Systems biology analysis reveals distinct molecular signatures associated with immune responsiveness to the BNT162b COVID-19 vaccine.

BACKGROUND: Human immune responses to COVID-19 vaccines display a large heterogeneity of induced immunity and the underlying immune mechanisms for this remain largely unknown. METHODS: Using a systems biology approach, we longitudinally profiled a unique cohort of female high and low responders to the BNT162b vaccine, who were known from previous COVID-19 vaccinations to develop maximum and minimum immune responses to the vaccine. We utilized high dimensional flow cytometry, bulk and single cell mRNA sequencing and 48-plex serum cytokine analyses. FINDINGS: We revealed early, transient immunological and molecular signatures that distinguished high from low responders and correlated with B and T cell responses measured 14 days later. High responders featured a distinct transcriptional activity of interferon-driven genes and genes connected to enhanced antigen presentation. This was accompanied by a robust cytokine response related to Th1 differentiation. Both transcriptome and serum cytokine signatures were confirmed in two independent confirmatory cohorts. INTERPRETATION: Collectively, our data contribute to a better understanding of the immunogenicity of mRNA-based COVID-19 vaccines, which might lead to the optimization of vaccine designs for individuals with poor vaccine responses. FUNDING: German Center for Infection Research, German Center for Lung Research, German Research Foundation, Excellence Strategy EXC 2155 "RESIST" and European Regional Development Fund.

Omicron infection-associated T- and B-cell immunity in antigen-naive and triple-COVID-19-vaccinated individuals.

Since early 2022, various Omicron variants have dominated the SARS-CoV-2 pandemic in most countries. All Omicron variants are B-cell immune escape variants, and antibodies induced by first-generation COVID-19 vaccines or by infection with earlier SARS-CoV-2 variants largely fail to protect individuals from Omicron infection. In the present study, we investigated the effect of Omicron infections in triple-vaccinated and in antigen-naive individuals. We show that Omicron breakthrough infections occurring 2-3.5 months after the third vaccination restore B-cell and T-cell immune responses to levels similar to or higher than those measured 14 days after the third vaccination, including the induction of Omicron-neutralizing antibodies. Antibody responses in breakthrough infection derived mostly from cross-reacting B cells, initially induced by vaccination, whereas Omicron infections in antigen-naive individuals primarily generated B cells binding to the Omicron but not the Wuhan spike protein. Although antigen-naive individuals mounted considerable T-cell responses after infection, B-cell responses were low, and neutralizing antibodies were frequently below the limit of detection. In summary, the detection of Omicron-associated B-cell responses in primed and in antigen-naive individuals supports the application of Omicron-adapted COVID-19 vaccines, but calls into question their suitability if they also contain/encode antigens of the original Wuhan virus.

BNT162b2-boosted immune responses six months after heterologous or homologous ChAdOx1nCoV-19/BNT162b2 vaccination against COVID-19.

Heterologous prime/boost vaccination with a vector-based approach (ChAdOx-1nCov-19, ChAd) followed by an mRNA vaccine (e.g. BNT162b2, BNT) has been reported to be superior in inducing protective immunity compared to repeated application of the same vaccine. However, data comparing immunity decline after homologous and heterologous vaccination as well as effects of a third vaccine application after heterologous ChAd/BNT vaccination are lacking. Here we show longitudinal monitoring of ChAd/ChAd (n = 41) and ChAd/BNT (n = 88) vaccinated individuals and the impact of a third vaccination with BNT. The third vaccination greatly augments waning anti-spike IgG but results in only moderate increase in spike-specific CD4 + and CD8 + T cell numbers in both groups, compared to cell frequencies already present after the second vaccination in the ChAd/BNT group. More importantly, the third vaccination efficiently restores neutralizing antibody responses against the Alpha, Beta, Gamma, and Delta variants of the virus, but neutralizing activity against the B.1.1.529 (Omicron) variant remains severely impaired. In summary, inferior SARS-CoV-2 specific immune responses following homologous ChAd/ChAd vaccination can be compensated by heterologous BNT vaccination, which might influence the choice of vaccine type for subsequent vaccination boosts.

Longitudinal Tracking of Immune Responses in COVID-19 Convalescents Reveals Absence of Neutralization Activity Against Omicron and Staggered Impairment to Other SARS-CoV-2 Variants of Concern.

