CD8+ T-cell Immune Response in SPF Chickens Induced by Fowlpox Vaccine.

Fowlpox (FP) is a viral disease that is widely distributed throughout the world. The disease has an economic impact on the poultry industry, and its prevalence has even been reported in vaccinated flocks. The present study used flow cytometry to evaluate the CD4+ and CD8+ T-cell immune response of chicks induced by FP vaccine. 120 specific pathogen-free (SPF) 21-day-old chicks were randomly divided into three groups of 40. One group was used as negative control with PBS inoculation, the other two groups were inoculated with the local fowlpox vaccine produced by Razi Institute and commercial FP vaccines, and they were kept for five weeks. Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll-Hypaque density gradients and the percentages of CD3+, CD3+, CD4+, and CD3+CD8+ T lymphocytes were analyzed with flow cytometry. Seven days post-immunization, a maximum (90-100%) swelling formation ("take") on the vaccination site was observed. The ratios of CD4+ to CD8+ T-lymphocytes in both vaccinated groups were significantly higher (p < 0.05) than the control group inoculated with PBS. The percentages of CD3+, CD3+CD4+, and CD3+CD8+ T-lymphocytes were increased in chickens vaccinated with commercial and local FP vaccines. There were no significant differences between the groups receiving commercial and local fowl pox vaccines. The present study showed that protective immunity could be associated with increased cellular immune responses, which has been interpreted as enhancing T-cell proliferation and increasing CD4+ to CD8+ ratios through vaccination with the FP vaccine. This study further suggests that the induction of enhanced immune responses is due mainly to the Th1-type response.

The role of sphingosine 1-phosphate receptor 2 on interleukin (IL)-4/IL-13-induced STAT6 phosphorylation in phorbol 12-myristate 13-acetate-stimulated THP-1 cells&lt;i&gt; &lt;/i&gt;

Previously we found that in bleomycin-administered sphingosine 1-phosphate receptor 2 (S1P2) knockout mice, phosphorylation of STAT6, a transcription factor which is activated downstream of interleukin (IL)-4/IL-13, in broncho-alveolar lavage fluids cells was diminished compared with wild-type mice (PLoS One, 2018). In this study, we examined the role of S1P2 on STAT6 activation using THP-1 cells differentiated with phorbol 12-myristate 13-acetate (PMA) as a model for macrophages (MΦs). Quantitative PCR analysis showed that THP-1 cells expresses S1P2. We examined the responses of PMA-treated THP-1 MΦs to chronic stimulation with IL-4/IL-13 in the presence of S1P. IL-4/IL-13 stimulation induced a strong increase in STAT6 phosphorylation in PMA-treated THP-1 MΦs. The major signaling pathway of S1P2 is a Rho–Rho kinase pathway. Pretreatment of PMA-treated THP-1 MΦs with the S1P2 antagonist JTE-013 and the Rho kinase inhibitor Y27632 inhibited IL-4/IL-13–induced increase in STAT6 phosphorylation. We established S1P2knockout THP-1 cells using CRISPR-Cas9 gene editing system. PMA-treated S1P2knockout THP-1 MΦs showed a lower extent of STAT6 phosphorylation in response to IL-4/IL-13 stimulation than PMA-treated wild-type THP-1 MΦs. S1P2 knockout markedly attenuated IL-4/IL-13-induced increase in the mRNA expression of the M2 marker arginase 1. These results suggested that S1P2/Rho kinase pathway is necessary for full activation of STAT6 by IL-4/IL-13 in MΦs. Recently, Th2-type cytokines IL-4/IL-13 have been reported to be important in the pathogenesis of allergic diseases such as bronchial asthma and atopic dermatitis. It is expected that S1P2 antagonist and Rho kinase inhibitor inhibit Th2-type immune response by IL-4/IL-13, thereby suppressing allergic diseases.

