Neutralization of SARS-CoV-2 Variants of Concern in Kidney Transplant Recipients after Standard COVID-19 Vaccination.

BACKGROUND AND OBJECTIVES: Antibody response after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination is impaired in kidney transplant recipients. Emerging variants, such as B.1.617.2 (δ), are of particular concern because of their higher transmissibility and partial immune escape. Little is known about protection against these variants in immunocompromised patients. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: In this prospective two-center study, antispike 1 IgG and surrogate neutralizing antibodies were measured in 173 kidney transplant recipients and 166 healthy controls with different vaccination schedules. In addition, different SARS-CoV-2 epitope antibodies from 135 vaccinated kidney transplant recipients were compared with antibodies in 25 matched healthy controls after second vaccination. In 36 kidney transplant recipients with seroconversion, neutralization against B.1.1.7 (α), B.1.351 (ß), and B.1.617.2 (δ) was determined on VeroE6 cells and compared with neutralization in 25 healthy controls. RESULTS: Kidney transplant recipients had significantly lower seroconversion rates compared with healthy controls. After the second vaccination, antispike 1, antireceptor-binding domain, and surrogate neutralizing antibodies were detectable in 30%, 27%, and 24% of kidney transplant recipients, respectively. This compares with 100%, 96%, and 100% in healthy controls, respectively (P<0.001). Neutralization against B.1.1.7 was detectable in all kidney transplant recipients with seroconversion, with a median serum dilution that reduces infection of cells by 50% of 80 (interquartile range, 80-320). In contrast, only 23 of 36 (64%) and 24 of 36 (67%) kidney transplant recipients showed neutralization against B.1.351 and B.1.617.2, respectively, with median serum dilutions that reduce infection of cells by 50% of 20 (interquartile range, 0-40) and 20 (interquartile range, 0-40), respectively. Neutralization against different variants was significantly higher in healthy controls (P<0.001), with all patients showing neutralization against all tested variants. CONCLUSIONS: Seroconverted kidney transplant recipients show impaired neutralization against emerging variants of concern after standard two-dose vaccination. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Observational study to assess the SARS-CoV-2 specific immune response in kidney transplant recipients (COVID-19 related immune response), DRKS00024668.

Humoral Responses to Single-Dose BNT162b2 mRNA Vaccination in Dialysis Patients Previously Infected With SARS-CoV-2.

Seroconversion rates following infection and vaccination are lower in dialysis patients compared to healthy controls. There is an urgent need for the characterization of humoral responses and success of a single-dose SARS-CoV-2 vaccination in previously infected dialysis patients. We performed a dual-center cohort study comparing three different groups: 25 unvaccinated hemodialysis patients after PCR-confirmed COVID-19 (Group 1), 43 hemodialysis patients after two-time BNT162b2 vaccination without prior SARS-CoV-2 infection (Group 2), and 13 single-dose vaccinated hemodialysis patients with prior SARS-CoV-2 infection (Group 3). Group 3 consists of seven patients from Group 1 and 6 additional patients with sera only available after single-dose vaccination. Anti-S1 IgG, neutralizing antibodies, and antibodies against various SARS-CoV-2 protein epitopes were measured 3 weeks after the first and 3 weeks after the second vaccination in patients without prior SARS-CoV-2 infection, 6 weeks after the onset of COVID-19 in unvaccinated patients, and 3 weeks after single-dose vaccination in patients with prior SARS-CoV-2 infection, respectively. Unvaccinated patients after COVID-19 showed a significantly higher neutralizing antibody capacity than two-time vaccinated patients without prior COVID-19 [median (IQR) percent inhibition 88.0 (71.5-95.5) vs. 50.7 (26.4-81.0); P = 0.018]. After one single vaccine dose, previously infected individuals generated 15- to 34-fold higher levels of anti-S1 IgG than age- and dialysis vintage-matched unvaccinated patients after infection or two-time vaccinated patients without prior SARS-CoV-2 infection with a median (IQR) index of 274 (151-791) compared to 18 (8-41) and 8 (1-21) (for both P < 0.001). With a median (IQR) percent inhibition of 97.6 (97.2-98.9), the neutralizing capacity of SARS-CoV-2 antibodies was significantly higher in single-dose vaccinated patients with prior SARS-CoV-2 infection compared to other groups (for both P < 0.01). Bead-based analysis showed high antibody reactivity against various SARS-CoV-2 spike protein epitopes after single-dose vaccination in previously infected patients. In conclusion, single-dose vaccination in previously infected dialysis patients induced a strong and broad antibody reactivity against various SARS-CoV-2 spike protein epitopes with high neutralizing capacity.