Evaluating long-term protection against SARS-CoV-2 variants of concern in convalescing individuals is of high clinical relevance. In this prospective study of a cohort of 46 SARS-CoV-2 patients infected with the Wuhan strain of SARS-CoV-2 we longitudinally analyzed changes in humoral and cellular immunity upon early and late convalescence. Antibody neutralization capacity was measured by surrogate virus neutralization test and cellular responses were investigated with 31-colour spectral flow cytometry. Spike-specific, isotype-switched B cells developed already during the disease phase, showed a memory phenotype and did not decrease in numbers even during late convalescence. Otherwise, no long-lasting perturbations of the immune compartment following COVID-19 clearance were observed. During convalescence anti-Spike (S1) IgG antibodies strongly decreased in all patients. We detected neutralizing antibodies against the Wuhan strain as well as the Alpha and Delta but not against the Beta, Gamma or Omicron variants for up to 7 months post COVID-19. Furthermore, correlation analysis revealed a strong association between sera anti-S1 IgG titers and their neutralization capacity against the Wuhan strain as well as Alpha and Delta. Overall, our data suggest that even 7 month after the clearance of COVID-19 many patients possess a protective layer of immunity, indicated by the persistence of Spike-specific memory B cells and by the presence of neutralizing antibodies against the Alpha and Delta variants. However, lack of neutralizing antibodies against the Beta, Gamma and Omicron variants even during the peak response is of major concern as this indicates viral evasion of the humoral immune response.

Robust induction of neutralizing antibodies against the SARS-CoV-2 Delta variant after homologous Spikevax or heterologous Vaxzevria-Spikevax vaccination.

Sera of vaccines were assessed by surrogate virus neutralization tests for their capacity to neutralize the SARS-CoV-2 Delta variant. Homologous prime-boost immunization with Moderna's Spikevax as well as heterologous immunization with AstraZeneca's Vaxzevria followed by Moderna's Spikevax were identified as highly potent vaccination regimens for the induction of Delta-neutralizing antibodies.

Imaging dendritic cell functions.

Dendritic cells (DCs) are crucial for the appropriate initiation of adaptive immune responses. During inflammation, DCs capture antigens, mature, and migrate to lymphoid tissues to present foreign material to naïve T cells. These cells get activated and differentiate either into pathogen-specific cytotoxic CD8+ T cells that destroy infected cells or into CD4+ T helper cells that, among other effector functions, orchestrate antibody production by B cells. DC-mediated antigen presentation is equally important in non-inflammatory conditions. Here, DCs mediate induction of tolerance by presenting self-antigens or harmless environmental antigens and induce differentiation of regulatory T cells or inactivation of self-reactive immune cells. Detailed insights into the biology of DCs are, therefore, crucial for the development of novel vaccines as well as the prevention of autoimmune diseases. As in many other life science areas, our understanding of DC biology would be extremely restricted without bioimaging, a compilation of methods that visualize biological processes. Spatiotemporal tracking of DCs relies on various imaging tools, which not only enable insights into their positioning and migration within tissues or entire organs but also allow visualization of subcellular and molecular processes. This review aims to provide an overview of the imaging toolbox and to provide examples of diverse imaging techniques used to obtain fundamental insights into DC biology.

Intranasal Delivery of MVA Vector Vaccine Induces Effective Pulmonary Immunity Against SARS-CoV-2 in Rodents.

Antigen-specific tissue-resident memory T cells (Trms) and neutralizing IgA antibodies provide the most effective protection of the lungs from viral infections. To induce those essential components of lung immunity against SARS-CoV-2, we tested various immunization protocols involving intranasal delivery of a novel Modified Vaccinia virus Ankara (MVA)-SARS-2-spike vaccine candidate. We show that a single intranasal MVA-SARS-CoV-2-S application in mice strongly induced pulmonary spike-specific CD8+ T cells, albeit restricted production of neutralizing antibodies. In prime-boost protocols, intranasal booster vaccine delivery proved to be crucial for a massive expansion of systemic and lung tissue-resident spike-specific CD8+ T cells and the development of Th1 - but not Th2 - CD4+ T cells. Likewise, very high titers of IgG and IgA anti-spike antibodies were present in serum and broncho-alveolar lavages that possessed high virus neutralization capacities to all current SARS-CoV-2 variants of concern. Importantly, the MVA-SARS-2-spike vaccine applied in intramuscular priming and intranasal boosting treatment regimen completely protected hamsters from developing SARS-CoV-2 lung infection and pathology. Together, these results identify intramuscular priming followed by respiratory tract boosting with MVA-SARS-2-S as a promising approach for the induction of local, respiratory as well as systemic immune responses suited to protect from SARS-CoV-2 infections.

Immune responses against SARS-CoV-2 variants after heterologous and homologous ChAdOx1 nCoV-19/BNT162b2 vaccination.