Efficient Immunization of BALB/c Mice against Pathogenic Brucella melitensis and B. ovis: Comparing Cell-Mediated and Protective Immune Responses Elicited by pCDNA3.1 and pVAX1 DNA Vaccines Coding for Omp31 of Brucella melitensis.

Background: Brucella spp. are intracellular pathogens, therefore cell-mediated immunity is the main response to inhibit survival and growth of the bacteria in vertebrate host.Objective: Many eukaryotic plasmid vectors are being used in setting up DNA vaccines which may show different efficiencies in same conditions. This is important in designing the vaccines and immunization strategies. We looked into the probable differences of immune responses induced by different eukaryotic DNA plasmid vectors (pcDNA3.1 and pVAX1) harboring the same Omp31 gene of B. melitensis.Materials and Methods: Female BALB/c mice were immunized with pcDNA-omp31 and pVAX-omp31 and further boosted with recombinant Omp31. Subclasses of specific serum IgG against the rOmp31 were measured by ELISA. Cytokines responses to rOmp31 in Splenocyte cultures of the immunized mice were evaluated by measuring the production of IL-4, IL-10, IL-12 and IFN-γ. Protective responses of the immunized mice were evaluated by intraperitoneal challenge with pathogenic Brucella melitensis 16M and Brucella ovis PA76250.Results: Both DNA vaccine candidates conferred potent Th1-type responses with higher levels of cytokines and immunoglobulins observed in mice immunized with pVAX-omp31. Although pcDNA-omp31 and pVAX-omp31 both elicited protective immunity, mice immunized with the latter showed a higher protection against both B. melitensis and B. ovis PA76250.Conclusion: The results of this study highlight the significant differences between efficiency of diverse plasmid backbones in DNA vaccines which code for an identical antigen. Comparing various plasmid vectors should be considered as an essential part of the studies aiming construction of DNA vaccines for intracellular pathogens.

TSLP Produced by Aspergillus fumigatus-Stimulated DCs Promotes a Th17 Response Through the JAK/STAT Signaling Pathway in Fungal Keratitis.

PurposeThe purpose of this study was to explore the role of thymic stromal lymphopoietin (TSLP) secreted by Aspergillus fumigatus–stimulated dendritic cells (DCs) during the T helper 17 (Th17) immune response, and further clarify the mechanisms contributing to the Th17 immune response of fungal keratitis (FK).MethodsA carboxyfluorescein diacetate succinimidyl ester assay, PCR, and flow cytometry were performed to detect Th17 differentiation of CD4+ T cells; PCR, ELISA, and Western blot were used to detect the expression of TSLP and JAK/STAT–related proteins; Signaling pathways involved in Th17 response was evaluated using RNA sequence; C57BL/6 mice were infected with A. fumigatus and treated with ruxolitinib or BBI608. Slit-lamp examination, fluorescein staining, and clinical scores were used to assess the clinical manifestation.ResultsA. fumigatus–infected DCs could drive naïve CD4+ T-cell proliferation and promote the production of Th17 cytokines IL-17A, IL-17F, and IL-22. A. fumigatus stimulation increased the expression of TSLP in DCs. DC-derived TSLP contributed to a Th17-type inflammatory response via the JAK/STAT signaling pathway. TSLP small interfering RNA, TSLPR small interfering RNA, or JAK/STAT inhibitors inhibited the Th17 immune response induced by A. fumigatus–infected DCs. Moreover, TSLP promoted A. fumigatus keratitis disease progression in a mouse model. However, inhibition of the JAK/STAT signaling pathway using a specific inhibitor reversed the development of FK by A. fumigatus infection.ConclusionsTSLP secreted by A. fumigatus–stimulated DCs played a significant role in the Th17-dominant immune response of FK through its JAK/STAT activation. Our findings may contribute to the elucidation of the molecular mechanisms of FK and to the development of novel therapeutic approaches.

