Mucosal boosting enhances vaccine protection against SARS-CoV-2 in macaques.

A limitation of current SARS-CoV-2 vaccines is that they provide minimal protection against infection with current Omicron subvariants1,2, although they still provide protection against severe disease. Enhanced mucosal immunity may be required to block infection and onward transmission. Intranasal administration of current vaccines has proven inconsistent3-7, suggesting that alternative immunization strategies may be required. Here we show that intratracheal boosting with a bivalent Ad26-based SARS-CoV-2 vaccine results in substantial induction of mucosal humoral and cellular immunity and near-complete protection against SARS-CoV-2 BQ.1.1 challenge. A total of 40 previously immunized rhesus macaques were boosted with a bivalent Ad26 vaccine by the intramuscular, intranasal and intratracheal routes, or with a bivalent mRNA vaccine by the intranasal route. Ad26 boosting by the intratracheal route led to a substantial expansion of mucosal neutralizing antibodies, IgG and IgA binding antibodies, and CD8+ and CD4+ T cell responses, which exceeded those induced by Ad26 boosting by the intramuscular and intranasal routes. Intratracheal Ad26 boosting also led to robust upregulation of cytokine, natural killer, and T and B cell pathways in the lungs. After challenge with a high dose of SARS-CoV-2 BQ.1.1, intratracheal Ad26 boosting provided near-complete protection, whereas the other boosting strategies proved less effective. Protective efficacy correlated best with mucosal humoral and cellular immune responses. These data demonstrate that these immunization strategies induce robust mucosal immunity, suggesting the feasibility of developing vaccines that block respiratory viral infections.

The Biodistribution of the Spike Protein after Ad26.COV2.S Vaccination Is Unlikely to Play a Role in Vaccine-Induced Immune Thrombotic Thrombocytopenia.

Ad26.COV2.S vaccination can lead to vaccine-induced immune thrombotic thrombocytopenia (VITT), a rare but severe adverse effect, characterized by thrombocytopenia and thrombosis. The mechanism of VITT induction is unclear and likely multifactorial, potentially including the activation of platelets and endothelial cells mediated by the vaccine-encoded spike protein (S protein). Here, we investigated the biodistribution of the S protein after Ad26.COV2.S dosing in three animal models and in human serum samples. The S protein was transiently present in draining lymph nodes of rabbits after Ad26.COV2.S dosing. The S protein was detected in the serum in all species from 1 day to 21 days after vaccination with Ad26.COV2.S, but it was not detected in platelets, the endothelium lining the blood vessels, or other organs. The S protein S1 and S2 subunits were detected at different ratios and magnitudes after Ad26.COV2.S or COVID-19 mRNA vaccine immunization. However, the S1/S2 ratio did not depend on the Ad26 platform, but on mutation of the furin cleavage site, suggesting that the S1/S2 ratio is not VITT related. Overall, our data suggest that the S-protein biodistribution and kinetics after Ad26.COV2.S dosing are likely not main contributors to the development of VITT, but other S-protein-specific parameters require further investigation.

Serum Immune Profiling in Paediatric Crohn's Disease Demonstrates Stronger Immune Modulation With First-Line Infliximab Than Conventional Therapy and Pre-Treatment Profiles Predict Clinical Response to Both Treatments.

BACKGROUND: Despite its efficacy, rational guidance for starting/stopping first-line biologic treatment in individual paediatric Crohn's disease [CD] patients is needed. We assessed how serum immune profiles before and after first-line infliximab [FL-IFX] or conventional [CONV] induction therapy associate with disease remission at week 52. METHODS: Pre- [n = 86], and 10-14-week post-treatment [n = 84] sera were collected from patients with moderate-to-severe paediatric CD in the TISKids trial, randomized to FL-IFX [n = 48; five 5-mg/kg infusions over 22 weeks] or CONV [n = 43; exclusive enteral nutrition or oral prednisolone]; both groups received azathioprine maintenance. The relative concentrations of 92 inflammatory proteins were determined with Olink Proteomics; fold changes [FC] with |log2FC| > 0.5 after false discovery rate adjustment were considered significant. RESULTS: FL-IFX modulated a larger number of inflammatory proteins and induced stronger suppression than CONV; 18/30 proteins modulated by FL-IFX were not regulated by CONV. Hierarchical clustering based on IFX-modulated proteins at baseline revealed two clusters of patients: CD-hi patients had significantly higher concentrations of 23/30 IFX-modulated proteins [including oncostatin-M, TNFSF14, HGF and TGF-α], and higher clinical disease activity, C-reactive protein and blood neutrophils at baseline than CD-lo patients. Only 24% of CD-hi FL-IFX-treated patients maintained remission without escalation at week 52 vs 58% of CD-lo FL-IFX-treated patients. Similarly, 6% of CD-hi CONV-treated patients achieved remission vs 20% of CONV-treated CD-lo patients. Clustering based on immune profiles post-induction therapy did not relate to remission at week 52. CONCLUSION: FL-IFX leads to stronger reductions and modulates more immune proteins than CONV. Stratification on pre-treatment profiles of IFX-modulated proteins directly relates to maintenance of remission without treatment escalation. TRIAL REGISTRATION NUMBER: NCT02517684.