Heterologous ChAdOx1 nCoV-19/BNT162b2 Prime-Boost Vaccination Induces Strong Humoral Responses among Health Care Workers.

Despite limited data on safety and immunogenicity, heterologous prime-boost vaccination is currently recommended for individuals with ChAdOx1 nCoV-19 prime immunization in certain age groups. In this prospective, single-center study we included 166 health care workers from Heidelberg University Hospital who received either heterologous ChAdOx1 nCoV-19/BNT162b2, homologous BNT162b2 or homologous ChAdOx1 nCoV-19 vaccination between December 2020 and May 2021. We measured anti-S1 IgG, SARS-CoV-2 specific neutralizing antibodies, and antibodies against different SARS-CoV-2 fragments 0-3 days before and 19-21 days after boost vaccination. Before boost, 55/70 (79%) ChAdOx1 nCoV-19-primed compared with 44/45 (98%) BNT162b2-primed individuals showed positive anti-S1 IgG with a median (IQR) anti-S1 IgG index of 1.95 (1.05-2.99) compared to 9.38 (6.26-17.12). SARS-CoV-2 neutralizing antibodies exceeded the threshold in 24/70 (34%) of ChAdOx1 nCoV-19-primed and 43/45 (96%) of BNT162b2-primed individuals. After boosting dose, median (IQR) anti-S1 IgG index in heterologous ChAdOx1 nCoV-19/BNT162b2 vaccinees was 116.2 (61.84-170), compared to 13.09 (7.03-29.02) in homologous ChAdOx1 nCoV-19 and 145.5 (100-291.1) in homologous BNT162b2 vaccinees. All boosted vaccinees exceeded the threshold for neutralization, irrespective of their vaccination scheme. Vaccination was well-tolerated overall. We show that heterologous ChAdOx1 nCoV-19/BNT162b2 vaccination is safe and induces a strong and broad humoral response in healthy individuals.

Early Humoral Responses of Hemodialysis Patients after COVID-19 Vaccination with BNT162b2.

BACKGROUND AND OBJECTIVES: Patients receiving hemodialysis are at high risk for both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and severe coronavirus disease 2019. A lifesaving vaccine is available, but sensitivity to vaccines is generally lower in patients on dialysis. Little is yet known about antibody responses after coronavirus disease 2019 (COVID-19) vaccination in this vulnerable group. DESIGN, SETTING, PARTICIPANTS, AND MEASUREMENTS: In this prospective single-center study, we included 22 patients on dialysis and 46 healthy controls from Heidelberg University Hospital between December 2020 and February 2021. We measured anti-S1 IgG with a threshold index for detection greater than one, neutralizing antibodies with a threshold for viral neutralization of ≥30%, and antibodies against different SARS-CoV2 fragments 17-22 days after the first dose and 18-22 days after the second dose of the mRNA vaccine BNT162b2. RESULTS: After the first vaccine dose, four of 22 (18%) patients on dialysis compared with 43 of 46 (93%) healthy controls developed positive anti-S1 IgG, with a median anti-S1 IgG index of 0.2 (interquartile range, 0.1-0.7) compared with nine (interquartile range, 4-16), respectively. SARS-CoV2 neutralizing antibodies exceeded the threshold for neutralization in four of 22 (18%) patients on dialysis compared with 43 of 46 (93%) healthy controls, with a median percent inhibition of 11 (interquartile range, 3-24) compared with 65 (interquartile range, 49-75), respectively. After the second dose, 14 of 17 (82%) patients on dialysis developed neutralizing antibodies exceeding the threshold for viral neutralization and antibodies against the receptor binding S1 domain of the spike protein, compared with 46 of 46 (100%) healthy controls, respectively. The median percent inhibition was 51 (interquartile range, 32-86) compared with 98 (interquartile range, 97-98) in healthy controls. CONCLUSIONS: Patients receiving long-term hemodialysis show a reduced antibody response to the first and second doses of the mRNA vaccine BNT162b2. The majority (82%) develop neutralizing antibodies after the second dose but at lower levels than healthy controls.

