Sublingual allergen immunotherapy prevents house dust mite inhalant type 2 immunity through dendritic cell-mediated induction of Foxp3+ regulatory T cells.

Sublingual allergen immunotherapy (SLIT) is an emerging treatment option for allergic asthma and a potential disease-modifying strategy for asthma prevention. The key cellular events leading to such long-term tolerance remain to be fully elucidated. We administered prophylactic SLIT in a mouse model of house dust mite (HDM)-driven allergic asthma. HDM extract was sublingually administered over 3 weeks followed by intratracheal sensitization and intranasal challenges with HDM. Prophylactic SLIT prevented allergic airway inflammation and hyperreactivity with a low lab-to-lab variation. The HDM-specific T helper (Th)2 (cluster of differentiation 4 Th) response was shifted by SLIT toward a regulatory and Th17 response in the lung and mediastinal lymph node. By using Derp1-specific cluster of differentiation 4+ T cells (1-DER), we found that SLIT blocked 1-DER T cell recruitment to the mediastinal lymph node and dampened IL-4 secretion following intratracheal HDM sensitization. Sublingually administered Derp1 protein activated 1-DER T cells in the cervical lymph node via chemokine receptor7+ migratory dendritic cells (DC). DCs migrating from the oral submucosa to the cervical lymph node after SLIT-induced Foxp3+ regulatory T cells. When mice were sensitized with HDM, prior prophylactic SLIT increased Derp1 specific regulatory T cells (Tregs) and lowered Th2 recruitment in the lung. By using Foxp3-diphtheria toxin receptor mice, Tregs were found to contribute to the immunoregulatory prophylactic effect of SLIT on type 2 immunity. These findings in a mouse model suggest that DC-mediated functional Treg induction in oral mucosa draining lymph nodes is one of the driving mechanisms behind the disease-modifying effect of prophylactic SLIT.

Type 2-polarized memory B cells hold allergen-specific IgE memory.

Allergen-specific immunoglobulin E (IgE) antibodies mediate pathology in diseases such as allergic rhinitis and food allergy. Memory B cells (MBCs) contribute to circulating IgE by regenerating IgE-producing plasma cells upon allergen encounter. Here, we report a population of type 2-polarized MBCs defined as CD23hi, IL-4Rαhi, and CD32low at both the transcriptional and surface protein levels. These MBC2s are enriched in IgG1- and IgG4-expressing cells while constitutively expressing germline transcripts for IgE. Allergen-specific B cells from patients with allergic rhinitis and food allergy were enriched in MBC2s. Furthermore, MBC2s generated allergen-specific IgE during sublingual immunotherapy, thereby identifying these cells as a major reservoir for IgE. The identification of MBC2s provides insights into the maintenance of IgE memory, which is detrimental in allergic diseases but could be beneficial in protection against venoms and helminths.

Allergenicity evaluation of quinoa proteins - A study in Brown Norway rats.

The popularity of quinoa seeds has increased in the last decade due to their high nutritional value and natural gluten-free composition. Consumption of new proteins may pose a risk of introducing new allergies. In the present study the immunogenicity and sensitising capacity of quinoa proteins were assessed in a dose-response experiment in Brown Norway rats in comparison to proteins from spinach and peanut. Cross-reactivity between quinoa proteins and known allergens was evaluated by in silico analyses followed by analyses with 11 selected protein extracts and their anti-sera by means of ELISAs and immunoblotting. Further, an in vitro simulated gastro-duodenal digestion was performed. Quinoa proteins were found to have an inherent medium to high immunogenicity and sensitising capacity, being able to induce specific IgG1 and IgE levels higher than spinach but lower than peanut and elicit reactions of clinical relevance similar to peanut. Quinoa proteins were generally shown to resist digestion and retain capacity to bind quinoa-specific antibodies. Quinoa proteins were shown to be cross-reactive with peanut and tree nut allergens as high sequence homology and antibody cross-binding were demonstrated. Present study suggests that quinoa pose a medium to high level of allergenicity that should be further investigated in human studies.

Dose and route of administration determine the efficacy of prophylactic immunotherapy for peanut allergy in a Brown Norway rat model.