Currently approved viral vector-based and mRNA-based vaccine approaches against coronavirus disease 2019 (COVID-19) consider only homologous prime-boost vaccination. After reports of thromboembolic events, several European governments recommended using AstraZeneca's ChAdOx1-nCov-19 (ChAd) only in individuals older than 60 years, leaving millions of already ChAd-primed individuals with the decision to receive either a second shot of ChAd or a heterologous boost with mRNA-based vaccines. However, such combinations have not been tested so far. We used Hannover Medical School's COVID-19 Contact Study cohort of healthcare professionals to monitor ChAd-primed immune responses before and 3 weeks after booster with ChAd (n = 32) or BioNTech/Pfizer's BNT162b2 (n = 55). Although both vaccines boosted prime-induced immunity, BNT162b2 induced significantly higher frequencies of spike-specific CD4+ and CD8+ T cells and, in particular, high titers of neutralizing antibodies against the B.1.1.7, B.1.351 and P.1 variants of concern of severe acute respiratory syndrome coronavirus 2.

Targeted delivery of regulatory macrophages to lymph nodes interferes with T cell priming by preventing the formation of stable immune synapses.

Immunosuppressive myeloid cells are frequently induced in tumors and attenuate anti-tumor effector functions. In this study, we differentiate immunosuppressive regulatory macrophages (Mregs) from hematopoietic progenitors and test their potential to suppress adaptive immune responses in lymph nodes. Targeted delivery of Mregs to lymph nodes is facilitated by retroviral overexpression of the chemokine receptor CCR7 and intra-lymphatic cell application. Delivery of Mregs completely abolishes the priming of cognate CD8 cells and strongly reduces delayed-type hypersensitivity reactions. Mreg-mediated T cell suppression requires cell-cell contact-regulated nitric oxide production. Two-photon microscopy reveals that nitric oxide produced by Mregs reduces the interaction duration between dendritic cells and T cells. Exposure of activated T cells to nitric oxide strongly reduces their binding to ICAM-1, indicating that nitrosylation of proteins involved in cell adhesion affects synapse formation. Thus, this study identifies a mechanism of myeloid cell-mediated immune suppression and provides an approach for its therapeutic use.

CRISPR/Cas9 Immunoengineering of Hoxb8-Immortalized Progenitor Cells for Revealing CCR7-Mediated Dendritic Cell Signaling and Migration Mechanisms in vivo.

To present antigens to cognate T cells, dendritic cells (DCs) exploit the chemokine receptor CCR7 to travel from peripheral tissue via afferent lymphatic vessels to directly enter draining lymph nodes through the floor of the subcapsular sinus. Here, we combined unlimited proliferative capacity of conditionally Hoxb8-immortalized hematopoietic progenitor cells with CRISPR/Cas9 technology to create a powerful experimental system to investigate DC migration and function. Hematopoietic progenitor cells from the bone marrow of Cas9-transgenic mice were conditionally immortalized by lentiviral transduction introducing a doxycycline-regulated form of the transcription factor Hoxb8 (Cas9-Hoxb8 cells). These cells could be stably cultured for weeks in the presence of doxycycline and puromycin, allowing us to introduce additional genetic modifications applying CRISPR/Cas9 technology. Importantly, modified Cas9-Hoxb8 cells retained their potential to differentiate in vitro into myeloid cells, and GM-CSF-differentiated Cas9-Hoxb8 cells showed the classical phenotype of GM-CSF-differentiated bone marrow-derived dendritic cells. Following intralymphatic delivery Cas9-Hoxb8 DCs entered the lymph node in a CCR7-dependent manner. Finally, we used two-photon microscopy and imaged Cas9-Hoxb8 DCs that expressed the genetic Ca2+ sensor GCaMP6S to visualize in real-time chemokine-induced Ca2+ signaling of lymph-derived DCs entering the LN parenchyma. Altogether, our study not only allows mechanistic insights in DC migration in vivo, but also provides a platform for the immunoengineering of DCs that, in combination with two-photon imaging, can be exploited to further dissect DC dynamics in vivo.

Dendritic cell migration in health and disease.

Dendritic cells (DCs) are potent and versatile antigen-presenting cells, and their ability to migrate is key for the initiation of protective pro-inflammatory as well as tolerogenic immune responses. Recent comprehensive studies have highlighted the importance of DC migration in the maintenance of immune surveillance and tissue homeostasis, and also in the pathogenesis of a range of diseases. In this Review, we summarize the anatomical, cellular and molecular factors that regulate the migration of different DC subsets in health and disease. In particular, we focus on new insights concerning the role of migratory DCs in the pathogenesis of diseases of the skin, intestine, lung, and brain, as well as in autoimmunity and atherosclerosis.

Chemokines and Chemokine Receptors in Lymphoid Tissue Dynamics.

The continuous migration of immune cells between lymphoid and nonlymphoid organs is a key feature of the immune system, facilitating the distribution of effector cells within nearly all compartments of the body. Furthermore, reaching their correct position within primary, secondary, or tertiary lymphoid organs is a prerequisite to ensure immune cells' unimpaired differentiation, maturation, and selection, as well as their activation or functional silencing. The superfamilies of chemokines and chemokine receptors are of major importance in guiding immune cells to and within lymphoid and nonlymphoid tissues. In this review we focus on the role of the chemokine system in the migration dynamics of immune cells within lymphoid organs at the steady state and on how these dynamics are affected by infectious and inflammatory processes.