Intravenous Administration of Ad26.COV2.S Does Not Induce Thrombocytopenia or Thrombotic Events or Affect SARS-CoV-2 Spike Protein Bioavailability in Blood Compared with Intramuscular Vaccination in Rabbits.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a very rare but serious adverse reaction that can occur after Ad26.COV2.S vaccination in humans, leading to thrombosis at unusual anatomic sites. One hypothesis is that accidental intravenous (IV) administration of Ad26.COV2.S or drainage of the vaccine from the muscle into the circulatory system may result in interaction of the vaccine with blood factors associated with platelet activation, leading to VITT. Here, we demonstrate that, similar to intramuscular (IM) administration of Ad26.COV2.S in rabbits, IV dosing was well tolerated, with no significant differences between dosing routes for the assessed hematologic, coagulation time, innate immune, or clinical chemistry parameters and no histopathologic indication of thrombotic events. For both routes, all other non-adverse findings observed were consistent with a normal vaccine response and comparable to those observed for unrelated or other Ad26-based control vaccines. However, Ad26.COV2.S induced significantly higher levels of C-reactive protein on day 1 after IM vaccination compared with an Ad26-based control vaccine encoding a different transgene, suggesting an inflammatory effect of the vaccine-encoded spike protein. Although based on a limited number of animals, these data indicate that an accidental IV injection of Ad26.COV2.S may not represent an increased risk for VITT.

Booster with Ad26.COV2.S or Omicron-adapted vaccine enhanced immunity and efficacy against SARS-CoV-2 Omicron in macaques.

Omicron spike (S) encoding vaccines as boosters, are a potential strategy to improve COVID-19 vaccine efficacy against Omicron. Here, macaques (mostly females) previously immunized with Ad26.COV2.S, are boosted with Ad26.COV2.S, Ad26.COV2.S.529 (encoding Omicron BA.1 S) or a 1:1 combination of both vaccines. All booster vaccinations elicit a rapid antibody titers increase against WA1/2020 and Omicron S. Omicron BA.1 and BA.2 antibody responses are most effectively boosted by vaccines including Ad26.COV2.S.529. Independent of vaccine used, mostly WA1/2020-reactive or WA1/2020-Omicron BA.1 cross-reactive B cells are detected. Ad26.COV2.S.529 containing boosters provide only slightly higher protection of the lower respiratory tract against Omicron BA.1 challenge compared with Ad26.COV2.S-only booster. Antibodies and cellular immune responses are identified as complementary correlates of protection. Overall, a booster with an Omicron-spike based vaccine provide only moderately improved immune responses and protection compared with the original Wuhan-Hu-1-spike based vaccine, which still provide robust immune responses and protection against Omicron.

Duplication of the IL2RA locus causes excessive IL-2 signaling and may predispose to very early onset colitis.

Single genetic mutations predispose to very early onset inflammatory bowel disease (VEO-IBD). Here, we identify a de novo duplication of the 10p15.1 chromosomal region, including the IL2RA locus, in a 2-year-old girl with treatment-resistant pancolitis that was brought into remission by colectomy. Strikingly, after colectomy while the patient was in clinical remission and without medication, the peripheral blood CD4:CD8 ratio was constitutively high and CD25 expression was increased on circulating effector memory, Foxp3+, and Foxp3neg CD4+ T cells compared to healthy controls. This high CD25 expression increased IL-2 signaling, potentiating CD4+ T-cell-derived IFNγ secretion after T-cell receptor (TCR) stimulation. Restoring CD25 expression using the JAK1/3-inhibitor tofacitinib controlled TCR-induced IFNγ secretion in vitro. As diseased colonic tissue, but not the unaffected duodenum, contained mainly CD4+ T cells with a prominent IFNγ-signature, we hypothesize that local microbial stimulation may have initiated colonic disease. Overall, we identify that duplication of the IL2RA locus can associate with VEO-IBD and suggest that increased IL-2 signaling predisposes to colonic intestinal inflammation.

Reovirus infection triggers inflammatory responses to dietary antigens and development of celiac disease.