Introduction: Allergen-specific immunotherapy (IT) is emerging as a viable option for treatment of peanut allergy. Yet, prophylactic IT remains unexplored despite early introduction of peanut in infancy was shown to prevent allergy. There is a need to understand how allergens interact with the immune system depending on the route of administration, and how different dosages of allergen may protect from sensitisation and a clinical active allergy. Here we compared peanut allergen delivery via the oral, sublingual (SL), intragastric (IG) and subcutaneous (SC) routes for the prevention of peanut allergy in Brown Norway (BN) rats. Methods: BN rats were administered PBS or three different doses of peanut protein extract (PPE) via either oral IT (OIT), SLIT, IGIT or SCIT followed by intraperitoneal (IP) injections of PPE to assess the protection from peanut sensitisation. The development of IgE and IgG1 responses to PPE and the major peanut allergens were evaluated by ELISAs. The clinical response to PPE was assessed by an ear swelling test (EST) and proliferation was assessed by stimulating splenocytes with PPE. Results: Low and medium dose OIT (1 and 10 mg) and all doses of SCIT (1, 10, 100 µg) induced sensitisation to PPE, whereas high dose OIT (100 mg), SLIT (10, 100 or 1000 µg) or IGIT (1, 10 and 100 mg) did not. High dose OIT and SLIT as well as high and medium dose IGIT prevented sensitisation from the following IP injections of PPE and suppressed PPE-specific IgE levels in a dose-dependent manner. Hence, administration of peanut protein via different routes confers different risks for sensitisation and protection from peanut allergy development. Overall, the IgE levels toward the individual major peanut allergens followed the PPE-specific IgE levels. Discussion: Collectively, this study showed that the preventive effect of allergen-specific IT is determined by the interplay between the specific site of PPE delivery for presentation to the immune system, and the allergen quantity, and that targeting and modulating tolerance mechanisms at specific mucosal sites may be a prophylactic strategy for prevention of peanut allergy.

Rescuing ESAT-6 Specific CD4 T Cells From Terminal Differentiation Is Critical for Long-Term Control of Murine Mtb Infection.

In most cases, Mycobacterium tuberculosis (Mtb) causes life-long chronic infections, which poses unique challenges for the immune system. Most of the current tuberculosis (TB) subunit vaccines incorporate immunodominant antigens and at this point, it is poorly understood how the CD4 T cell subsets recognizing these antigens are affected during long-term infection. Very little is known about the requirements for sustainable vaccine protection against TB. To explore this, we screened 62 human-recognized Mtb antigens during chronic murine Mtb infection and identified the four most immunodominant antigens in this setting (MPT70, Rv3020c, and Rv3019c and ESAT-6). Combined into a subunit vaccine, this fusion protein induced robust protection both in a standard short-term model and in a long-term infection model where immunity from BCG waned. Importantly, replacement of ESAT-6 with another ESAT-6-family antigen, Rv1198, led to similar short-term protection but a complete loss of bacterial control during chronic infection. This observation was further underscored, as the ESAT-6 containing vaccine mediated sustainable protection in a model of post-exposure vaccination, where the ESAT-6-replacement vaccine did not. An individual comparison of the CD4 T cell responses during Mtb infection revealed that ESAT-6-specific T cells were more terminally differentiated than the other immunodominant antigens and immunization with the ESAT-6 containing vaccine led to substantially greater reduction in the overall T cell differentiation status. Our data therefore associates long-term bacterial control with the ability of a vaccine to rescue infection-driven CD4T cell differentiation and future TB antigen discovery programs should focus on identifying antigens with the highest accompanying T cell differentiation, like ESAT-6. This also highlights the importance of long-term readouts in both preclinical and clinical studies with TB vaccines.

Immunization with Mycobacterium tuberculosis-Specific Antigens Bypasses T Cell Differentiation from Prior Bacillus Calmette-Guérin Vaccination and Improves Protection in Mice.

Despite the fact that the majority of people in tuberculosis (TB)-endemic areas are vaccinated with the Bacillus Calmette-Guérin (BCG) vaccine, TB remains the leading infectious cause of death. Data from both animal models and humans show that BCG and subunit vaccines induce T cells of different phenotypes, and little is known about how BCG priming influences subsequent booster vaccines. To test this, we designed a novel Mycobacterium tuberculosis-specific (or "non-BCG") subunit vaccine with protective efficacy in both mice and guinea pigs and compared it to a known BCG boosting vaccine. In naive mice, this M. tuberculosis-specific vaccine induced similar protection compared with the BCG boosting vaccine. However, in BCG-primed animals, only the M. tuberculosis-specific vaccine added significantly to the BCG-induced protection. This correlated with the priming of T cells with a lower degree of differentiation and improved lung-homing capacity. These results have implications for TB vaccine design.

The Skin Reservoir Model: A Tool for Evaluating Microdialysis Sampling of Large Biomarkers from Human Skin.

Microdialysis is a well-established technique for sampling of small molecules from the human skin, but larger molecules are more difficult to recover. Consequently, sampling feasibility must be evaluated before microdialysis is used in vivo. This report presents a tool for estimating the recovery of large biomarkers from human skin by microdialysis, using previously frozen human skin specimens as reservoirs for biomarker reference solutions. Recovery of the following 17 biomarkers was assessed: CCL27/CTACK, CXCL1/GROα, CXCL7/NAP-2, CXCL10/IP-10, EGF, GM-CSF, IFN-γ, IL-1α, IL-6, IL-8, IL-17, IL-22, IL-23, MIF, TNF-α, TSLP and VEGF. The relative skin recoveries of 13/17 biomarkers were successfully determined in the range 4.0-18.4%. Sampling in the skin reservoir model was not associated with probe leakage, as fluid recovery was stable, at between 80% and 110%. Furthermore, the skin reservoir model enabled studies and optimization of different parameters known to affect biomarker recovery, including flow rate and perfusate composition.