Characterization of experimental diabetic neuropathy using multicontrast magnetic resonance neurography at ultra high field strength.

In light of the limited treatment options of diabetic polyneuropathy (DPN) available, suitable animal models are essential to investigate pathophysiological mechanisms and to identify potential therapeutic targets. In vivo evaluation with current techniques, however, often provides only restricted information about disease evolution. In the study of patients with DPN, magnetic resonance neurography (MRN) has been introduced as an innovative diagnostic tool detecting characteristic lesions within peripheral nerves. We developed a novel multicontrast ultra high field MRN strategy to examine major peripheral nerve segments in diabetic mice non-invasively. It was first validated in a cross-platform approach on human nerve tissue and then applied to the popular streptozotocin(STZ)-induced mouse model of DPN. In the absence of gross morphologic alterations, a distinct MR-signature within the sciatic nerve was observed mirroring subtle changes of the nerves' fibre composition and ultrastructure, potentially indicating early re-arrangements of DPN. Interestingly, these signal alterations differed from previously reported typical nerve lesions of patients with DPN. The capacity of our approach to non-invasively assess sciatic nerve tissue structure and function within a given mouse model provides a powerful tool for direct translational comparison to human disease hallmarks not only in diabetes but also in other peripheral neuropathic conditions.

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry.

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated ≥ 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Gastric bypass simultaneously improves adipose tissue function and insulin-dependent type 2 diabetes mellitus.

OBJECTIVE: The underlying causes of type 2 diabetes (T2DM) remain poorly understood. Adipose tissue dysfunction with high leptin, inflammation, and increased oxidative stress may play a pivotal role in T2DM development in obese patients. Little is known about the changes in the adipose tissue after Roux-Y gastric bypass (RYGB) in non-severely obese patients (BMI < 35 kg/m2) and since these patients have more T2DM-associated complications than obese patients ("obesity paradox"), we investigated changes in adipose tissue function in a cohort of BMI <35 kg/m2 with insulin-dependent T2DM after RYGB surgery which resolves T2DM. METHODS: Twenty patients with insulin-dependent T2DM and BMI <35 kg/m2 underwent RYGB. Insulin-resistance, leptin, oxidative stress, and cytokines were determined over 24 months. Expression of cytokines and NF-kappaB pathway genes were measured in leukocytes (PBMC). Adipose tissue inflammation was examined histologically preoperatively and 24 months after RGYB in subcutaneous adipose tissue. RESULTS: Insulin-resistance, leptin, oxidative stress as well as adipose tissue inflammation decreased significantly after RYGB. Similarly, systemic inflammation was reduced and peripheral blood mononuclear cells (PBMCs) were reprogrammed towards an M2-type inflammation. Loss of BMI correlated with leptin levels (r = 0.891, p < 0.0001), insulin resistance (r = 0.527, p = 0.003), and oxidative stress (r = 0.592, p = 0.016). Leptin correlated with improved insulin resistance (r = 0.449, p = 0.032) while reduced leptin showed a strong association with improved oxidative stress (r = 0.809, p = 0.001). Lastly, reduced oxidative stress correlated strongly with improved insulin-resistance (r = 0.776, p = 0.001). CONCLUSIONS: RYGB improves adipose tissue function and inflammation. Leptin as marker for adipose tissue dysfunction may be the mediating factor between insulin resistance and oxidative stress and thereby likely improving T2DM.

