Recent advances in epicutaneous immunotherapy and potential applications in food allergy.

Given the potent immunological properties of the skin, epicutaneous immunotherapy (EPIT) emerges as a promising treatment approach for inducing immune tolerance, particularly for food allergies. Targeting the highly immunocompetent, non-vascularized epidermis allows for the application of microgram amounts of allergen while significantly reducing the risk of allergen passage into the bloodstream, thus limiting systemic allergen exposure and distribution. This makes EPIT highly suitable for the treatment of potentially life-threatening allergies such as food allergies. Multiple approaches to EPIT are currently under investigation for the treatment of food allergy, and these include the use of allergen-coated microneedles, application of allergen on the skin pretreated by tape stripping, abrasion or laser-mediated microperforation, or the application of allergen on the intact skin using an occlusive epicutaneous system. To date, the most clinically advanced approach to EPIT is the Viaskin technology platform. Viaskin is an occlusive epicutaneous system (patch) containing dried native allergen extracts, without adjuvants, which relies on frequent application for the progressive passage of small amounts of allergen to the epidermis through occlusion of the intact skin. Numerous preclinical studies of Viaskin have demonstrated that this particular approach to EPIT can induce potent and long-lasting T-regulatory cells with broad homing capabilities, which can exert their suppressive effects in multiple organs and ameliorate immune responses from different routes of allergen exposure. Clinical trials of the Viaskin patch have studied the efficacy and safety for the treatment of life-threatening allergies in younger patients, at an age when allergic diseases start to occur. Moreover, this treatment approach is designed to provide a non-invasive therapy with no restrictions on daily activities. Taken together, the preclinical and clinical data on the use of EPIT support the continued investigation of this therapeutic approach to provide improved treatment options for patients with allergic disorders in the near future.

Epicutaneous immunotherapy protects cashew-sensitized mice from anaphylaxis.

BACKGROUND: The prevalence of tree nut allergy has increased worldwide, and cashew has become one of the most common food allergens. More critically, cashew allergy is frequently associated with severe anaphylaxis. Despite the high medical need, no approved treatment is available and strict avoidance and preparedness for prompt treatment of allergic reactions are considered dual standard of care. In the meantime, Phase III study results suggest investigational epicutaneous immunotherapy (EPIT) may be a relevant and safe treatment for peanut allergy and may improve the quality of life for many peanut allergic children. OBJECTIVE: We aimed to evaluate the capacity of EPIT to provide protection against cashew-induced anaphylaxis in a relevant mouse model. METHODS: The efficacy of EPIT was evaluated by applying patches containing cashew allergens to cashew-sensitized mice. As negative control, sham mice received patches containing excipient. Following treatment, mice were challenged orally to cashew and anaphylactic symptoms, as well as plasmatic levels of mast-cell proteases (mMCP)-1/7, were quantified. RESULTS: Of 16 weeks of EPIT significantly protects against anaphylaxis by promoting a faster recovery of challenged mice. This protection was characterized by a significant reduction of temperature drop and clinical symptoms, 60 minutes after challenge. This was associated with a decrease in mast-cell reactivity as attested by the reduction of mMCP-1/7 in plasma, suggesting that EPIT specifically decrease IgE-mediated anaphylaxis. CONCLUSION: We demonstrate that EPIT markedly reduced IgE-mediated allergic reactions in a mouse model of cashew allergy, which suggests that EPIT may be a relevant approach to treating cashew allergy.

Reducing V3 Antigenicity and Immunogenicity on Soluble, Native-Like HIV-1 Env SOSIP Trimers.