An attenuated Mycobacterium tuberculosis clinical strain with a defect in ESX-1 secretion induces minimal host immune responses and pathology.

Although Mycobacterium tuberculosis (M.tb) DK9897 is an attenuated strain, it was isolated from a patient with extrapulmonary tuberculosis and vaccination with a subunit vaccine (H56) induced poor protection against it. Both attenuation and lack of protection are because M.tb DK9897 cannot secrete the EsxA virulence factor nor induce a host response against it. Genome sequencing identified a frameshift mutation in the eccCa1 gene. Since the encoded EccCa1 protein provides energy for ESX-1 secretion, it suggested a defect in the ESX-1 type VII secretion system. Genetic complementation with a plasmid carrying the M.tb H37Rv sequence of eccCa1-eccCb1-pe35 re-established EsxA secretion, host specific EsxA T-cell responses, and increased strain virulence. The ESX-1 secretion defect prevents several virulence factors from being functional during infection and therefore attenuates M.tb. It precludes specific T-cell responses against strong antigens and we found very little in vivo cytokine production, gross pathology or granuloma formation in lungs from M.tb DK9897 infected animals. This coincides with M.tb DK9897 being unable to disrupt the phagosome membrane and make contact to the cytosol.

Different human vaccine adjuvants promote distinct antigen-independent immunological signatures tailored to different pathogens.

The majority of vaccine candidates in clinical development are highly purified proteins and peptides relying on adjuvants to enhance and/or direct immune responses. Despite the acknowledged need for novel adjuvants, there are still very few adjuvants in licensed human vaccines. A vast number of adjuvants have been tested pre-clinically using different experimental conditions, rendering it impossible to directly compare their activity. We performed a head-to-head comparison of five different adjuvants Alum, MF59®, GLA-SE, IC31® and CAF01 in mice and combined these with antigens from M. tuberculosis, influenza, and chlamydia to test immune-profiles and efficacy in infection models using standardized protocols. Regardless of antigen, each adjuvant had a unique immunological signature suggesting that the adjuvants have potential for different disease targets. Alum increased antibody titers; MF59® induced strong antibody and IL-5 responses; GLA-SE induced antibodies and Th1; CAF01 showed a mixed Th1/Th17 profile and IC31® induced strong Th1 responses. MF59® and GLA-SE were strong inducers of influenza HI titers while CAF01, GLA-SE and IC31® enhanced protection to TB and chlamydia. Importantly, this is the first extensive attempt to categorize clinical-grade adjuvants based on their immune profiles and protective efficacy to inform a rational development of next generation vaccines for human use.

Tuberculosis vaccine with high predicted population coverage and compatibility with modern diagnostics.

A central goal in vaccine research is the identification of relevant antigens. The Mycobacterium tuberculosis chromosome encodes 23 early secretory antigenic target (ESAT-6) family members that mostly are localized as gene pairs. In proximity to five of the gene pairs are ESX secretion systems involved in the secretion of the ESAT-6 family proteins. Here, we performed a detailed and systematic investigation of the vaccine potential of five possible Esx dimer substrates, one for each of the five ESX systems. On the basis of gene transcription during infection, immunogenicity, and protective capacity in a mouse aerosol challenge model, we identified the ESX dimer substrates EsxD-EsxC, ExsG-EsxH, and ExsW-EsxV as the most promising vaccine candidates and combined them in a fusion protein, H65. Vaccination with H65 gave protection at the level of bacillus Calmette-Guérin, and the fusion protein exhibited high predicted population coverage in high endemic regions. H65 thus constitutes a promising vaccine candidate devoid of antigen 85 and fully compatible with current ESAT-6 and culture filtrate protein 10-based diagnostics.

Control of chronic mycobacterium tuberculosis infection by CD4 KLRG1- IL-2-secreting central memory cells.

The bacille Calmette-Guérin vaccine provides very efficient protection in standard animal models of Mycobacterium tuberculosis challenge. We show in this article that although bacille Calmette-Guérin controlled M. tuberculosis growth for 7 wk of infection, the protection was gradually lost as the infection entered the chronic phase. The regrowth of M. tuberculosis coincided with an almost complete disappearance of IL-2-producing CD4 T cells. Booster vaccination with a subunit vaccine (Ag85B-ESAT-6+CAF01) expanded IL-2(+) CD4(+) T cell coexpressing either TNF-α or TNF-α/IFN-γ, and the maintenance of this population in the late stage of infection was associated with enhanced control of bacterial growth. The IL-2(+) CD4(+) T cell subsets were KLRG1(-) (nonterminally differentiated), were found to be CD62L(high), and further maintained a pronounced proliferative and cytokine-producing potential in the draining lymph nodes, when the animals were challenged 2 y postvaccination. These results suggest that the CD4(+) KLRG1(-) IL-2-secreting subsets are central memory T cells with the potential to continuously replenish the T cells at the site of infection and prevent attrition and functional exhaustion.