Viral infections have been proposed to elicit pathological processes leading to the initiation of T helper 1 (TH1) immunity against dietary gluten and celiac disease (CeD). To test this hypothesis and gain insights into mechanisms underlying virus-induced loss of tolerance to dietary antigens, we developed a viral infection model that makes use of two reovirus strains that infect the intestine but differ in their immunopathological outcomes. Reovirus is an avirulent pathogen that elicits protective immunity, but we discovered that it can nonetheless disrupt intestinal immune homeostasis at inductive and effector sites of oral tolerance by suppressing peripheral regulatory T cell (pTreg) conversion and promoting TH1 immunity to dietary antigen. Initiation of TH1 immunity to dietary antigen was dependent on interferon regulatory factor 1 and dissociated from suppression of pTreg conversion, which was mediated by type-1 interferon. Last, our study in humans supports a role for infection with reovirus, a seemingly innocuous virus, in triggering the development of CeD.

IL-10 control of CD11c+ myeloid cells is essential to maintain immune homeostasis in the small and large intestine.

Although IL-10 promotes a regulatory phenotype of CD11c+ dendritic cells and macrophages in vitro, the role of IL-10 signaling in CD11c+ cells to maintain intestinal tolerance in vivo remains elusive. To this aim, we generated mice with a CD11c-specific deletion of the IL-10 receptor alpha (Cd11ccreIl10rafl/fl). In contrast to the colon, the small intestine of Cd11ccreIl10rafl/fl mice exhibited spontaneous crypt hyperplasia, increased numbers of intraepithelial lymphocytes and lamina propria T cells, associated with elevated levels of T cell-derived IFNγ and IL-17A. Whereas naive mucosal T-cell priming was not affected and oral tolerance to ovalbumin was intact, augmented T-cell function in the lamina propria was associated with elevated numbers of locally dividing T cells, expression of T-cell attracting chemokines and reduced T-cell apoptosis. Upon stimulation, intestinal IL-10Rα deficient CD11c+ cells exhibited increased activation associated with enhanced IL-6 and TNFα production. Following colonization with Helicobacter hepaticus Cd11ccreIl10rafl/fl mice developed severe large intestinal inflammation characterized by infiltrating T cells and increased levels of Il17a, Ifng, and Il12p40. Altogether these findings demonstrate a critical role of IL-10 signaling in CD11c+ cells to control small intestinal immune homeostasis by limiting reactivation of local memory T cells and to protect against Helicobacter hepaticus-induced colitis.

The role of animal models in unravelling therapeutic targets in coeliac disease.

Coeliac disease is a complex small intestinal enteropathy that develops consequently to a breach of tolerance to gliadin, a storage protein abundantly found in cereals such as wheat, rye and barley. The understanding of the mechanisms underlying the development of coeliac disease in HLA-DQ2 and HLA-DQ8 genetically susceptible individuals has greatly improved during the last decades but so far did not allow to develop curative therapeutics, leaving a long-life gluten free diet as the only treatment option for the patients. In order to bring new therapeutic targets to light and to test the safety and efficacy of putative drugs, animal models recapitulating features of the disease are needed. Here, we will review the existing animal models and the clinical features of coeliac disease they reflect and discuss their relevance for modelling immune pathways that may lead to potential therapeutic approaches.

IL22/IL-22R pathway induces cell survival in human glioblastoma cells.

Interleukin-22 (IL-22) is a member of the IL-10 cytokine family that binds to a heterodimeric receptor consisting of IL-22 receptor 1 (IL-22R1) and IL-10R2. IL-22R expression was initially characterized on epithelial cells, and plays an essential role in a number of inflammatory diseases. Recently, a functional receptor was detected on cancer cells such as hepatocarcinoma and lung carcinoma, but its presence was not reported in glioblastoma (GBM). Two GBM cell lines and 10 primary cell lines established from patients undergoing surgery for malignant GBM were used to investigate the expression of IL-22 and IL-22R by using quantitative RT-PCR, western blotting and confocal microscopy studies. The role of IL-22 in proliferation and survival of GBM cell lines was investigated in vitro by BrdU and ELISA cell death assays. We report herein that the two subunits of the IL-22R complex are expressed on human GBM cells. Their activation, depending on exogenous IL-22, induced antiapoptotic effect and cell proliferation. IL-22 treatment of GBM cells resulted in increased levels of phosphorylated Akt, STAT3 signaling protein and its downstream antiapoptotic protein Bcl-xL and decreased level of phosphorylated ERK1/2. In addition, IL-22R subunits were expressed in all the 10 tested primary cell lines established from GBM tumors. Our results showed that IL-22R is expressed on GBM established and primary cell lines. Depending on STAT3, ERK1/2 and PI3K/Akt pathways, IL-22 induced GBM cell survival. These data are consistent with a potential role of IL-22R in tumorigenesis of GBM. Since endogenous IL-22 was not detected in all studied GBM cells, we hypothesize that IL-22R could be activated by immune microenvironmental IL-22 producing cells.