Native-like trimers of the SOSIP design are being developed as immunogens in human immunodeficiency virus type 1 (HIV-1) vaccine development programs. These trimers display the epitopes for multiple broadly neutralizing antibodies (bNAbs) but can also expose binding sites for some types of nonneutralizing antibodies (non-NAbs). Among the latter are epitopes in the gp120 V3 region that are highly immunogenic when SOSIP trimers are evaluated in animal models. It is presently uncertain whether antibodies against V3 can interfere with the induction of NAbs, but there are good arguments in favor of suppressing such "off-target" immune responses. Accordingly, we have assessed how to minimize the exposure of V3 non-NAb epitopes and thereby reduce their immunogenicity by introducing N-glycans within the V3 region of BG505 SOSIP trimers. We found that inserting glycans at positions 306 and 314 (termed M1 and M7) markedly reduced V3 antigenicity while improving the presentation of trimer apex bNAb epitopes. Both added glycans were shown to be predominantly of the Man6GlcNAc2 form. The additional introduction of the E64K ground-state stabilizing substitution markedly reduced or ablated soluble CD4 (sCD4) induction of non-NAb epitopes in V3 and/or associated with the coreceptor binding site. When a V3 glycan- and E64K-modified trimer variant, BG505 SOSIP.664-E64K.M1M7, was tested in rabbits, V3 immunogenicity was eliminated while the autologous NAb response was unchanged.IMPORTANCE Trimeric proteins are being developed for future HIV-1 vaccine trials in humans, with the goal of eliciting broadly active neutralizing antibodies (NAbs) that are active against a wide variety of circulating strains. In animal models, the present generation of native-like trimer immunogens, exemplified by the BG505 SOSIP.664 construct, induces narrow-specificity antibodies against the neutralization-resistant (tier-2), sequence-matched virus and more broadly active antibodies against sequence-divergent atypically neutralization-sensitive (tier-1) viruses. A concern in the trimer immunogen design field has been whether the latter off-target antibodies might interfere with the induction of the more desired responses to tier-2 epitopes. Here, we have inserted two glycans into the dominant site for tier-1 NAbs, the gp120 V3 region, to block the induction of off-target antibodies. We characterized the new trimers, tested them as immunogens in rabbits, and found that the blocking glycans eliminated the induction of tier-1 NAbs to V3-epitopes.

Pruritus in pediatric burn survivors: defining the clinical course.

Pruritus is a frequent and severe symptom and a significant cause of distress for adult burn patients. Its effects in children are largely unstudied. The aim of this study is to characterize postburn itch in the pediatric population. This is a retrospective review from 2006 to 2013 for pediatric burn survivors who were enrolled in a longitudinal multicenter outcomes study. Demographic data, injury characteristics, associated symptoms (skin-related problems, pain, and sleep), and incidence and intensity (Numerical Rating Scale) of itch were examined. Measures were completed at hospital discharge and at 6, 12, and 24 months after injury. Spearman's correlations were used to examine the correlation between itch intensity and associated symptoms. Multivariate regression analyses examined the impact of associated symptoms on itch intensity. There were 430 pediatric burn survivors with a mean age of 7.8 years and a mean TBSA of 40.8%. Pruritus is present in most children (93%) and is of moderate intensity (5.7 ± 3.1) at discharge. The frequency and intensity of pruritus decreases over time; a majority of children continue to report symptoms at 2 years (63%). Itch was significantly correlated with associated symptoms. Regression analyses showed a correlation between itch intensity and pain at each time point. There was no association between itch intensity and burn etiology, age, gender, or burn size. Pruritus is a frequent complication that lasts for at least 2 years after injury in a majority of pediatric burn survivors. This information will enable better tracking of outcomes and will serve as a baseline for assessing interventions.

Stable 293 T and CHO cell lines expressing cleaved, stable HIV-1 envelope glycoprotein trimers for structural and vaccine studies.

