Bernhard Kratzer

Background: Acute peanut-allergic reactions often occur early in life and may follow first known ingestion, indicating that sensitization can precede gastrointestinal exposure. Cutaneous exposure through a disrupted skin barrier is considered an important route of primary sensitization. Langerhans cells (LCs), immune sentinels of stratified epithelia, may contribute to this process.Objective: To characterize peanut-allergen uptake by human LCs and its functional consequences.Methods: Uptake of Alexa Fluor 488-labelled natural Ara h 1 and Ara h 2 was assessed in a human skin explant model and MUTZ-3-derived LC-like cells. Binding of glycosylated and deglycosylated allergens to selected C-type lectin receptors (CLRs) was examined by CLR-based ELISA. For functional analysis, allergen-exposed primary LCs emigrating from human epidermal sheets were co-cultured with allogeneic naive CD4⁺ T cells, followed by multiplex cytokine analysis.Results: Natural Ara h 1 was internalized by resident LCs in barrier-disrupted ex vivo skin and by MUTZ-3-derived LC-like cells in a time- and concentration-dependent manner. Blocking CD207 significantly reduced Ara h 1 uptake. CLR-based ELISA demonstrated glycosylation-dependent binding of Ara h 1 to CD207 Ara h 1-exposed primary LCs induced modest but significant increases in IL-5 and IL-13 production by CD4⁺ T cells, whereas Ara h 2 had no significant effect.Conclusion: By identifying glycan-dependent recognition of Ara h 1 by CD207-expressing LCs as a mechanism of allergen uptake and demonstrating that Ara h 1-exposed LCs promote Th2-associated T cell responses, this study provides mechanistic and functional insight into how human LCs may contribute to peanut sensitization through barrier-disrupted skin.

Virus-like nanoparticles (VNPs) based on Moloney murine leukemia virus represent a well-established platform for the expression of heterologous molecules such as cytokines, cytokine receptors, peptide MHC (pMHC) and major allergens, but their application for inducing protective anti-viral immunity has remained understudied as of yet. Here, we variably fused the wildtype SARS-CoV-2 spike, its receptor-binding domain (RBD) and nucleocapsid (NC) to the minimal CD16b-GPI anchor acceptor sequence for expression on the surface of VNP. Moreover, a CD16b-GPI-anchored single-chain version of IL-12 was tested for its adjuvanticity. VNPs expressing RBD::CD16b-GPI alone or in combination with IL-12::CD16b-GPI were used to immunize BALB/c mice intramuscularly and subsequently to investigate virus-specific humoral and cellular immune responses. CD16b-GPI-anchored viral molecules and IL-12-GPI were well-expressed on HEK-293T-producer cells and purified VNPs. After the immunization of mice with VNPs, RBD-specific antibodies were only induced with RBD-expressing VNPs, but not with empty control VNPs or VNPs solely expressing IL-12. Mice immunized with RBD VNPs produced RBD-specific IgM, IgG_2a and IgG₁ after the first immunization, whereas RBD-specific IgA only appeared after a booster immunization. Protein/peptide microarray and ELISA analyses confirmed exclusive IgG reactivity with folded but not unfolded RBD and showed no specific IgG reactivity with linear RBD peptides. Notably, booster injections gradually increased long-term IgG antibody avidity as measured by ELISA. Interestingly, the final immunization with RBD–Omicron VNPs mainly enhanced preexisting RBD Wuhan Hu-1-specific antibodies. Furthermore, the induced antibodies significantly neutralized SARS-CoV-2 and specifically enhanced cellular cytotoxicity (ADCC) against RBD protein-expressing target cells. In summary, VNPs expressing viral proteins, even in the absence of adjuvants, efficiently induce functional SARS-CoV-2-specific antibodies of all three major classes, making this technology very interesting for future vaccine development and boosting strategies with low reactogenicity.