IL-27 regulates IL-4-induced chemokine production in human bronchial epithelial cells

IL-4 coordinates the Th2-type immune response in inflammatory diseases such as asthma. IL-27 can inhibit the development of both Th2 and Th1 cells. However, IL-27 can also drive naïve T cells to differentiate toward the Th1 phenotype. In this study, we investigated the effects of IL-27 on the activation of IL-4-induced human bronchial epithelial cells (BEAS-2B). Compared to controls, both IL-4 and IL-27 (25–100 ng/mL) increased the concentrations of CCL2 and IL-8 in a dose-dependent manner. However, compared to cells stimulated individually with IL-4 or IL-27, treatment with a combination of both cytokines reduced CCL2 and IL-8 concentrations in a dose- and time-dependent manner. IL-4 increased the activation of p38 MAPK, ERK1/2, STAT6 and NF-κB, while IL-27 increased the activation of p38 MAPK and ERK1/2 but not STAT6 and NF-κB. Compared to IL-4-stimulated cells, cells treated with both IL-27 and IL-4 displayed decreased activation of STAT6 and NF-κB but not ERK1/2 and p38 MAPK. Taken together, these results suggest that IL-27 plays a pro-inflammatory role when administered alone but downregulates bronchial epithelial cell activation when combined with IL-4. Therefore, IL-27 may be an interesting target for the treatment of Th2 inflammatory diseases.

Oral administration of Hsp65-producing Lactococcus lactis attenuates allergic asthma in a murine model.

Allergic asthma is a chronic inflammatory lung disease characterized by a Th2-type immune response pattern. The development of nonspecific immunotherapy is one of the primary goals for the control of this disease. In this study, we evaluated the therapeutic effects of Lactococcus lactis-producing mycobacterial heat shock protein 65 (LLHsp65) in an ovalbumin (OVA)-induced allergic asthma model. OVA-challenged BALB/c mice were orally administrated with LLHsp65 for 10 consecutive days. The results demonstrate that LLhsp65 attenuates critical features of allergic inflammation, like airway hyperresponsiveness and mucus production. Likewise, the treatment decreases the pulmonary eosinophilia and the serum level of OVA-specific IgE. In addition to deviating immune responses towards Th1-cytokine profile, increase regulatory T cells, and cytokine levels, such as IL-6 and IL-10. Our results reveal that the mucosal immunotherapy of LLHsp65 significantly reduces the overall burden of airway allergic inflammation, suggesting a promising therapeutic strategy for allergic asthma treatment. This research reveals new perspectives on nonspecific immunotherapy based on the delivery of recombinant proteins by lactic acid bacteria to treat of allergic disorders.

Digitoxigenin presents an effective and selective antileishmanial action against Leishmania infantum and is a potential therapeutic agent for visceral leishmaniasis.

Treatment for visceral leishmaniasis (VL) is hampered mainly by drug toxicity, their high cost, and parasite resistance. Drug development is a long and pricey process, and therefore, drug repositioning may be an alternative worth pursuing. Cardenolides are used to treat cardiac diseases, especially those obtained from Digitalis species. In the present study, cardenolide digitoxigenin (DIGI) obtained from a methanolic extract of Digitalis lanata leaves was tested for its antileishmanial activity against Leishmania infantum species. Results showed that 50% Leishmania and murine macrophage inhibitory concentrations (IC50 and CC50, respectively) were of 6.9 ± 1.5 and 295.3 ± 14.5 μg/mL, respectively. With amphotericin B (AmpB) deoxycholate, used as a control drug, values of 0.13 ± 0.02 and 0.79 ± 0.12 μg/mL, respectively, were observed. Selectivity index (SI) values were of 42.8 and 6.1 for DIGI and AmpB, respectively. Preliminary studies suggested that the mechanism of action for DIGI is to cause alterations in the mitochondrial membrane potential, to increase the levels of reactive oxygen species and induce accumulation of lipid bodies in the parasites. DIGI was incorporated into Pluronic® F127-based polymeric micelles, and the formula (DIGI/Mic) was used to treat L. infantum–infected mice. Miltefosine was used as a control drug. Results showed that animals treated with either miltefosine, DIGI, or DIGI/Mic presented significant reductions in the parasite load in their spleens, livers, bone marrows, and draining lymph nodes, as well as the development of a specific Th1-type response, when compared with the controls. Results obtained 1 day after treatment were corroborated with data corresponding to 15 days after therapy. Importantly, treatment with DIGI/Mic induced better parasitological and immunological responses when compared with miltefosine- and DIGI-treated mice. In conclusion, DIGI/Mic has the potential to be used as a therapeutic agent to protect against L. infantum infection, and it is therefore worth of consideration in future studies addressing VL treatment.