Electrical Muscle Stimulation Induces an Increase of VEGFR2 on Circulating Hematopoietic Stem Cells in Patients With Diabetes.

PURPOSE: External electric muscle stimulation (EMS) of the thigh muscles was found to reduce pain resulting from diabetic neuropathy (DN), a vascular complication of diabetes. This study investigated circulating hematopoietic stem cells (HSCs) after EMS treatment. Impaired function of HSCs and the subpopulation endothelial progenitor cells (EPCs), important for neovascularization and endothelial repair, has been associated with DN. METHODS: Twenty-four patients with painful DN were treated 3 times with EMS over a period of 1 week. Blood samples were collected before and after the first EMS treatment. Before a fourth treatment, neuropathic pain was evaluated and a third blood sample was collected. Cells were used for flow cytometry. FINDINGS: Patients with painful DN reported that the pain decreased after 3 times of 1-hour treatments with EMS (Neuropathy Symptom Score: from 8 to 6, P = 0.001; Neuropathy Disability Score: from 5.5 to 5, P = 0.027, n = 24). At the end of the study, diastolic blood pressure had decreased from 80 to 70 mm Hg (P = 0.043), and plasma adrenaline and noradrenaline metabolites metanephrine and normetanephrine were reduced (both P ≤ 0.01; n = 21). A single EMS treatment caused an immediate and transient decrease in the frequency of CD34+ HSCs in circulation (-20%; P < 0.001; n = 27). In 9 of the patients with DN, the proportion of HSCs expressing vascular endothelial growth factor receptor 2 (VEGFR2; defining the HSCs as EPCs) increased by 36% (P = 0.011) after EMS treatment. Proteins required for binding to endothelium (junctional adhesion molecule A and CD31), homing toward hypoxic tissue (C-X-C chemokine receptor type 4), and endothelial differentiation (CD31) were increased on HSCs immediately after EMS treatment. An increased frequency of VEGFR2 expression was also observed on HSCs of 6 healthy control volunteers (34%; P = 0.046) after EMS treatment, but not after sham treatment. IMPLICATIONS: Three EMS treatments decreased symptoms of pain caused by DN and reduced diastolic blood pressure and biomarkers of stress. A single EMS treatment increased molecules mediating attachment and differentiation on the surface of HSCs in circulation. We hypothesize that the EMS-induced increase in surface attachment molecules on the HSCs caused the HSCs to leave circulation and that EMS treatment improves the function of HSCs and EPCs in vivo.

STZ causes depletion of immune cells in sciatic nerve and dorsal root ganglion in experimental diabetes.

Streptozotocin (STZ) treatment, a common model for inducing diabetes in rodent models, induces thermal hyperalgesia and neuronal toxicity independently of hyperglycemia by oxidizing and activating TRPA1 and TRPV1. Following treatment with STZ, CD45+ immune cells were found to be depleted in sciatic nerve (SN) and DRG in mice, prior to hyperglycemia. Macrophages were also lost in DRG and NFκB-p65-activation was increased in SN macrophages. Immune cells were significantly reduced in both SN and DRG up to three weeks, post-treatment. Loss of PNS-resident macrophages in response to STZ-mediated toxicity may affect the regenerative capacity of the nerve in response to further injury caused by diabetes.

Influence of insulin and glargine on outgrowth and number of circulating endothelial progenitor cells in type 2 diabetes patients: a partially double-blind, randomized, three-arm unicenter study.