BACKGROUND: Recombinant soluble, cleaved HIV-1 envelope glycoprotein SOSIP.664 gp140 trimers based on the subtype A BG505 sequence are being studied structurally and tested as immunogens in animals. For these trimers to become a vaccine candidate for human trials, they would need to be made in appropriate amounts at an acceptable quality. Accomplishing such tasks by transient transfection is likely to be challenging. The traditional way to express recombinant proteins in large amounts is via a permanent cell line, usually of mammalian origin. Making cell lines that produce BG505 SOSIP.664 trimers requires the co-expression of the Furin protease to ensure that the cleavage site between the gp120 and gp41 subunits is fully utilized. RESULTS: We designed a vector capable of expressing Env and Furin, and used it to create Stable 293 T and CHO Flp-In™ cell lines through site-specific recombination. Both lines produce high quality, cleaved trimers at yields of up to 12-15 mg per 1 × 109 cells. Trimer expression at such levels was maintained for up to 30 days (10 passages) after initial seeding and was consistently superior to what could be achieved by transient transfection. Electron microscopy studies confirm that the purified trimers have the same native-like appearance as those derived by transient transfection and used to generate high-resolution structures. They also have appropriate antigenic properties, including the presentation of the quaternary epitope for the broadly neutralizing antibody PGT145. CONCLUSIONS: The BG505 SOSIP.664 trimer-expressing cell lines yield proteins of an appropriate quality for structural studies and animal immunogenicity experiments. The methodology is suitable for making similar lines under Good Manufacturing Practice conditions, to produce trimers for human clinical trials. Moreover, any env gene can be incorporated into this vector system, allowing the manufacture of SOSIP trimers from multiple genotypes, either by transient transfection or from stable cell lines.

An HIV-1 envelope glycoprotein trimer with an embedded IL-21 domain activates human B cells.

Broadly neutralizing antibodies (bNAbs) that target the HIV-1 envelope glycoproteins (Env) can prevent virus acquisition, but several Env properties limit its ability to induce an antibody response that is of sufficient quantity and quality. The immunogenicity of Env can be increased by fusion to co-stimulatory molecules and here we describe novel soluble Env trimers with embedded interleukin-4 (IL-4) or interleukin-21 (IL-21) domains, designed to activate B cells that recognize Env. In particular, the chimeric Env(IL-21) molecule activated B cells efficiently and induced the differentiation of antibody secreting plasmablast-like cells. We studied whether we could increase the activity of the embedded IL-21 by designing a chimeric IL-21/IL-4 (ChimIL-21/4) molecule and by introducing amino acid substitutions in the receptor binding domain of IL-21 that were predicted to enhance its binding. In addition, we incorporated IL-21 into a cleavable Env trimer and found that insertion of IL-21 did not impair Env cleavage, while Env cleavage did not impair IL-21 activity. These studies should guide the further design of chimeric proteins and Env(IL-21) may prove useful in improving antibody responses against HIV-1.

Clinical adjuvant combinations stimulate potent B-cell responses in vitro by activating dermal dendritic cells.

CD14(+) dermal DCs (CD14(+) DDCs) have a natural capacity to activate naïve B-cells. Targeting CD14(+) DDCs is therefore a rational approach for vaccination strategies aimed at improving humoral responses towards poorly immunogenic antigens, for example, HIV-1 envelope glycoproteins (Env). Here, we show that two clinically relevant TLR ligand combinations, Hiltonol plus Resiquimod and Glucopyranosyl lipid A plus Resiquimod, potently activate CD14(+) DDCs, as shown by enhanced expression of multiple cytokines (IL-6, IL-10, IL-12p40 and TNF-α). Furthermore, the responses of CD14(+) DDCs to these TLR ligands were not compromised by the presence of HIV-1 gp120, which can drive immunosuppressive effects in vitro and in vivo. The above TLR ligand pairs were better than the individual agents at boosting the inherent capacity of CD14(+) DDCs to induce naïve B-cells to proliferate and differentiate into CD27(+) CD38(+) B-cells that secrete high levels of immunoglobulins. CD14(+) DDCs stimulated by these TLR ligand combinations also promoted the differentiation of Th1 (IFN-γ-secreting), but not Th17, CD4(+) T-cells. These observations may help to identify adjuvant strategies aimed at inducing better antibody responses to vaccine antigens, including, but not limited to HIV-1 Env.