A murine model of eosinophilic chronic rhinosinusitis using the topical application of a vitamin D3 analog.

Eosinophilic chronic rhinosinusitis (ECRS) is a chronic inflammatory disease, characterized by eosinophilic infiltration, T-helper type 2 (Th2-type) response, and olfactory dysfunction. A master regulator of Th2-type inflammation, thymic stromal lymphopoietin (TSLP), is important for basophil activation. TSLP-elicited basophils are a key factor in the pathogenesis of ECRS. In order to elucidate the mechanisms of ECRS in humans, we aimed to establish a murine model of ECRS based on TSLP production in response to the topical application of MC903 (a vitamin D3 analog) and the subsequent TSLP-induced basophil activation. Histological analyses were performed to assess immune cell infiltration into the nasal mucosa and to explore the impact of eosinophilic inflammation on the olfactory epithelium. The status of Th2-type inflammation was evaluated by quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA). Eosinophils, basophils, and M2 macrophages increased significantly in the nasal mucosa of the mice treated with MC903 and ovalbumin (OVA), compared to those treated with OVA alone or the controls. Quantitative real-time PCR and ELISA revealed elevated expression of interleukin (IL)-4, IL-5, IL-13, TSLP, the chemokine CCL11, and CCL24 in the nasal mucosa of the ECRS mice. In parallel, thinned olfactory epithelium and decreased mature olfactory sensory neurons were observed in the ECRS mice. Our model of ECRS displayed Th2-type inflammation in the sinonasal region, including both eosinophil infiltration and basophil infiltration. Additionally, olfactory epithelium turned out to be affected by eosinophilic inflammation. These features are consistent with the characteristics of the human ECRS.

LmjF.22.0810 from Leishmania major Modulates the Th2-Type Immune Response and Is Involved in Leishmaniasis Outcome.

A novel serine/threonine protein kinase, LmjF.22.0810, was recently described in Leishmania major. After generating an L. major cell line overexpressing LmjF.22.0810 (named LmJ3OE), the ability of this novel protein to modulate the Th2-type immune response was analyzed. Our results suggest that the protein kinase LmjF.22.0810 might be involved in leishmaniasis outcomes. Indeed, our study outlined the LmJ3OE parasites infectivity in vitro and in vivo. Transgenic parasites displayed lower phagocytosis rates in vitro, and their promastigote forms exhibited lower expression levels of virulence factors compared to their counterparts in control parasites. In addition, LmJ3OE parasites developed significantly smaller footpad swelling in susceptible BALB/c mice. Hematoxylin–eosin staining allowed the observation of a lower inflammatory infiltrate in the footpad from LmJ3OE-infected mice compared to animals inoculated with control parasites. Gene expression of Th2-associated cytokines and effectors revealed a dramatically lower induction in interleukin (IL)-4, IL-10, and arginase 1 (ARG1) mRNA levels at the beginning of the swelling; no expression change was found in Th1-associated cytokines except for IL-12. Accordingly, such results were validated by immunohistochemistry studies, illustrating a weaker expression of ARG1 and a similar induction for inducible NO synthase (iNOS) in footpads from LmJ3OE-infected mice compared to control L. major infected animals. Furthermore, the parasite burden was lower in footpads from LmJ3OE-infected mice. Our analysis indicated that such significant smaller footpad swellings might be due to an impairment of the Th2 immune response that subsequently benefits Th1 prevalence. Altogether, these studies depict LmjF.22.0810 as a potential modulator of host immune responses to Leishmania. Finally, this promising target might be involved in the modulation of infection outcome.