BACKGROUND: Endothelial progenitor cells (EPC) are bone marrow-derived cells which can undergo differentiation into endothelial cells and participate in endothelial repair and angiogenesis. Insulin facilitates this in vitro mediated by the IGF-1 receptor. Clinical trials showed that the number of circulating EPCs is influenced by glucose control and EPC are a predictor of cardiovascular death. To study direct effects of insulin treatment on EPCs in type 2 diabetes patients, add-on basal insulin treatment was compared to an escalation of oral medication aiming at similar glucose control between the groups. METHODS: 55 patients with type 2 diabetes (61.6±5.9 years) on oral diabetes medication were randomized in a 2:2:1 ratio in 3 groups. Patients were treated additionally with insulin glargine (n=20), NPH insulin (n=22) or escalated with oral medication (n=13). Number of circulating EPC, EPC-outgrowth, intima media thickness, skin microvascular function and HbA1c were documented at baseline and/or after 4 weeks and 4 months. RESULTS: HbA1c at baseline was, 7.3+/-0.7% in the oral group, 7.3+/-0.9% and 7.5+/-0.7% in the glargine and NPH insulin respectively (p=0.713). HbA1c after 4 months decreased to 6.8+/-0.8%, 6.6+/-0.7% and 6.7+/-0.6%, in the oral, glargine and NPH insulin group respectively (p=0.61). FACS analysis showed no difference in number of circulating EPC between the groups after 4 weeks and 4 months. However, the outgrowth of EPCs as detected by colony forming assay was increased in the NPH insulin and glargine groups (29.2+/-6.4 and 29.4+/- 6.7 units respectively) compared to the group on oral medication (23.2+/-6.3, p=0.013) after 4 months of treatment. A significant decrease of IMT from 0.80mm (+/-0.14) at baseline to 0.76mm (+/-0.12) after 4 months could be observed in all patients only (p=0.03) with a trend towards a reduction of IMT after 4 months when all patients on insulin treatment were compared to the oral treatment group (p=0.06). Skin microvascular function revealed no differences between the groups (p=0.74). CONCLUSION: The study shows that a 4-month treatment with add-on insulin significantly increases the outgrowth of EPC in patients with type 2 diabetes mellitus. TRIAL REGISTRATION: (Clinical Trials Identifier: NCT00523393).

Cutting edge: TLR13 is a receptor for bacterial RNA.

Bacterial RNA (bRNA) can induce cytokine production in macrophages and dendritic cells (DCs) through a previously unidentified receptor. Gene expression analysis of murine DCs showed that bRNA induced gene regulation similar to that induced by stimulation of TLR7 with R848. Although TLR7 was dispensable for cytokine induction by bRNA, TLR-associated proteins MyD88 and UNC93B were required. TLR13 is an endosomal murine TLR that has been described to interact with UNC93B with, so far, no characterized ligand. Small interfering RNA against TLR13 reduced cytokine induction by bRNA in DCs. Moreover, Chinese hamster ovary cells transfected with TLR13, but not with TLR7 or 8, could activate NF-κB in response to bRNA or Streptococcus pyogenes in an RNA-specific manner. TLR7 antagonist IRS661 could, in addition, inhibit TLR13 signaling and reduced recognition of whole Gram-positive bacteria by DCs, also in the absence of TLR7. The results identify TLR13 as a receptor for bRNA.

A model system for in vitro studies of bank vole borne viruses.