Potent induction of antibody-secreting B cells by human dermal-derived CD14+ dendritic cells triggered by dual TLR ligation.

Targeting CD14(+) dermal-derived dendritic cells (DDCs) is a rational approach for vaccination strategies aimed at improving humoral immune responses, because of their natural ability to stimulate naive B cells. In this study, we show that CD14(+) DDCs express mRNA for TLRs 1-9, but respond differentially to single or paired TLR ligands. Compared to single ligands, some combinations were particularly effective at activating CD14(+) DDCs, as shown by enhanced expression of B cell stimulatory cytokines (IL-6, IL-10, and TNF-α) and more pronounced phenotypic maturation. These combinations were resiquimod (R-848) plus polyinosinic-polycytidylic acid [Poly(I:C)], R-848 plus LPS, Pam3CSK4 plus Poly(I:C), and LPS plus Poly(I:C). We also found that selected TLR ligand pairs [R-848 plus either LPS or Poly(I:C)] were superior to individual agents at boosting the inherent capacity of CD14(+) DDCs to induce naive B cells to proliferate and differentiate into CD27(+) CD38(+) B cells that secrete high levels of IgG and IgA. When treated with the same TLR ligand combinations, CD14(+) DDCs also promoted the differentiation of Th1 (IFN-γ-secreting) CD4(+) T cells, but not of Th2 or Th17 CD4(+) T cells. These observations may help to identify adjuvant strategies aimed at inducing protective immune responses to various pathogens, including but not limited to HIV-1.

HIV-1 gp120 impairs the induction of B cell responses by TLR9-activated plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) play a central role in innate and adaptive immune responses to viral infections, including HIV type 1 (HIV-1). pDCs produce substantial quantities of type I IFN and proinflammatory cytokines upon stimulation via TLRs, specifically TLR7 or TLR9. The HIV-1 envelope glycoproteins, exemplified by the gp120 monomer, are the focus of vaccines aimed at inducing B cell responses. We have studied how the interactions of gp120 with various receptors on human pDCs affect the activation of these cells via TLR9 and their subsequent ability to stimulate B cells. We observed that IFN-α production by pDCs in response to TLR9, but not TLR7, stimulation was reduced by exposure to gp120. Specifically, gp120 inhibited the CpG-induced maturation of pDCs and their expression of TNF-α, IL-6, TLR9, IFN regulatory factor 7, and BAFF. Receptor-blocking and cross-linking studies showed that these inhibitory effects of gp120 were mediated by interactions with CD4 and mannose-binding C-type lectin receptors, but not with the chemokine receptors CCR5 and CXCR4. Of note is that gp120 inhibited the activation of B cells by TLR9-stimulated pDCs. Taken together, our data show that HIV-1 gp120 impairs pDC functions, including activation of B cell responses, and imply that TLR9 ligands may not be good adjuvants to use in combination with envelope glycoprotein vaccines.

Env-glycoprotein heterogeneity as a source of apparent synergy and enhanced cooperativity in inhibition of HIV-1 infection by neutralizing antibodies and entry inhibitors.

We measured the inhibition of infectivity of HIV-1 isolates and derivative clones by combinations of neutralizing antibodies (NAbs) and other entry inhibitors in a single-cycle-replication assay. Synergy was analyzed both by the current linear and a new non-linear method. The new method reduced spurious indications of synergy and antagonism. Synergy between NAbs was overall weaker than between other entry inhibitors, and no stronger where one ligand is known to enhance the binding of another. However, synergy was stronger for a genetically heterogeneous HIV-1 R5 isolate than for its derivative clones. Enhanced cooperativity in inhibition by combinations, compared with individual inhibitors, correlated with increased synergy at higher levels of inhibition, while being less variable. Again, cooperativity enhancement was stronger for isolates than clones. We hypothesize that genetic, post-translational or conformational heterogeneity of the Env protein and of other targets for inhibitors can yield apparent synergy and increased cooperativity between inhibitors.