IL-4Rα signaling in CD4+CD25+FoxP3+ T regulatory cells restrains airway inflammation via limiting local tissue IL-33.

Impaired tolerance to innocuous particles during allergic asthma has been linked to increased plasticity of FoxP3+ regulatory T cells (Tregs) reprogramming into pathogenic effector cells, thus exacerbating airway disease. However, failure of tolerance mechanisms is driven by Th2 inflammatory signals. Therefore, the in vivo role of canonical IL-4 receptor α (IL-4Rα) signaling, an essential driver of Th2-type airway responses to allergens, on the regulatory function of FoxP3+ Tregs in allergic asthma was explored. Here, we used transgenic Foxp3cre IL-4Rα–/lox and littermate control mice to investigate the role of IL-4 and IL-13 signaling via Tregs in house dust mite–induced (HDM-induced) allergic airway disease. We sensitized mice intratracheally on day 0, challenged them on days 6–10, and analyzed airway hyperresponsiveness (AHR), airway inflammation, mucus production, and cellular profile on day 14. In the absence of IL-4Rα responsiveness on FoxP3+ Tregs, exacerbated AHR and airway inflammation were shown in HDM-sensitized mice. Interestingly, reduced induction of FoxP3+ Tregs accompanied increased IL-33 alarmin production and type 2 innate lymphoid cell activation in the lung, exacerbating airway hyperreactivity and lung eosinophilia. Taken together, our findings indicate that IL-4Rα–unresponsive FoxP3+ Tregs result in exaggerated innate Th2-type, IL-33–dependent airway inflammation and a break in tolerance during allergic asthma.

Impact de la grossesse sur le psoriasis.

During pregnancy there is spontaneous improvement in various inflammatory diseases, including psoriasis for the majority of women. Hormonal changes during pregnancy may play a role in improving psoriasis by promoting a state of transient immune tolerance, promoting a TH2-type response, and lowering the inflammatory cascades of psoriasis. It is more the ratio of estrogens to progesterone than the respective relative levels of the circulating hormones themselves that may improve psoriasis. There is a risk of psoriasis flare in the first few months postpartum. Breastfeeding seems to have less influence on the natural history of skin lesions.

Chronic Stress Physically Spares But Functionally Impairs Innate-Like Invariant T Cells

Long-term stress disrupts the neuroimmune interface, often resulting in immunosuppression. The deleterious effects of stress on mainstream T cells are well-documented. However, innate-like invariant T cells have been overlooked. Chief among these are invariant natural killer T (iNKT) and mucosa-associated invariant T (MAIT) cells, which fulfill important functions in anticancer and antimicrobial immunity. We found stress to surprisingly abrogate both TH1- and TH2-type responses mediated by iNKT cells. This was not due to cell death since iNKT cells were unusually refractory to stress-inflicted apoptosis. Activated iNKT cells in stressed mice triggered an abnormal response characterized by a ‘split’ inflammatory signature along with a sudden and robust induction of IL-10, IL-23 and IL-27. Pharmacological inhibitors, conditional knockout systems and gene expression analyses revealed that dysregulated iNKT cell responses were driven by iNKT cell-intrinsic glucocorticoid receptor signaling. Accordingly, the inhibitory effects of chronic stress on iNKT cell functions were lost upon habituation to predictable stressors. Importantly, iNKT cells in stressed mice failed to potentiate cytotoxicity against lymphoma targets or promote immune surveillance against metastatic melanoma. Using a congenic mouse model in which MAIT cells are abundant, these cells remained resistant to stress-induced death but demonstrated hindered TH1- and TH2-type cytokine responses, reminiscent of their iNKT cell counterparts. This was corroborated in culture systems in which we interrogated human peripheral blood and hepatic iNKT and MAIT cells. Our findings uncover a previously unappreciated mechanism of stress-induced immunosuppression and implicate the stress response as a potential barrier to iNKT cell- and MAIT cell-based immunotherapies.