The bank vole (Myodes glareolus) is a common small mammal in Europe and a natural host for several important emerging zoonotic viruses, e.g. Puumala hantavirus (PUUV) that causes hemorrhagic fever with renal syndrome (HFRS). Hantaviruses are known to interfere with several signaling pathways in infected human cells, and HFRS is considered an immune-mediated disease. There is no in vitro-model available for infectious experiments in bank vole cells, nor tools for analyses of bank vole immune activation and responses. Consequently, it is not known if there are any differences in the regulation of virus induced responses in humans compared to natural hosts during infection. We here present an in vitro-model for studies of bank vole borne viruses and their interactions with natural host cell innate immune responses. Bank vole embryonic fibroblasts (VEFs) were isolated and shown to be susceptible for PUUV-infection, including a wild-type PUUV strain (only passaged in bank voles). The significance of VEFs as a model system for bank vole associated viruses was further established by infection studies showing that these cells are also susceptible to tick borne encephalitis, cowpox and Ljungan virus. The genes encoding bank vole IFN-ß and Mx2 were partially sequenced and protocols for semi-quantitative RT-PCR were developed. Interestingly, PUUV did not induce an increased IFN-ß or Mx2 mRNA expression. Corresponding infections with CPXV and LV induced IFN-ß but not Mx2, while TBEV induced both IFN-ß and Mx2. In conclusion, VEFs together with protocols developed for detection of bank vole innate immune activation provide valuable tools for future studies of how PUUV and other zoonotic viruses affect cells derived from bank voles compared to human cells. Notably, wild-type PUUV which has been difficult to cultivate in vitro readily infected VEFs, suggesting that embryonic fibroblasts from natural hosts might be valuable for isolation of wild-type hantaviruses.

Nonhematopoietic cells control the outcome of infection with Listeria monocytogenes in a nucleotide oligomerization domain 1-dependent manner.

We analyzed the defensive role of the cytosolic innate recognition receptor nucleotide oligomerization domain 1 (NOD1) during infection with Listeria monocytogenes. Mice lacking NOD1 showed increased susceptibility to systemic intraperitoneal and intravenous infection with high or low doses of L. monocytogenes, as measured by the bacterial load and survival. NOD1 also controlled dissemination of L. monocytogenes into the brain. The increased susceptibility to reinfection of NOD1(-/-) mice was not associated with impaired triggering of listeria-specific T cells, and similar levels of costimulatory molecules or activation of dendritic cells was observed. Higher numbers of F480(+) Gr1(+) inflammatory monocytes and lower numbers of F480(-) Gr1(+) neutrophils were recruited into the peritoneum of infected WT mice than into the peritoneum of infected NOD1(-/-) mice. We determined that nonhematopoietic cells accounted for NOD1-mediated resistance to L. monocytogenes in bone marrow radiation chimeras. The levels of NOD1 mRNA in fibroblasts and bone marrow-derived macrophages (BMM) were upregulated after infection with L. monocytogenes or stimulation with different Toll-like receptor ligands. NOD1(-/-) BMM, astrocytes, and fibroblasts all showed enhanced intracellular growth of L monocytogenes compared to WT controls. Gamma interferon-mediated nitric oxide production and inhibition of L. monocytogenes growth were hampered in NOD1(-/-) BMM. Thus, NOD1 confers nonhematopoietic cell-mediated resistance to infection with L. monocytogenes and controls intracellular bacterial growth in different cell populations in vitro.

Increased human immunodeficiency virus type 1 Env expression and antibody induction using an enhanced alphavirus vector.

Viral vectors encoding heterologous vaccine antigens are potent inducers of cellular immune responses, but they are generally less efficient at stimulating humoral immunity. To improve the induction of antibody responses by Semliki Forest virus-based vaccines, a vector encoding a translation-enhancer element and a novel internal signal sequence for increased expression and secretion of soluble antigens was designed. Approximately tenfold more human immunodeficiency virus type 1 gp120 was secreted into culture supernatants of infected cells using the enhanced vector compared with the parental vector. This translated into a significant increase in gp120-specific antibodies in immunized mice, suggesting that antigen-expression levels from the parental vector are limiting for induction of antibody responses. These data encourage the use of the enhanced vector for elicitation of immune responses against heterologous antigens during vaccination.

Role of interferon regulatory factor 3 in type I interferon responses in rotavirus-infected dendritic cells and fibroblasts.