A stabilized HIV-1 envelope glycoprotein trimer fused to CD40 ligand targets and activates dendritic cells.

BACKGROUND: One reason why subunit protein and DNA vaccines are often less immunogenic than live-attenuated and whole-inactivated virus vaccines is that they lack the co-stimulatory signals provided by various components of the more complex vaccines. The HIV-1 envelope glycoprotein complex (Env) is no exception to this rule. Other factors that limit the induction of neutralizing antibodies against HIV-1 lie in the structure and instability of Env. We have previously stabilized soluble trimeric mimics of Env by introducing a disulfide bond between gp120 and gp41 and adding a trimer stabilizing mutation in gp41 (SOSIP.R6 gp140). RESULTS: We further stabilized the SOSIP.R6 gp140 using a GCN4-based isoleucine zipper motif, creating SOSIP.R6-IZ gp140. In order to target SOSIP.R6-IZ to immune cells, including dendritic cells, while at the same time activating these cells, we fused SOSIP.R6-IZ to the active domain of CD40 ligand (CD40L), which may serve as a 'cis-adjuvant'. The Env component of the SOSIP.R6-IZ-CD40L fusion construct bound to CD4 and neutralizing antibodies, while the CD40L moiety interacted with CD40. Furthermore, the chimeric molecule was able to signal efficiently through CD40 and induce maturation of human dendritic cells. Dendritic cells secreted IL-6, IL-10 and IL-12 in response to stimulation by SOSIP.R6-IZ-CD40L and were able to activate naïve T cells. CONCLUSIONS: Chimeric HIV-1 gp140 - CD40L trimers can target and activate dendritic cells. Targeting and activating immune cells using CD40L and other 'cis-adjuvants' may improve subunit protein vaccine immunogenicity for HIV-1 and other infectious diseases.

A chimeric HIV-1 envelope glycoprotein trimer with an embedded granulocyte-macrophage colony-stimulating factor (GM-CSF) domain induces enhanced antibody and T cell responses.

An effective HIV-1 vaccine should ideally induce strong humoral and cellular immune responses that provide sterilizing immunity over a prolonged period. Current HIV-1 vaccines have failed in inducing such immunity. The viral envelope glycoprotein complex (Env) can be targeted by neutralizing antibodies to block infection, but several Env properties limit the ability to induce an antibody response of sufficient quantity and quality. We hypothesized that Env immunogenicity could be improved by embedding an immunostimulatory protein domain within its sequence. A stabilized Env trimer was therefore engineered with the granulocyte-macrophage colony-stimulating factor (GM-CSF) inserted into the V1V2 domain of gp120. Probing with neutralizing antibodies showed that both the Env and GM-CSF components of the chimeric protein were folded correctly. Furthermore, the embedded GM-CSF domain was functional as a cytokine in vitro. Mouse immunization studies demonstrated that chimeric Env(GM-CSF) enhanced Env-specific antibody and T cell responses compared with wild-type Env. Collectively, these results show that targeting and activation of immune cells using engineered cytokine domains within the protein can improve the immunogenicity of Env subunit vaccines.

Rapid recovery of lymphocyte subsets is not associated with protection from relapse of myelodysplastic syndromes and acute myeloid leukaemia after haematopoietic stem cell transplantation using a reduced intensity conditioning regimen and alemtuzumab.

Graft-versus-leukaemia (GvL) and graft-versus-host disease (GvHD) are both caused by alloreactive lymphocytes. We previously reported that GvHD correlated with higher numbers of effector CD4 T cells and Natural Killer cells early after allogeneic transplantation using a regimen comprising fludarabine, busulphan and alemtuzumab. Here, we assessed immune cell subset recovery in these patients in the context of early myeloid malignant disease relapse. Despite the close relationship between the GvL and GvHD immune responses, rapid recovery of lymphocyte subsets was not associated with protection from disease relapse. These results indicated that GvL may be weak in this treatment setting for patients with myelodysplastic syndromes and acute myeloid leukaemia. Consistent with low GvL activity, we previously reported that mixed T cell chimaerism had no detrimental effect on relapse in this treatment setting and instead correlated with better outcome because of reduced GvHD incidence. We now report that patients with significantly higher lymphocyte numbers prior to transplantation subsequently maintained the mixed T cell chimaeric state. This pre-transplant profile, together with absence of the early post-transplant signature indicative of GvHD predisposition, could potentially be used to identify patients suitable for early withdrawal of immunosuppression and prophylactic donor leucocyte infusion to boost GvL activity.