Neuro-anatomical evidence indicating indirect modulation of macrophages by vagal efferents in the intestine but not in the spleen.

BACKGROUND: Electrical stimulation of the vagus nerve suppresses intestinal inflammation and normalizes gut motility in a mouse model of postoperative ileus. The exact anatomical interaction between the vagus nerve and the intestinal immune system remains however a matter of debate. In the present study, we provide additional evidence on the direct and indirect vagal innervation of the spleen and analyzed the anatomical evidence for neuroimmune modulation of macrophages by vagal preganglionic and enteric postganglionic nerve fibers within the intestine. METHODS: Dextran conjugates were used to label vagal preganglionic (motor) fibers projecting to the small intestine and spleen. Moreover, identification of the neurochemical phenotype of the vagal efferent fibers and enteric neurons was performed by immunofluorescent labeling. F4/80 antibody was used to label resident macrophages. RESULTS: Our anterograde tracing experiments did not reveal dextran-labeled vagal fibers or terminals in the mesenteric ganglion or spleen. Vagal efferent fibers were confined within the myenteric plexus region of the small intestine and mainly endings around nNOS, VIP and ChAT positive enteric neurons. nNOS, VIP and ChAT positive fibers were found in close proximity of intestinal resident macrophages carrying α7 nicotinic receptors. Of note, VIP receptors were found on resident macrophages located in close proximity of VIP positive nerve fibers. CONCLUSION: In the present study, we show that the vagus nerve does not directly interact with resident macrophages in the gut or spleen. Instead, the vagus nerve preferentially interacts with nNOS, VIP and ChAT enteric neurons located within the gut muscularis with nerve endings in close proximity of the resident macrophages.

The spleen responds to intestinal manipulation but does not participate in the inflammatory response in a mouse model of postoperative ileus.

BACKGROUND: Postoperative ileus is characterized by a transient impairment of the gastrointestinal motility after abdominal surgery. The intestinal inflammation, triggered by handling of the intestine, is the main factor responsible for the prolonged dysmotility of the gastrointestinal tract. Secondary lymphoid organs of the intestine were identified as essential components in the dissemination of inflammation to the entire gastrointestinal tract also called field effect. The involvement of the spleen, however, remains unclear. AIM: In this study, we investigated whether the spleen responds to manipulation of the intestine and participates in the intestinal inflammation underlying postoperative ileus. METHODS: Mice underwent Laparotomy (L) or Laparotomy followed by Intestinal Manipulation (IM). Twenty-four hours later, intestinal and colonic inflammation was assessed by QPCR and measurement of the intestinal transit was performed. Analysis of homeostatic chemokines in the spleen was performed by QPCR and splenic cell populations analysed by Flow Cytometry. Blockade of the egress of cells from the spleen was performed by administration of the Sphingosine-1-phosphate receptor 1 (S1P1) agonist CYM-5442 10 h after L/IM. RESULTS: A significant decrease in splenic weight and cellularity was observed in IM mice 24 h post-surgery, a phenomenon associated with a decreased splenic expression level of the homeostatic chemokine CCL19. Splenic denervation restored the expression of CCL19 and partially prevented the reduction of splenocytes in IM mice. Treatment with CYM-5442 prevented the egress of splenocytes but did not ameliorate the intestinal inflammation underlying postoperative ileus. CONCLUSIONS: Intestinal manipulation results in two distinct phenomena: local intestinal inflammation and a decrease in splenic cellularity. The splenic response relies on an alteration of cell trafficking in the spleen and is partially regulated by the splenic nerve. The spleen however does not participate in the intestinal inflammation during POI.

Neural networks in intestinal immunoregulation.

Key physiological functions of the intestine are governed by nerves and neurotransmitters. This complex control relies on two neuronal systems: an extrinsic innervation supplied by the two branches of the autonomic nervous system and an intrinsic innervation provided by the enteric nervous system. As a result of constant exposure to commensal and pathogenic microflora, the intestine developed a tightly regulated immune system. In this review, we cover the current knowledge on the interactions between the gut innervation and the intestinal immune system. The relations between extrinsic and intrinsic neuronal inputs are highlighted with regards to the intestinal immune response. Moreover, we discuss the latest findings on mechanisms underlying inflammatory neural reflexes and examine their relevance in the context of the intestinal inflammation. Finally, we discuss some of the recent data on the identification of the gut microbiota as an emerging player influencing the brain function.