The main pathway for the induction of type I interferons (IFN) by viruses is through the recognition of viral RNA by cytosolic receptors and the subsequent activation of interferon regulatory factor 3 (IRF-3), which drives IFN-alpha/beta transcription. In addition to their role in inducing an antiviral state, type I IFN also play a role in modulating adaptive immune responses, in part via their effects on dendritic cells (DCs). Many viruses have evolved mechanisms to interfere with type I IFN induction, and one recently reported strategy for achieving this is by targeting IRF-3 for degradation, as shown for rotavirus nonstructural protein 1 (NSP1). It was therefore of interest to investigate whether rotavirus-exposed DCs would produce type I IFN and/or mature in response to the virus. Our results demonstrate that IRF-3 was rapidly degraded in rotavirus-infected mouse embryonic fibroblasts (MEFs) and type I IFN was not detected in these cultures. In contrast, rotavirus induced type I IFN production in myeloid DCs (mDCs), resulting in their activation. Type I IFN induction in response to rotavirus was reduced in mDCs from IRF-3(-/-) mice, indicating that IRF-3 was important for mediating the response. Exposure of mDCs to UV-treated rotavirus induced significantly higher type I IFN levels, suggesting that rotavirus-encoded functions also antagonized the response in DCs. However, in contrast to MEFs, this action was not sufficient to completely abrogate type I IFN induction, consistent with a role for DCs as sentinels for virus infection.

DAP12 signaling regulates plasmacytoid dendritic cell homeostasis and down-modulates their function during viral infection.

DAP12 is an ITAM-containing adaptor molecule conveying activating properties to surface receptors on many cell types. We show here that DAP12 paradoxically down-modulates plasmacytoid dendritic cell (pDC) cytokine production in vivo during murine CMV (MCMV) infection. Higher levels of IFN-alphabeta and IL-12 were detected upon MCMV infection or CpG treatment in DAP12-deficient (DAP12(o)) mice as compared with wild-type (WT) mice. This resulted from altered homeostasis and enhanced responsiveness of pDCs in DAP12(o) animals. Increased numbers of pDCs were observed in the periphery of both naive and MCMV-infected DAP12(o) mice. A higher proportion of pDCs was activated in infected DAP12(o) mice, as demonstrated by intracellular staining using an optimized protocol for simultaneous detection of IFN-alpha and IFN-beta. The homeostasis of WT and DAP12(o) pDCs did not differ in mixed bone marrow chimeric mice. In addition, a similar efficiency of pDC differentiation was observed in vitro in Fms-like tyrosine kinase receptor 3 ligand cultures of WT and DAP12(o) bone marrow cells. This suggests that DAP12 signaling effects on pDC homeostasis are indirect. In contrast, in response to CpG, DAP12-mediated effects on both IL-12 and IFN-alphabeta production were intrinsic to the pDCs. However, in response to MCMV, only IL-12 but not IFN-alphabeta production was affected by pDC-intrinsic DAP12 signaling. Thus, DAP12 signaling in pDCs can mediate different regulatory effects on their functions, depending on the mechanisms of pDC activation. The potential implications of the regulation of pDC functions by DAP12 for promoting health over disease are discussed.

Humoral responses against coimmunized protein antigen but not against alphavirus-encoded antigens require alpha/beta interferon signaling.

Viruses typically elicit potent adaptive immune responses, and live-virus-based vaccines are among the most efficient human vaccines known. The mechanisms by which viruses stimulate adaptive immune responses are not fully understood, but activation of innate immune signaling pathways in the early phase of the infection may be of importance. In addition to stimulating immune responses to viral antigens expressed in infected cells, viruses can also provide adjuvant signals to coimmunized protein antigens. Using recombinant Semliki Forest virus (rSFV)-based vaccines, we show that rSFV potently enhanced antibody responses against coimmunized protein antigens in the absence of other exogenously added adjuvants. Elicitation of antibody responses against both virus-encoded antigens and coimmunized protein antigens was independent of the signaling via Toll-like receptors (TLRs) previously implicated in antiviral responses. In contrast, the adjuvant effect of rSFV on coimmunized protein was completely abolished in mice lacking the alpha/beta interferon (IFN-alpha/beta) receptor (IFN-AR1), demonstrating that IFN-alpha/beta signaling was critical for mediating this effect. Antibody responses directed against virus-encoded antigens were intact in IFN-AR1(-/-) mice, suggesting that other signals are sufficient to drive immune responses against virally encoded antigens. These data provide a basis for the adjuvant effect of rSFV and show that different signals are required to stimulate antibody responses to virally encoded antigens and to antigens administered as purified protein vaccines, together with viral particles.