Imbalance of effector and regulatory CD4 T cells is associated with graft-versus-host disease after hematopoietic stem cell transplantation using a reduced intensity conditioning regimen and alemtuzumab.

BACKGROUND: A variety of immune pathways can lead to graft-versus-host disease. A better understanding of the type of immune response causing graft-versus-host disease in defined clinical hematopoietic stem cell transplant settings is required to inform development of methods for monitoring patients and providing them tailored care. DESIGN AND METHODS: Twenty-five patients were recruited presenting with myeloid malignancies and treated with a reduced intensity conditioning transplant regimen with graft-versus-host disease prophylaxis comprising in vivo lymphocyte depletion with alemtuzumab and cyclosporin. A prospective study was performed of lymphocyte subset reconstitution in peripheral blood in relation to the incidence of graft-versus-host disease. RESULTS: Acute graft-versus-host disease was associated with significantly higher numbers of natural killer cells and donor-derived effector CD4 T cells (CD45RO(+) CD27(-)) early (day 30) after transplantation (p=0.04 and p=0.02, respectively). This association was evident before the emergence of clinical pathology in six out of seven patients. Although numbers of regulatory CD4 T cells (CD25(high) Foxp3(+)) were similar at day 30 in all patients, a significant deficit in those who developed acute graft-versus-host disease was apparent relative to effector CD4 T cells (median of 41 effectors per regulatory cell compared to 12 to 1 for patients without graft-versus-host disease) (p=0.03). By day 180, a functional regulatory CD4 T-cell population had expanded significantly in patients who developed chronic graft-versus-host disease, reversing the imbalance (median of 3 effectors per regulatory cell compared to 9.6 to 1 for patients without graft-versus-host disease) (p=0.018) suggesting no overt absence of immune regulation in the late onset form of the disease. CONCLUSIONS: Imbalance of effector and regulatory CD4 T cells is a signature of graft-versus-host disease in this transplantation protocol.

IL-17-producing CD4(+) T cells, pro-inflammatory cytokines and apoptosis are increased in low risk myelodysplastic syndrome.

Immunological responses are increasingly recognised as being important in the initiation and progression of myelodysplastic syndrome (MDS). Indeed, autoimmune diseases commonly occur in association with MDS, particularly in subtypes with a low risk of leukaemic transformation. This study showed for the first time that the numbers of CD3(+) CD4(+) IL-17 producing T cells (Th17) were markedly increased in low risk MDS compared with high risk MDS (P < 0.01). An inverse relationship between the numbers of Th17 cells and naturally occurring CD4(+)CD25(high) FoxP3(+) regulatory T cells (Tregs) were also described. The Th17:Tregs ratio was significantly higher in low risk disease (P < 0.005) compared with high risk MDS and was correlated with increased bone marrow (BM) apoptosis (P < 0.01). Tregs from MDS patients suppressed interferon-gamma (IFN-gamma) secretion by effector CD4(+) T cells but had no effect on interleukin (IL)-17 production. In addition, the serum levels of IL-7, IL-12, RANTES and IFN-gamma are significantly elevated in low risk MDS, while inhibitory factors, such as IL-10 and soluble IL-2 receptor, are significantly higher in high risk disease. The 'unfavourable' Th17:Tregs ratio in low risk MDS may explain the higher risk of autoimmunity and the improved response to immune suppression in patients with low risk MDS compared to those with high risk disease.