Protein expression profile of Taenia crassiceps cysticerci related to Th1- and Th2-type responses in the mouse cysticercosis model

The intraperitoneal cysticercosis model with the Taenia crassiceps ORF strain in female BALB/cAnN mice has been widely used to study the immune response in cysticercosis. During early infection (2 weeks), the host develops a non-permissive Th1 response, whereas during late infection (8 weeks), molecules from the cysticerci induce a Th2 response that is permissive to parasite growth. The modulation of the Th2 response is induced by molecules excreted/secreted by the larval stage of the parasite. However, there is limited information regarding the response of cysticerci to the mouse immunological environment during infection. The proteomic profiles in T. crassiceps ORF cysticerci when faced with the mouse Th1 and Th2 responses were analyzed through two-dimensional gel electrophoresis (2DE), and the differential expression of proteins was evaluated. Thirteen proteins, whose differential expression varied between 70% and 100%, were selected randomly. Protein identification by MALDI-TOF MS and BLAST showed that the proteins were related to folding, signaling, enzymatic activities, cell-movement regulation, cell-cell interactions, motility, carbohydrate metabolism, detoxification, and redox regulation processes. Notably, some of the proteins can act as antigenic-protective molecules and elicit a weak Th1 response; however, most are involved in the avoidance of the immune system, which leads to a Th2 response, or apoptosis. The findings indicate the process by which T. crassiceps cysticerci responds based on the host environment and provides novel insights into the mechanism by which this facilitates its establishment and persistence in the mouse. Furthermore, these proteins could be used as targets for drug and vaccine development.

Leishmania infantum transfected with toxic plasmid induces protection in mice infected with wild type L. infantum or L. amazonensis

Leishmania infantum infection may cause visceral leishmaniasis (VL), a fatal disease having worldwide distribution, that may be silent or asymptomatic. The latter indicates that immunity is naturally developed in some individuals, and, therefore, a vaccine against VL would be possible. Molecular mechanisms of gene expression are being understood in Leishmania, and this knowledge may be useful for vaccine development. The aim of this study was developing an attenuated strain by regulating the expression of toxic proteins in a stage specific manner. For that purpose, the 3’ UTR of an amastin gene, known by its increased expression in the amastigote phase, was selected for direct the expression of exogenous proteins. This construct (pFL-AMA), firstly, was proved effective for the expression of mCherry specifically in the intracellular form of L. infantum, as demonstrated by fluorescence microscopy, flow cytometry and Western blotting. Afterwards, mCherry coding sequence was replaced, in the pFL-AMA plasmid, by either egg avidin or the active form of bovine trypsin. Viability of transfected parasites was evaluated in promastigote axenic cultures and in in vitro infection of macrophages. Both lines of transfected parasites showed a limited capacity to multiply inside macrophages. BALB/c mice were inoculated intraperitoneally (i.p.) with a single dose consisting of 2 × 106L. infantum promastigotes transfected with plasmids bearing the toxic genes. After 10 weeks post-inoculation, no parasites were recovered by limiting dilution in either liver or spleen, but a specific immunological response was detected. The immunization with transfected parasites induced cellular and humoral immune responses with activation of TCD4+, TCD8+ and B cells, having a TH1-type response with increased levels of pro-inflammatory cytokines such as IFN-γ, TNF-α and IL-6. In parallel groups of mice, a challenge consisting on 1 × 106 virulent parasites of either L. infantum (inoculated i.p.) or L. amazonensis subcutaneously (s.c.) was performed. Vaccinated mice, challenged with L. infantum, showed lower parasite burdens in liver, spleen and bone marrow than infected mice with WT L. infantum (non-vaccinated); similarly, vaccinated mice developed smaller footpad inflammation than control group. These data support this strategy as an efficient immunization system aimed to the development of vaccines against different forms of leishmaniasis.