STAT1 regulates IFN-alpha beta- and IFN-gamma-dependent control of infection with Chlamydia pneumoniae by nonhemopoietic cells.

STAT1 mediates signaling in response to IFN-alpha, -beta, and -gamma, cytokines required for protective immunity against several viral, bacterial, and eukaryotic pathogens. The protective role of STAT1 in the control of intranasal infection with the obligate intracellular bacterium Chlamydia pneumoniae was analyzed. IFN-gamma-/- or IFN-gamma receptor (R)-/- mice were highly susceptible to infection with C. pneumoniae. We found that STAT1-/- mice were even more susceptible to C. pneumoniae than IFN-gamma-/- or IFN-gammaR-/- mice. Phosphorylation of STAT1 was detected in the lungs of C. pneumoniae-infected wild-type, IFN-gammaR-/-, and IFN-alphabetaR-/- mice, but not in mice lacking both IFN-alphabetaR and IFN-gammaR. In line with this, IFN-alphabetaR-/-/IFN-gammaR-/- mice showed increased susceptibility to infection compared with IFN-gammaR-/- mice. However, C. pneumoniae-infected IFN-alphabetaR-/- or IFN regulatory factor 3-/- mice showed no increased susceptibility and similar IFN-gamma expression compared with wild-type mice. CD4+ or CD8+ cells released IFN-gamma in vivo and conferred protection against C. pneumoniae in a STAT1-independent manner. In contrast, STAT1 mediated a nonredundant protective role of nonhemopoietic cells but not of hemopoietic cells. Nonhemopoietic cells accounted for the expression of STAT1-mediated indoleamine 2, 3-dioxygenase and the p47 GTPase LRG-47, but not inducible NO synthase mRNA. In summary, we demonstrate that STAT1 mediates a cooperative effect of IFN-alphabeta and IFN-gamma on nonhemopoietic cells, resulting in protection against C. pneumoniae.

Early alpha/beta interferon production by myeloid dendritic cells in response to UV-inactivated virus requires viral entry and interferon regulatory factor 3 but not MyD88.

Alpha/beta interferons (IFN-alpha/beta) are key mediators of innate immunity and important modulators of adaptive immunity. The mechanisms by which IFN-alpha/beta are induced are becoming increasingly well understood. Recent studies showed that Toll-like receptors 7 and 8 expressed by plasmacytoid dendritic cells (pDCs) mediate the endosomal recognition of incoming viral RNA genomes, a process which requires myeloid differentiation factor 88 (MyD88). Here we investigate the requirements for virus-induced IFN-alpha/beta production in cultures of bone marrow-derived murine myeloid DCs (mDCs). Using recombinant Semliki Forest virus blocked at different steps in the viral life cycle, we show that replication-defective virus induced IFN-alpha/beta in mDCs while fusion-defective virus did not induce IFN-alpha/beta. The response to replication-defective virus was largely intact in MyD88-/- mDC cultures but was severely reduced in mDC cultures from mice lacking IFN regulatory factor 3. Our observations suggest that mDCs respond to incoming virus via a pathway that differs from the fusion-independent, MyD88-mediated endosomal pathway described for the induction of IFN-alpha/beta in pDCs. We propose that events during or downstream of viral fusion, but prior to replication, can activate IFN-alpha/beta in mDCs. Thus, mDCs may contribute to the antiviral response activated by the immune system at early time points after infection.