Virus-like nanoparticles (VNP) are regarded as efficient vaccination platforms and have proven to be useful for the non-anaphylactogenic delivery of allergen-specific immunotherapy in preclinical models previously. Herein, we sought to determine the mode of VNP uptake by antigen presenting cells (APC). Accordingly, we screened a collection of substances known to inhibit different uptake pathways by APC. The human leukemia monocytic cell line THP-1 and the murine dendritic cell line DC 2.4 were examined for the uptake of fluorescently labelled VNP in the presence or absence of inhibitors. The inhibitory effect of candidate substances that blocked VNP uptake in APC lines was subsequently evaluated in studies with primary APC present in splenocyte and lung cell homogenates in vitro and upon intratracheal application of VNP in vivo. The uptake of allergen-specific VNP in vitro and in vivo was mainly observed by macrophages and CD103⁺ dendritic cells and was sensitive to inhibitors that block macropinocytosis, such as hyperosmolarity induced by sucrose or the polyphenol compound Rottlerin at low micromolar concentrations but not by other inhibitors. Also, T-cell proliferation induced by allergen-specific VNP was significantly reduced by both substances. In contrast, substances that stimulate macropinocytosis, such as Heparin and phorbol myristate acetate (PMA), increased VNP-uptake and may, thus, help modulate allergen-specific T-cell responses. We have identified macropinocytosis as the principal uptake mechanism of APC for allergen-specific VNP in vitro and in vivo, paving the way for further improvement of VNP-based therapies, especially those that can be used for tolerance induction in allergy, in the future.

Background: SARS‐CoV‐2 has triggered a pandemic and contributes to long‐lasting morbidity. Several studies have investigated immediate cellular and humoral immune responses during acute infection. However, little is known about long‐term effects of COVID‐19 on the immune system.Methods: We performed a longitudinal investigation of cellular and humoral immune parameters in 106 non‐vaccinated subjects ten weeks (10 w) and ten months (10 m) after their first SARS‐CoV‐2 infection. Peripheral blood immune cells were analyzed by multiparametric flow cytometry, serum cytokines were examined by multiplex technology. Antibodies specific for the Spike protein (S), the receptor‐binding domain (RBD) and the nucleocapsid protein (NC) were determined. All parameters measured 10 w and 10 m after infection were compared with those of a matched, noninfected control group (n = 98).Results: Whole blood flow cytometric analyses revealed that 10 m after COVID‐19, convalescent patients compared to controls had reduced absolute granulocyte, monocyte, and lymphocyte counts, involving T, B, and NK cells, in particular CD3⁺CD45RA⁺CD62L⁺CD31⁺ recent thymic emigrant T cells and non‐class‐switched CD19⁺IgD⁺CD27⁺ memory B cells. Cellular changes were associated with a reversal from Th1‐ to Th2‐dominated serum cytokine patterns. Strong declines of NC‐ and S‐specific antibody levels were associated with younger age (by 10.3 years, p < .01) and fewer CD3⁻CD56⁺ NK and CD19⁺CD27⁺ B memory cells. Changes of T‐cell subsets at 10 m such as normalization of effector and Treg numbers, decline of RTE, and increase of central memory T cell numbers were independent of antibody decline pattern.Conclusions: COVID‐19 causes long‐term reduction of innate and adaptive immune cells which is associated with a Th2 serum cytokine profile. This may provide an immunological mechanism for long‐term sequelae after COVID‐19.

Background: COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has now become endemic and is currently one of the important respiratory virus infections regularly affecting mankind. The assessment of immunity against SARS-CoV-2 and its variants is important for guiding active and passive immunization and SARS-CoV-2-specific treatment strategies. Methods: We here devised a novel flow cytometry-based diagnostic platform for the assessment of immunity against cell-bound virus antigens. This platform is based on a collection of HEK-293T cell lines which, as exemplified in our study, stably express the receptor-binding domains (RBDs) of the SARS-CoV-2 S-proteins of eight major SARS-CoV-2 variants, ranging from Wuhan-Hu-1 to Omicron. Results: RBD-expressing cell lines stably display comparable levels of RBD on the surface of HEK-293T cells, as shown with anti-FLAG-tag antibodies directed against a N-terminally introduced 3x-FLAG sequence while the functionality of RBD was proven by ACE2 binding. We exemplify the usefulness and specificity of the cell-based test by direct binding of IgG and IgA antibodies of SARS-CoV-2-exposed and/or vaccinated individuals in which the assay shows a wide linear performance range both at very low and very high serum antibody concentrations. In another application, i.e., antibody adsorption studies, the test proved to be a powerful tool for measuring the ratios of individual variant-specific antibodies. Conclusion: We have established a toolbox for measuring SARS-CoV-2-specific immunity against cell-bound virus antigens, which may be considered as an important addition to the armamentarium of SARS-CoV-2-specific diagnostic tests, allowing flexible and quick adaptation to new variants of concern.