Purified Splenic amastigotes of Leishmania donovani-Immunoproteomic approach for exploring Th1 stimulatory polyproteins.

Visceral leishmaniasis (VL) represents one of the most challenging infectious diseases worldwide. The reason that once infected, patient develops immunity against Leishmania parasite has paved way to develop prophylacticvaccinesagainst disease, but only some of these have moved ahead for clinical trials. Herein, the study to explore novel and potential vaccine candidates was extended to pathogenic form of parasite, that is, amastigote form which is less explored due to complexity of its purification process. Methods and results. Classical protocol of purification of splenic amastigotes was modified to obtain highly pure amastigotes which was confirmed by Western blotting in support with proteomics studies. Fractionation and sub-fractionation of purified splenic amastigotes revealed four sub-fractions, belonging to 97 to 68kDa and 68 to 43kDa ranges, which showed long-lasting protection with remarkable Th1-type cellular responses in hamsters vaccinated with these sub-fractions (LTT, NO, QRT-PCR). Further proteomics analysis, to identify and understand the precise nature and function of these protective protein sub-fractions, identified a total of 47 proteins including twenty-five hypothetical proteins/unknowns. Amastigote stage has potential Th1-stimulatory vaccine candidates, notably, among identified proteins, major were uncharacterized proteins/hypothetical proteins, which once characterized may serve as novel and potential vaccine candidates/drug targets.

1537P Phase I/II study with CXCL12 inhibitor NOX-A12 and pembrolizumab in patients with microsatellite-stable, metastatic colorectal or pancreatic cancer

NOX-A12 is a CXCL12 inhibitor that prevents activation of receptors CXCR4 and CXCR7 and blocks formation of CXCL12 concentration gradients. The Opera study is a phase I/II study to investigate pharmacodynamics of NOX-A12 monotherapy and safety and efficacy of a combination with pembrolizumab in metastatic MSS colorectal (CRC) and pancreatic (PaC) cancer where PD-1 inhibition alone has no clinical benefit. Patients received NOX-A12 twice weekly for 2 weeks. Biopsies were taken from liver metastases before and after treatment to analyze immune cells and cytokines. Subsequently, patients received repeated 21-day cycles of NOX-A12 and pembrolizumab. 11 patients had CRC and 9 PaC. All patients were heavily pretreated with 5 (CRC) and 3 mean lines (PaC) of prior treatment. Best response to last prior treatment was progressive disease in 95% of cases. The AE profile was comparable with the pembrolizumab profile and typical for the underlying diseases. 2 weeks of NOX-A12 monotherapy induced changes in the tumor microenvironment: an upregulated cassette of Th1 cytokines in approx. 50% of patients, being associated with better clinical stabilization. Biopsy analysis before and after NOX-A12 monotherapy showed agglomeration of T cells and migration of T cells towards cancer cells in patients where NOX-A12 induced a Th1-type response. These trends suggest that CXCL12 inhibition increased effector immune cell infiltration of the tumor, resulting in a more effective immune response. 25% of patients achieved stable disease (SD) and long term disease control. Remarkably, 7 patients showed prolonged time on treatment vs. prior therapy. Median PFS was 1.87 months, OS was 42% at 6 and 22% at 12 months. Agglomeration of T cells within tumors was seen in ∼half of the patients where NOX-A12 had induced a Th1-type cytokine response. This was accompanied by reduced distances between T and cancer cells, i.e. more engaged T cells. Increased infiltration of effector immune cells into tumor was associated with SD in 25% of patients and prolonged time on treatment vs. prior therapy for 35% of pts. Safety profile of the combination was consistent with that of pembrolizumab in advanced cancer patients.