Lymph node T cell homeostasis relies on steady state homing of dendritic cells.

Little is known about mechanisms determining the homeostasis of lymphocytes within lymphoid organs. Applying different mouse models, including conditionally proficient Ccr7 gene-targeted mice, we now show that semimature steady state dendritic cells (sDCs) constitutively trafficking into lymph nodes (LNs) were essential contributors to T cell homeostasis in these organs. sDCs provided vascular endothelial growth factor known to support high endothelial venule formation, thus facilitating enhanced homing of T cells to LNs. The presence of sDCs led to increased CCL21 production in T-zone fibroblastic reticular cells. CCL21 is a ligand for CCR7 known to regulate homing as well as retention of T cells in LNs. In addition, we provide evidence that CCL21 binds to the surface of DCs via its heparin-binding domain, further explaining why T cells leave LNs more rapidly in the absence of sDCs. Together, these data reveal multiple roles for sDCs in regulating T cell homeostasis in LNs.

Retinoic acid induces homing of protective T and B cells to the gut after subcutaneous immunization in mice.

Diarrheal diseases represent a major health burden in developing countries. Parenteral immunization typically does not induce efficient protection against enteropathogens because it does not stimulate migration of immune cells to the gut. Retinoic acid (RA) is critical for gut immunity, inducing upregulation of gut-homing receptors on activated T cells. In this study, we have demonstrated that RA can redirect immune responses elicited by s.c. vaccination of mice from skin-draining inguinal LNs (ingLNs) to the gut. When present during priming, RA induced robust upregulation of gut-homing receptors in ingLNs, imprinting gut-homing capacity on T cells. Concurrently, RA triggered the generation of gut-tropic IgA+ plasma cells in ingLNs and raised the levels of antigen-specific IgA in the intestinal lumen and blood. RA applied s.c. in vivo induced autonomous RA production in ingLN DCs, further driving efficient induction of gut-homing molecules on effector cells. Importantly, RA-supplemented s.c. immunization elicited a potent immune response in the small intestine that protected mice from cholera toxin­induced diarrhea and diminished bacterial loads in Peyer patches after oral infection with Salmonella. Thus, the use of RA as a gut-homing navigator represents a powerful tool to induce protective immunity in the intestine after s.c. immunization, offering what we believe to be a novel approach for vaccination against enteropathogens.

Mesenteric lymph node stroma cells in the generation of intestinal immune responses.

Lymph nodes at different anatomical locations share similar architecture and operate on the basis of identical principles. Still, the quality of immune responses is modified substantially by the local peculiarities at the site of its induction. Here, we discuss how lymph node stroma cells contribute to functional differences between various lymph nodes, thus helping to explain why and how an immune response induced in skin draining peripheral lymph nodes differs from that elicited in the gut draining mesenteric lymph nodes. Stroma cells constitute a major part of the lymph node scaffold and control the flow of immune cells as well as soluble substances within the organ. Moreover, stroma cells express cytokines, chemokines as well as adhesion factors and thereby actively influence immune status. Lymph node transplantations and adoptive transfers of dendritic cells demonstrated that regional lymph node stroma cells differ in their ability to support mucosal tolerance, the induction of tissue tropism, and humoral immunity. This suggests that stroma cells shape tissue-specific immune responses and equip lymph nodes with unique functional properties that might originate during lymph node organogenesis.

Stromal mesenteric lymph node cells are essential for the generation of gut-homing T cells in vivo.

T cells primed in the gut-draining mesenteric lymph nodes (mLN) are imprinted to express alpha4beta7-integrin and chemokine receptor CCR9, thereby enabling lymphocytes to migrate to the small intestine. In vitro activation by intestinal dendritic cells (DC) or addition of retinoic acid (RA) is sufficient to instruct expression of these gut-homing molecules. We report that in vivo stroma cells, but not DC, allow the mLN to induce the generation of gut tropism. Peripheral LN (pLN) transplanted into the gut mesenteries fail to support the generation of gut-homing T cells, even though gut-derived DC enter the transplants and prime T cells. DC that fail to induce alpha4beta7-integrin and CCR9 in vitro readily induce these factors in vivo upon injection into mLN afferent lymphatics. Moreover, uniquely mesenteric but not pLN stroma cells express high levels of RA-producing enzymes and support induction of CCR9 on activated T cells in vitro. These results demonstrate a hitherto unrecognized contribution of stromal cell delivered signals, including RA, on the imprinting of tissue tropism in vivo.