Background: COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a recurrent endemic disease affecting the whole world. Since November 2021, Omicron and its subvariants have dominated in the spread of the disease. In order to prevent severe courses of disease, vaccines are needed to boost and maintain antibody levels capable of neutralizing Omicron. Recently, we produced and characterized a SARS-CoV-2 vaccine based on a recombinant fusion protein consisting of hepatitis B virus (HBV)-derived PreS and two SARS-CoV-2 wild-type RBDs. Objectives: To develop a PreS-RBD vaccine which induces high levels of Omicron-specific neutralizing antibodies. Methods: We designed, produced, characterized and compared strain-specific (wild-type: W-PreS-W; Omicron: O-PreS-O), bivalent (mix of W-PreS-W and O-PreS-O) and chimeric (i.e., W-PreS-O) SARS-CoV-2 protein subunit vaccines. Immunogens were characterized in vitro using protein chemical methods, mass spectrometry, and circular dichroism in combination with thermal denaturation and immunological methods. In addition, BALB/c mice were immunized with aluminum–hydroxide-adsorbed proteins and aluminum hydroxide alone (i.e., placebo) to study the specific antibody and cytokine responses, safety and Omicron neutralization. Results: Defined and pure immunogens could be produced in significant quantities as secreted and folded proteins in mammalian cells. The antibodies induced after vaccination with different doses of strain-specific, bivalent and chimeric PreS-RBD fusion proteins reacted with wild-type and Omicron RBD in a dose-dependent manner and resulted in a mixed Th1/Th2 immune response. Interestingly, the RBD-specific IgG levels induced with the different vaccines were comparable, but the W-PreS-O-induced virus neutralization titers against Omicron (median VNT50: 5000) were seven- and twofold higher than the W-PreS-W- and O-PreS-O-specific ones, respectively, and they were six-fold higher than those of the bivalent vaccine. Conclusion: Among the tested immunogens, the chimeric PreS-RBD subunit vaccine, W-PreS-O, induced the highest neutralizing antibody titers against Omicron. Thus, W-PreS-O seems to be a highly promising COVID-19 vaccine candidate for further preclinical and clinical evaluation.

Background: COVID-19 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a recurrent endemic disease affecting the whole world. Since November 2021, Omicron and its subvariants are dominating. In order to prevent severe courses of disease, vaccines are needed to boost and maintain antibody levels capable of neutralizing Omicron. Recently we produced and characterized a SARS-CoV-2 vaccine based on a recombinant fusion protein consisting of hepatitis B virus (HBV)-derived PreS and two SARS-CoV-2 wild-type RBDs. Objectives: To develop a PreS-RBD vaccine which induces high levels of Omicron-specific neutralizing antibodies. Methods: We designed, produced, characterized and compared strain-specific (wild-type: W-PreS-W; Omicron: O-PreS-O), bivalent (mix of W-PreS-W and O-PreS-O) and chimeric (i.e., W-PreS-O) SARS-CoV-2 protein subunit vaccines. Immunogens were characterized in vitro by protein chemical methods, mass-spectrometry, circular dichroism in combination with thermal denaturation and immunological methods. In addition, BALB/c mice were immunized with aluminum hydroxide-adsorbed proteins and aluminum hydroxide alone (i.e., placebo) to study specific antibody and cytokine responses, safety and Omicron neutralization. Results: Defined and pure immunogens could be produced in large amounts as secreted and folded proteins in mammalian cells. Antibodies induced after vaccination with different doses of strain-specific, bivalent and chimeric PreS-RBD fusion proteins reacted with wild-type and Omicron RBD in a dose-dependent manner and resulted in a mixed Th1/Th2 immune response. Interestingly, RBD-specific IgG levels induced by the different vaccines were comparable but the W-PreS-O-induced virus neutralization titers against Omicron (median VNT50: 5000) were 7- and 2-fold higher than the W-PreS-W- and O-PreS-O-specific ones, respectively and 6-fold higher than those of the bivalent vaccine. Conclusion: Among the tested immunogens, the chimeric PreS-RBD subunit vaccine, W-PreS-O, induced the highest neutralizing antibody titers against Omicron. Thus, W-PreS-O seems to be a highly promising COVID-19 vaccine candidate for further preclinical and clinical evaluation.

Loss-of-function variants in AP3D1 have been linked to Hermansky–Pudlak syndrome (HPS) 10, a severe multisystem disorder characterized by oculocutaneous albinism, immunodeficiency, neurodevelopmental delay, hearing loss (HL), and neurological abnormalities, fatal in early childhood. Here, we report a consanguineous family who presented with presumably isolated autosomal recessive (AR) HL. Whole-exome sequencing was performed on all core family members, and selected patients were screened using array-based copy-number analysis and karyotyping. Candidate variants were validated by Sanger sequencing and assessed in silico. A homozygous, likely pathogenic p.V711I missense variant in AP3D1 segregated with the HL. The family was characterized by thorough medical and laboratory examination. The HL was consistent across patients and accompanied by neurological manifestations in two brothers. The sole female patient was diagnosed with premature ovarian failure. Further findings, including mild neutropenia and reduced NK-cell cytotoxicity in some as well as brain alterations in all homozygous patients, were reminiscent of HPS10, though milder and lacking the characteristic albinism. Previously unrecognized, milder, isolated HL was identified in all heterozygous carriers. A protein model indicates that the variant interferes with protein–protein interactions. These results suggest that a missense variant alters inner-ear-specific functions leading to HL with mild HPS10-like symptoms of variable penetrance. Milder HL in heterozygous carriers may point towards semi-dominant inheritance of this trait. Since all previously reported HPS10 cases were pediatric, it is unknown whether the observed primary ovarian insufficiency recapitulates the subfertility in Ap3d1-deficient mice.

Background: Antibody‐based tests are available for measuring SARS‐CoV‐2‐specific immune responses but fast T‐cell assays remain scarce. Robust T cell‐based tests are needed to differentiate specific cellular immune responses after infection from those after vaccination.Methods: One hundred seventeen individuals (COVID‐19 convalescent patients: n = 40; SARS‐CoV‐2 vaccinees: n = 41; healthy controls: n = 36) were evaluated for SARS‐CoV‐2‐specific cellular immune responses (proliferation, Th1, Th2, Th17, and inflammatory cytokines, activation‐induced marker [AIM] expression) by incubating purified peripheral blood mononuclear cells (PBMC) or whole blood (WB) with SARS‐CoV‐2 peptides (S, N, or M), vaccine antigens (tetanus toxoid, tick borne encephalitis virus) or polyclonal stimuli (Staphylococcal enterotoxin, phytohemagglutinin).Results: N‐peptide mix stimulation of WB identified the combination of IL‐2 and IL‐13 secretion as superior to IFN‐γ secretion to discriminate between COVID‐19‐convalescent patients and healthy controls (p < .0001). Comparable results were obtained with M‐ or S‐peptides, the latter almost comparably recalled IL‐2, IFN‐γ, and IL‐13 responses in WB of vaccinees. Analysis 10 months as opposed to 10 weeks after COVID‐19, but not allergic disease status, positively correlated with IL‐13 recall responses. WB cytokine responses correlated with cytokine and proliferation responses of PBMC. Antigen‐induced neo‐expression of the C‐type lectin CD69 on CD4⁺ (p < .0001) and CD8⁺ (p = .0002) T cells informed best about the SARS‐CoV‐2 exposure status with additional benefit coming from CD25 upregulation.Conclusion: Along with N‐ and S‐peptide‐induced IL‐2 and CD69 neo‐expression, we suggest to include the type 2 cytokine IL‐13 as T‐cellular recall marker for SARS‐CoV‐2 specific T‐cellular immune responses after infection and vaccination.